ES2376665T3 - SELECTIVE PRODUCTION PROCEDURE FOR MALE OR FEMALE STERILE PLANTS. - Google Patents

Abstract

A method of producing sterile male or female plants comprising the steps of transforming plant material with a polynucleotide encoding at least one enzyme that reacts with a non-phytotoxic substance to produce a cytotoxic and regenerate the material thus transformed into a plant, in which said non-phytotoxic substance is applied to the plant until the formation and / or maturation of male or female gametes, so that the non-phytotoxic substance enables the production of a phytotoxic that selectively prevents the formation of or acts as a otherwise to said non-functional gametes, in which the enzyme is preferentially expressed in male or female reproductive structures and the non-phytotoxic substance is a D-alpha amino acid, characterized in that the enzyme is a D-amino acid oxidase obtained from Rhodotorula gracilis and encoded by the nucleic acid sequence as represented in SEQ ID NO: 3, said enzyme being a mutated version of the protein encoded by SEQ ID NO: 3 and comprising a lysine at position 58 and at position 213 an amino acid selected from the group consisting of Thr, Gln and Gly; at position 238 an amino acid selected from the group consisting of Thr, Gln and Gly and / or at position 223 an amino acid selected from the group consisting of Thr, Cys, Gly, Gln and / or Asn.

Description

Selective production procedure for male or female sterile plants

Heterosis in crop plants can have a noticeable effect on improving performance. In general, hybrids show increased yields compared to non-hybrid varieties. Hybrids usually provide a greater return unit for growth factors such as water and fertilizers. Hybrids often offer superior strain tolerance, product uniformity and maturity, and also provide a simple breeding opportunity to combine traits or traits that may be difficult to combine in other ways. The vigor of the hybrid in plants is generally of sufficient magnitude to guarantee commercial exploitation. Commercial hybrids are used extensively on many crops including corn, sorghum, sugar beet, sunflower, and rapeseed. However, mainly due to the lack of inexpensive hybrid seed production procedures, wheat, barley and rice are still mainly cultivated as inbreeds.

Traditionally, hybrid seed production involves planting separate blocks of male and female parental lines with only the seed being collected from the female parent. To ensure that this seed is hybrid, self-pollination of the female parental line should be minimized by making the male line sterile. Procedures for making the female parent line sterile include mechanical, chemical, and genetic procedures. In dioecious plants, such as corn, male sterility can be easily achieved mechanically by detachment of the male inflorescence. However most crops are monoecious and having male and female organs within the same flower makes such physical emasculation impractical. Therefore genetic approaches have been used on occasion.

Genetic male sterility traits that normally appear are controlled by nuclear genes in which alleles associated with the sterile phenotype are generally expressed recessively relative to the corresponding alleles associated with fertility. When genetic male sterility occurs it is normally associated with a single recessive gene that must be homozygous for male sterility to be expressed. To make practical use of such genetic male sterility traits, breeders typically develop a phenotypically uniform female line that secretes into male sterile and male fertile plants. Male fertile plants, once identified, need to be refined which requires a lot of work. There is always a problem with maintaining the parental line since male fertile plants cannot be removed from the population because they are essential for population maintenance. Instead of relying on the existence of natural male sterility alleles, it is also possible to use molecular biological procedures. Plants can be engineered to express, for example, antisense or ribozyme genes that decrease or eliminate the expression of key genes necessary for the formation of viable pollen. Such transgenic plant lines are male sterile and are used for hybrid seed production by cross-pollen from male fertile plants. The main problem with such lines is that they can only be kept in a heterozygous state in subsequent generations, by crossing with the isogenic fertile lines. This can be a problem in hybrid seed production where performance is critical. Although, for example by linking herbicide resistance to male sterility, it may be possible to selectively debug male fertile plants this still requires plants to be initially planted at very high densities.

The use of cytoplasmic male sterility for commercial hybrid production requires a stable male sterile cytoplasm and a source of pollen. The male sterility cytoplasmic genetic system requires the existence of three types of line for hybrid production: line A (male cytoplasmic sterile), line B (male fertile maintainer) and line R (male fertile with restorative genes). Three-way crossings produced with this system involve the maintenance and production of four lines, one line A and one B of one inbred and fertile male inbred of the other two. Dependence on a single source of male sterile cytoplasm can minimize culture flexibility and lead to offspring with widespread susceptibility to particular diseases.

Hybrid seed can also be produced through the use of chemical agents that inhibit the formation of viable pollen. These chemical agents, called gametocides, are used to transmit transient male sterility. Whatever the expense, the registrability and reliability of gametocides has limited their use.

A shortcoming of traditional hybrid seed production systems is the need to plant separate rows or blocks of the male and female parent lines. Here low-efficiency pollination is an especially acute problem in crop species, such as wheat, that release small amounts of pollen that does not travel far in the wind. In such crops up to two-thirds of the hybrid-producing field needs to be devoted to male pollen-donor plants, and then the production of hybrid seed is therefore wasteful.

To achieve cheaper seed production in wheat and other crops it is necessary to move the male and female plants closer to each other for a more efficient pollen transfer; more effectively by interleaving males and females at a distance of centimeters from each other in the same rows. In a

Such a system would not be practical to collect only the seed from the female (male sterile) parents. Contamination with non-hybrid seed originating from the male parent can be minimized by using as low a percentage of such male parent plants in the planting mixture as possible and / or by using male plants that are female sterile. A procedure for constructing a dominant female sterile line has been described (EP 412.006 A1 (1990); Goldman et al., (1994) EMBO. J., 13, 2976-2984) but, as with male sterile lines, the line has to be kept heterozygous.

Consequently, there remains a need for simple inexpensive hybrid seed production procedures. In particular, to efficiently produce hybrid seed there remains a need to provide both male sterile parental female lines and female sterile male parental lines that can be easily maintained as pure homozygous lines and that are useful for efficient hybrid seed production. Procedures described in the art to achieve this include procedures in which hybrid seed is produced from male and female parental lines at least one of which comprises a heterologous chimeric gene, preferably expressed in floral tissue, which makes the conditionally sterile line depending on the exogenous application of a non-phytotoxic substance that can be specifically and locally converted to a phytotoxin by an enzyme that is encoded by the heterologous chimeric gene and that is preferentially expressed in the male or female reproductive structures. The non-phytotoxic substance can be a proherbicide. The advantage of having such conditionally sterile parental lines is that it allows them to remain homozygous with respect to the sterility trait. Fertility is interrupted only after exogenous application of the non-phytotoxic substance. In such an example of a conditional male sterility system a gene encoding a deacetylase enzyme is preferentially expressed in cells of the male flower tissue mat in which it converts the proherbicide N-acetyl L phosphinothricin applied exogenously to phytotoxin L phosphinothricin and thus avoids the formation of viable pollen. In additional similar examples: (i) the preferred expression on the mat of a bacterial cytochrome P450 catalyzed the conversion of proherbicide R7402 to a sulfonylurea phytotoxin that prevents the production of viable pollen and (ii) the preferred expression on the mat of a phosphonate Monoester hydrolase catalyzes the conversion of glyceryl glyphosate proherbicide to phytotoxin glyphosate which also prevents the production of viable pollen. WO 98/03838 describes examples of a conditional female sterility system in which enzymes capable of converting proherbicides to phytotoxins are preferably expressed in female reproductive structures.

Despite the existence of these procedures for making male and female parental lines that are conditionally sterile, hybrid seed production remains far from routine in crops such as wheat. The current inventions relate, among others, to improvements in the technique with respect to the generation of female parental lines that are sterile male conditional and male parental lines that are sterile female conditional.

The present invention relates to improvements in processes for the production of hybrid crop seed. In particular, the invention relates to a method of producing hybrid seed from male and female parental lines, at least one of which is conditionally sterile female or male depending on the exogenous application of a substance that is not phytotoxic for cultivation and that includes proherbicides. The invention further relates to a process in which said non-phytotoxic substance is applied at a time and in sufficient quantity so that self-fertilization is minimized or avoided in the conditionally sterile parental line or lines. The present invention also relates to a method of generating conditionally sterile male or female plants i) by transforming plant material with one or more chimeric genes that, individually or together, encode one or more enzymes capable of reacting with a non-phytotoxic substance, preferably in the form of a proherbicide, to produce a phytotoxic one. Enzymes are expressed under the operational control of one or more promoters that, in the case of conditionally male sterile plants, cause the enzyme or enzymes to be preferentially expressed in male reproductive structures or that, in the case of conditionally female sterile plants, it causes said enzyme or enzymes to be preferentially expressed in the female reproductive structures. Plant material is regenerated into fertile, morphologically normal plants that are male or female sterile conditionals. The invention also includes the use of conditionally male sterile plants in combination with conditionally female sterile plants to produce hybrid seed more effectively, the use, as non-phytotoxic substances, of certain proherbicides, and the use of chimeric genes to more efficiently produce hybrid seeds, genes chimerics and enzymes useful for the invention. The invention also provides conditionally male sterile plants, conditionally female sterile plants, seeds of these plants, and hybrid seeds produced by the method. In preferred embodiments of the invention, the crop plants to which the process for making hybrid seeds is applied are corn, rice, sorghum, wheat, millet, oats, rapeseed and barley.

In accordance with the present invention there is provided a method of producing sterile male or female plants comprising the steps of transforming plant material with a polynucleotide encoding at least one enzyme that reacts with a non-phytotoxic substance to produce a phytotoxic and regenerate the material. thus transformed into a plant, in which said non-phytotoxic substance is applied to the plant until the moment of formation of male or female gametes and / or maturation, so that the non-phytotoxic substance enables the production of a phytotoxic that selectively avoids the formation of or otherwise such non-functional gametes, in which the enzyme is preferentially expressed in male or female reproductive structures, characterized in that (i) the non-phytotoxic substance is selected from the group consisting of D-alpha amino acids,

peptide derivatives of non-protein D-alpha amino acids and (ii) the enzyme is a mutant form of a D-amino acid oxidase that has a lysine at the position corresponding to phenylalanine at position 58 of Rhodotorula gracilis type D-amino acid oxidase wild.

Since it is a desirable goal to maximize the yield of the hybrid seed and therefore to minimize any damage to the crop, in preferred embodiments, the non-phytotoxic substance is a proherbicide selected from compounds that are relatively non-phytotoxic to the crop. In order to have an effect against floral tissues it is also desirable that proherbicides be parents of phytotoxins that are effective in "non-green" tissues. Therefore, in preferred embodiments of the invention, proherbicides are selected from those that are parents of phytotoxins that are directly phytotoxic to non-green tissues rather than those that have a major site of action in photosynthesis or in the generation of photosynthetic pigments. It is also a desirable goal to minimize hybrid seed production costs. Therefore, in preferred embodiments, proherbicides are selected from those chemicals for which the approval of appropriate regulatory authorities for use in crops has already been granted or is pending.

Nomenclature: Definitions

"Gene" as used herein refers to any DNA sequence comprising several operably linked DNA fragments such as a promoter and a 5 'regulatory region, a coding sequence and a 3' untranslated region that comprises a site of polyadenylation.

"Chimeric" when referring to a gene or DNA sequence is used to refer to the fact that in nature, the coding sequence is not associated with the promoter or with at least one other DNA regulatory region in the gene.

"Chimeric gene" as used herein refers to a gene in which, in nature, the coding sequence is not associated with the promoter or with at least one other DNA regulatory region in the gene.

"Expression cassette" as used herein refers to a transferable region of DNA that comprises a chimeric gene that is flanked by one or more restriction sites or others that facilitate precise excision of a DNA locus and insertion into other.

"Non-phytotoxic substances" are, in the context of the present invention, substances that are relatively non-phytotoxic to plants, cells or tissues of any particular culture to which the method of the invention is applied. Non-phytotoxic substances need not be non-phytotoxic in all plant tissues of all plants. Non-phytotoxic substances include proherbicides which are substances with no appreciable direct toxic effect on plant tissues but which are progenitors of active phytotoxins. In susceptible plant species, such proherbicides act indirectly as herbicides through the action of endogenous enzymes that convert them into a phytotoxin in the plant.

"Phytotoxins" are, in the context of the present invention, substances that are toxic to plants, plant tissues and plant cells of the particular culture for which the method of the invention is applied. Such phytotoxins need not be phytotoxins for all plant tissues of all plant species.

"Female reproductive structure" means the female gametes and the parts of the plant that specialize in the production, maturation, and viability of female gametes. Normally this includes the parts of a plant that comprise the carpel or gyneceus ("pistil"). Carpel of a plant includes, but is not limited to, a stigma, style, ovary, and cells or tissues that are comprised of the stigma, style, and ovary.

"Male reproductive structure" means the male gametes and the parts of the plant that are specialized in the production, maturation, and viability of male gametes. This comprises the parts of a plant comprising, for example, microspores, stamens, rugs, anthers, and pollen.

"Female sterile plant" as used herein is a plant that is unable to withstand viable seed formation when pollinated with functional or viable pollen. Such female sterility may be the result of reproductive selection or the presence of a transgene. A "conditionally female sterile plant" refers to a plant that under normal growing conditions is female fertile and can be made female sterile under specific conditions. In the context of the present invention, said conditions comprise the exogenous application of a proherbicide or other non-phytotoxic substance. In the context of the present invention, said "female sterile plant" or "conditionally female sterile plant" continues to be male fertile and capable of producing viable pollen.

"Male sterile plant" as used herein is a plant that is unable to withstand the formation of viable pollen. Such male sterility may be the result of reproductive selection or the presence of a transgene. A "conditionally male sterile plant" refers to a plant that under normal growing conditions is male fertile and can be made male sterile under specific conditions. For example, conditions could include physical emasculation or application of a specific chemical gametocide. In the context of the present invention, said conditions particularly comprise the exogenous application of a proherbicide or other non-phytotoxic substance. In the context of the present invention a

Such a "male sterile plant" or "conditionally male sterile plant" remains fertile female and capable of producing viable seeds when pollinated with functional or viable pollen.

"Promoter region" as used herein is a DNA region comprising at least one functional promoter and, optionally, some or all of its upstream associated regulatory sequences including enhancer sequences and / or downstream associated sequences including part of or the entire 5 'untranslated region of the endogenous promoter gene.

"Intercropping" as used herein refers to a method of planting seeds or plants in a field that ensures adequate cross-pollination of male sterile or conditionally male sterile plants by male fertile plants. This can be accomplished by randomly mixing male and female parent seed in different mixes (80/20; 90/10; etc.) before planting or planting in specific field patterns where different seeds alternate. When separate collection of different plants is required, planting in alternating blocks or rows is preferred.

In the process according to the invention, said non-phytotoxic substance can be applied in mixture together with at least one additional substance that can be selected from the group consisting of amino acids, healers, gametocides, glutathione-S-transferase inducers, cytochrome P450 inducers, fertilizers, herbicides, nematocides, synergists, insecticides, fungicides, hormones, plant growth regulators and cytochrome P450 inhibitors. In particular embodiments, said non-phytotoxic substance can be applied in a mixture with the same phytotoxic substance from which the non-phytotoxic substance is a parent.

The non-phytotoxic substance is a D amino acid and in particular it may be the D enantiomer of phosphinothricin, the D enantiomer of bialaphos or selected from the group consisting of D aspartate and D glutamate.

D-amino acid oxidases are mutant forms of the Rhodotorula gracilis enzyme. Such mutants always have a lysine at position 58 (ie they are F58K mutants of the wild type sequence) and comprise single, double or triple additional amino acid substitutions at positions 213, 223 and 238 compared to the wild type sequence . At position 213 the wild-type methionine is replaced with Thr, Gly, or Gln and / or the wild-type tyrosine at position 223 is replaced with Thr, Gly, Gln, Cys or Asn and / or the wild-type tyrosine at position 238 it is replaced with Thr, Gly, or Gln. In a particularly preferred embodiment the methionine at position 213 is replaced with threonine.

When the non-phytotoxic substance is a D-amino acid other than D-phosphinothricin or D-bialaphos then the D-amino acid is preferably not an endogenous plant metabolite and is selected to be mobile in the phloem, metabolically stable in the plant (preferably having a t 1/2 in the plant over ∼ 1 week) and an effective substrate for D-amino acid oxidase. The oxidation of D amino acid by the enzyme is combined with the reduction of oxygen to phytotoxic peroxide anions.

In a preferred embodiment the enzyme oxidase targets a subcellular location other than the peroxisome. This is achieved, for example, by modifying the gene so that three C-terminal amino acids are deleted or modified (for example SKL in the case of the amino acid oxidase D derived from Rhodotorula gracilis) and / or by adding sequence to add a chloroplast or Mitochondrial transit peptide to the N-terminus.

Additional suitable D-amino acid oxidases can be obtained preferably from fungal sources, by mutation and selective procedures known to those skilled in the art and augmented by the present disclosure.

Mutant D-amino acid oxidase enzymes and additional DNA coding sequences suitable for making the invention are obtained by expressing candidate mutant D-amino acid oxidase libraries in a suitable host cell such as E. coli or yeast (suitable host strains lack activity oxidase

or endogenous dehydrogenase against D-phosphinothricin) for transformation to a phenotype with increased sensitivity to growth inhibition by D-phosphinothricin in minimal medium. This procedure is based on the ability of transformed E. coli clones to produce L-PPT from D-PPT through the combined action of their endogenous L transaminase activity and heterologously expressed oxidase. Alternatively, suitable and improved genes are selected based on in vitro assay of the expressed enzyme for the desired ability to oxidize D-phosphinothricin. There are many procedures for directly assaying for D-amino acid oxidase activities such as based on peroxide detection (Enzyme Microb. Technol., (2000), 27 (8), 605-611), oxygen depletion using an oxygen electrode or based on direct detection of ammonia or the keto acid product.

It is disclosed that a fungus-derived DAMOX gene is cloned into a shuttle vector under the operational control of a promoter (eg, GAL promoter) capable of expression in the host organism in which the selection will take place (preferably yeast). This gene is then subjected to mutagenesis, for example by Mn2 + poisoned PCR; replication of plasmid DNA in a strain that is defective in DNA repair / editing processes such as the E. coli XL1 red strain; or by replication of plasmid DNA in a host strain that undergoes mutagenesis using for example X-rays, ultraviolet light, addition of a chemical mutagen and is transformed into a host organism (preferably yeast). The desired DNA encoding a

DAMOX having the desired property of an enhanced ability to oxidize D-PPT is selected (following an optional initial selection step for transformants based on selectable markers present in the shuttle vector that allow, for example, selection by prototrophy restoration or growth in presence of hygromycin etc.) by, for example

a) Selection of transformed cells that have the ability to use amino acids that are chemically similar to D-phosphinothricin as the sole source of nitrogen. For example, transformed yeast colonies that are capable of growing on D-PPT analogs (and their esters) in which the phosphinic acid residue is replaced with a carboxylate (i.e., D-glutamate), sulfonate, phosphonate, are selected. sulfone or sulfoxide residues (or esters thereof) as the sole source of N. For example,

b) Selection of transformed cells capable of using D-PPT itself as the sole source of N. For this selection, the host cell is also transformed with a gene capable of negating the inhibitory effect of Lphosphinotricin on glutamine synthetase. For example, the shuttle vector also comprises a gene encoding an enzyme such as PAT that inactivates L-PPT.

Mutation and selection cycles can be repeated. D-amino acid oxidases can additionally be cloned, expressed, partially purified and kinetically characterized to identify genes and DAMOX with the most suitable properties (for example enzymatic stability, high kcat / Km value for D-PPT oxidation, minimal oxidation of any substrate endogenous plant, optimal pH, etc.).

When the non-phytotoxic substance is D-phosphinothricin (PPT), it can be obtained from a mixture of D and L PPT. For example, DL PPT can be added to a culture medium (preferably minimal) of E. coli cells (optionally an arg E mutant to minimize the background level of N-acetyl PPT deacetylase activity) when E. coli is transformed to express a PAT gene (encoding an enzyme that transfers an acetyl group from acetyl CoA to L-PPT) at a high level (eg inducibly, after addition of IPTG). Preferably, E. coli is also genetically engineered to express acetyl CoA synthetase. After allowing adequate time for the L-component of the phosphinothricin to become substantially complete N-acetyl (judged, for example, by controlling the conversion using 31-P NMR) D-PPT is recovered and purified from the cell-free medium using successive steps of , eg, high and low pH solvent extraction, anion and cation exchange chromatography, selective crystallization with chiral cations such as quincocin, or other procedures known in the art such as liquid / liquid extraction with two aqueous non-miscible phases such as phase system (see procedures in USP 5,153,355). Typically a later step is cation exchange chromatography from which D-PPT is recovered as the ammonium salt.

Alternatively, D-PPT can be obtained by an enzymatic procedure in which DL PPT + 2-ketoglutarate is converted to primarily a mixture of D-PPT, 2-oxo PPT (and its decarboxylation products), and GABA by the combined actions of (I) L-aminotransferase (eg from E. coli) and (II) glutamate decarboxylase. The desired pure D-PPT is resolved from the reaction mixture using procedures known in the art and as described above.

D-PPT can also be obtained using an enzymatic procedure in which DL PPT + 2-ketoglutarate + NAD

it is converted to mainly a mixture of D-PPT, 2-oxo PPT (and its decarboxylation products) NADH and ammonia by the combined actions of (I) L-aminotransferase and (II) glutamate dehydrogenase. The desired D-PPT is purified from the reaction mixture.

In a still further procedure to prepare D-PPT, DL PPT is treated with an L-amino acid oxidase so that the only remaining amino acid is the desired D-form. This D-PPT is purified after the reaction mixture.

A still further procedure involves (I) conversion of DL PPT to N-acetyl DL PPT (using acetic anhydride or other acetylating reagents and procedures well known in the art) and (II) treatment of N-acetyl DL PPT with D-aminoacylase from so only N-acetyl-D-PPT is deacetylated. The resulting D-PPT is purified from the reaction mixture. For example, D-PPT is resolved from N-acetyl-L-PPT by binding to Dowex anion exchange resin and elution with 40mM formic acid. Under suitable loading conditions this acid elutes D-PPT leaving N-acetyl L-PPT bound to the column.

A still further procedure involves treatment of DL PPT with L-amino acylase and an acylating agent in a nonaqueous solvent so that only the desired D-PPT is left in a non-acetylated form.

A still further procedure to prepare pure D-PPT involves enantioselective crystallization of DL PPT using a chiral base such as quincocin and addition of a crystallization nucleus of the chiral base with pure D-PPT.

A further additional procedure to prepare pure D-PPT from DL-PPT by direct chiral chromatography using a chiral base column.

A detailed procedure for the production of D-PPT is provided in one of the Examples below.

The DNA sequences encoding the enzymes used in the present invention can, optionally, be further mutated and selected to generate additional useful enzymes that have improved utility. Many enzyme characteristics are improved in this way including catalytic activity (kcat / Km) against the desired substrate, temperature stability and optimal pH. Procedures for generating, exploring and selecting for such improved variants are well known. For example, suitable variant DNA sequences are generated by a mutagenesis procedure (eg, by passing DNA through bacterial or yeast strains with error-prone DNA replication such as E. coli XL1 red, by PCR mutagenesis of target oligonucleotides. , chemical or UV). In particular such genes are produced by any of several alternative DNA shuffling or "sexual PCR" procedures as, for example, document WO 00/61740 on pages 28-41 is summarized. Many procedures are suitable for selecting such improved genes. Genes can be adequately expressed in a suitable host cell such as E. coli or yeast and selected for improvement using suitable assays such as, for example, those described herein.

Chimeric genes encoding enzymes for use in the invention that are capable, individually or in combination with one another, of converting a non-phytotoxic to a phytotoxic substance, may each comprise a DNA sequence encoding one of said enzymes operably linked to a 5 'promoter region that preferentially directs expression to male or female reproductive structures. This specificity of expression ensures that the effect of the expressed enzyme or enzymes will be exerted only within the locality of the tissues and cells necessary for the formation of viable seed or viable pollen and will not be deleterious to the plant beyond its effect on the fertility in the presence of a suitable non-phytotoxic substance, perhaps a proherbicide. In addition to chimeric genes from promoter regions according to the present invention it also comprises a 3 'transcription terminator sequence. This is responsible for the termination of transcription and correct polyadenylation of mRNA. Many such 3 'transcription terminator sequences are known in the art and are suitable for use in the chimeric genes of the present invention. In particular embodiments the 3 'transcription terminator sequence is selected from the CMV 35S terminator, the tml terminator, the nopaline synthase (nos) terminator, and the pea rbcS E0 terminator.

5 ’promoter regions suitable for use in certain embodiments of such chimeric genes include 5’ regions of genes that are preferentially expressed in female flower tissues. In certain embodiments the 5 'promoter region is selected from the group consisting of the tobacco stig 1 promoter (Goldman et al., (1994) EMBO J., 13, 2976-2984), a modified S13 promoter (Dzelkalns et al ( 1993) Plant Cell, 5, 8555), the AGL5 promoter (Savidge et al (1995) Plant Cell, 7, 721-733, and the 5 'promoter region of the corn carpel-specific ZAG2 gene (Thiessen et al (1995) Gene , 156, 155-166) Optionally, additional suitable promoter regions are obtained from regions upstream of the genomic DNA coding sequences that correspond to cDNA sequences known in the art that are preferentially expressed in female reproductive structures. Embodiments such probe cDNAs are selected from the group consisting of Arabidopsis Fbp7 and Fbp11 genes (Angenent et al., (1995) Plant Cell, 7, 1569-1582) and orchid-specific cDNAs O40, O108, 039, O126 and O141 (Nadeau et al., (1996) Plant Cell, 8, 213-239.) In particular embodiments en 5 'promoter regions are selected which comprise genomic DNA associated with preferential expression in female reproductive structures of DNA regions comprised within the group consisting of the genomic DNA clone pSH64 having accession number NRRL B-21920, genomic clone, pCIB10302 that hybridizes with the

P26-A4 cDNA having accession number NRRL B-21655 and genomic DNA clone X2-1 that hybridizes to cDNA clone P 19-QA having accession number NRRL B-21919. In further particular embodiments this promoter region comprises nucleotides 1 to 1390 of SEQ ID NO: 11, SEQ ID NO: 2 and nucleotides 1 to 1093 of SEQ ID NO: 4 in WO 98/39462. In further embodiments, additional 5 'promoter regions suitable for use in the chimeric genes of the invention are isolated and cloned by procedures familiar to those skilled in the art. For example, new transcripts expressed in female reproductive structures are identified by isolating RNA from tissues such as corn silk or wheat pistil followed by differential scanning using techniques such as differential presentation, subtraction of selected cDNA by PCR, and construction of subtractive cDNA library. CDNA clones that are preferentially expressed in female tissues and not in other parts of the plant such as leaves, roots and tufts are isolated. The tissue specificity of the expression is optionally further confirmed by Northern blotting. The cDNA clones are used as probes for genomic library scanning. 5 ’promoter regions and, optionally, 3’ untranslated DNA regions associated with preferential tissue expression of genomic DNA clones are obtained and used in the construction of chimeric genes for preferential expression in female reproductive structures.

5 ’promoter regions suitable for use in certain embodiments of such chimeric genes include 5’ regions of genes that are preferentially expressed in male flower tissues. These include promoter regions for expression in pollen, the mat, or other structures in the anther. In certain embodiments, these 5 'promoter regions are selected from the group consisting of the LAT52 promoter (Twell et al., (1989) Dev., 109, 705-713), the tomato A127 promoter (Dotson et al., ( 1996) Plant J., 10, 383-392), the maize Zmg promoter (Hamilton et al., (1989) Sex. Plant Reprod. 2, 208-212), the maize CDPK promoter (Guerro et al., (1990) Mol. Gen. Genet., 224, 161-168) and the anther-specific ant32 and ant43D promoters disclosed in USP 5477002. In certain further embodiments the 5 'promoter region is selected from the group consisting of the promoter maize specific CA55 mat ("Pca55" described in WO 92/13956), the mat-specific promoter E1 of rice (described in USP 5639948), the T72 mat-specific promoter of rice (described in document USP 5639948), the rice-anther RA8-specific promoter (Genbank / EMBL accession number AF042275; Jeon et al, (1999) PMB, 39, 35-44; WO 00 / 26389) the anther specific Tap1 promoter (Spena et al (1992) Theor Appl Genet 84, 520-527) and the maize ZmC5 pollen specific promoter (Genbank / EMBL accession number Y13285; Wakeley et al, (1998) PMB, 37, 187-192). Optionally, additional suitable promoter regions are obtained from upstream regions of the genomic DNA coding sequences corresponding to cDNA sequences, which are known in the art to be preferentially expressed in male reproductive structures. In certain embodiments such probe cDNAs are selected from the group consisting of the orchid pollin tube specific cytochrome P450 gene (Nadeau et al., (1996) Plant Cell, 8,213-239), the Arabidopsis Bcp1 gene (Xu et al (1995) PNAS, 92, 2106-2110) and the male corn flower specific MFS14 gene (Wright et al., (1993) Plant J, 3, 41-49). In further embodiments, additional 5 'promoter regions suitable for use in the chimeric genes of the invention are isolated and cloned by procedures familiar to those skilled in the art. For example, new transcripts expressed in male reproductive structures are identified by isolating RNA from tissues such as tufts, pollen tubes, anther or mat followed by differential scanning by techniques such as differential presentation, subtraction of selected cDNA by PCR, and construction of subtractive cDNA library . CDNA clones that are preferentially expressed in male tissues and not in other parts of the plant such as leaves, roots and stigma are isolated. The tissue specificity of the expression is optionally confirmed by Northern blotting. The cDNA clones are used as probes for genomic library scanning. 5 ’promoter regions and 3’ untranslated DNA regions associated with preferential tissue expression are obtained from genomic DNA clones and used in the construction of chimeric genes for preferential expression in male reproductive structures.

Additional promoter regions useful in the chimeric genes of the invention include the upstream regions of the rice Osmads 13 gene, the rice anther OSG gene and the rice YY2 gene. Generally, promoter regions that produce high, early, prolonged, and preferential expression in male and female reproductive structures are selected as the most suitable. The promoter regions may also additionally comprise chimeric combinations with each other and with additional enhancer regions.

Chimeric genes can optionally comprise a region, immediately preceding the DNA sequence encoding the enzyme involved in the conversion of non-phytotoxic substance to phytotoxin, encoding a peptide sequence capable of directing said enzyme to subcellular organelles such as the chloroplast, peroxisome (other than when the phytotoxin is a peroxide or superoxide anion) or mitochondria and said targeting protein may have the sequence of (i) a chloroplast transit peptide or (ii) an N-terminal part of chloroplast transit peptide from a chloroplast protein-chloroplast transit peptide. In particular, to target the mitochondria, said DNA region immediately preceding the enzyme-encoding DNA sequence, encodes a peptide sequence for transit of mitochondria. In certain embodiments, the transit peptide sequence can be selected from the group consisting of endogenous transit peptide sequences of the NADP beta subunit mitochondrial Nicotinia plumbaginifolia, mitochondria-specific NADP-dependent isocitrate dehydrogenase, NADPH-binding subunit of the complex of respiratory chain I and yeast mitochondrial tryptophanylRNA-synthetase.

The polynucleotides for use in the present method of the invention may comprise one or more genes.

chimeric encoding enzymes that catalyze reactions involved in the generation of phytotoxins from non-phytotoxic substances. Optionally such polynucleotides comprise more additional genes and chimeric genes, such as a chimeric marker gene. A chimeric marker gene as used herein comprises a marker DNA under the control of expression of a promoter that is active in plant cells. Marker DNA encodes an RNA, protein, or polypeptide that, when expressed in a plant, plant tissue, or plant cell, allows such plant material to be distinguished from plant material that does not express the marker DNA. Examples of marker genes are genes that provide a specific color to a cell such as the A1 gene (Meyer et al. (1987) Nature 330, 667) or genes that make plant cells resistant to selection otherwise lethal with antibiotics ( for example the aac (6 ') gene encoding resistance to gentamicin, WO 94/01560 or hygromycin phosphotransferase genes that provide resistance to hygromycin) or herbicides such as glyphosate (for example EPSPS genes such as in USP 5510471 or WO 00/66748), fenmedipham (eg pmph gene USP 5347047; USP 5543306), bromoxynyl (eg genes described in USP 4810648) sulfonylureas (eg genes described in EP 0360750), dalapon ( genes described in WO 99/48023), cyanamide (genes described in WO 98/48023; WO 98/56238) and genes encoding resistance to glutamine synthetase inhibitors such as Lphosphinothricin (t as, for example, N-acetyl transferase genes described in EP 0242246, EP 0242246 and EP 0257542). In a preferred embodiment of the polynucleotide of the present invention comprising a herbicide resistance gene as a marker gene, said herbicide is a herbicide that is useful for weed control in cultivation and, additionally, the herbicide resistance gene is expressed sufficiently to provide robust tolerance to field rates of said herbicide. In a further preferred embodiment the herbicide is glyphosate and the herbicide resistance gene is EPSP synthase. However, the marker gene may be a gene that provides positive selection in which the marker gene encodes an enzyme that provides, in the context of a particular medium, a transformed metabolic advantage to transformed plant cells. USP 5767378 describes several suitable positive selection genes and systems.

When the polynucleotide of the present invention comprises a herbicide resistance gene, the herbicide is applied exogenously to crop plants that are interleaved at a density sufficient to eliminate non-hybrid seed production originating from parent plants non-transgenic fertile. In a preferred embodiment the herbicide is glyphosate or an agronomically useful salt thereof and said herbicide resistance marker gene is selected from the glyphosate resistance conferring genes described in WO 00166748.

When a marker gene is present, means may also be provided for the removal of said marker gene. This is desirable when, for example, you decide to combine traits. Furthermore it is also desirable to remove herbicide resistance marker genes that could interfere with the functioning of the proherbicide dependent conditional fertility mechanism of the present invention. For example, it would be desirable to remove a phosphinothricin N-acetyl transferase (PAT) herbicide resistance marker gene from a polynucleotide that also comprises a chimeric gene, useful for providing conditional sterility male or female dependent on exogenous application of D-phosphinothricin proherbicide . The presence of the PAT gene could potentially interfere with successful conditional sterility by inactivating phytotoxin L-phosphinothricin. Therefore, polynucleotides comprising marker genes can optionally comprise specific recognition sites for specific recombinases at positions that flank the marker gene and allow the sequence to be "knocked out". Crossing a plant carrying the thus flanked marker gene with a plant carrying a gene encoding the corresponding specific recombinase results in offspring plants from which the marker is specifically cleaved. Examples of such suitable site-specific homologous recombination systems are the flp / frt system (Lyznik et al., (1996), Nucleic Acids Res. 24, 3784-3789) and the Cre / Lox system (Bayley, CC et al. , (1992) PMB, 18, 353-361).

The polynucleotides used in the present method of the invention may optionally comprise one or more translational enhancers located within the 5 'untranslated regions of the protein coding sequences. The person skilled in the art is aware of the identity of such suitable translational enhancers such as the Omega and Omega prime sequences derived from TMV and derived from the tobacco engraving virus and how such translational enhancers can be introduced into the polynucleotide to provide the desired result. of increased protein expression. Additional examples include translational enhancers derived from corn chlorotic mottle virus and alfalfa mosaic virus (Gallie et al., (1987) Nucl. Acids Res., 15, 8693-8711; Skuzeski et al., (1990) PMB., 15, 65-79). To further optimize the expression of chimeric gene proteins and chimeric marker genes, said polynucleotides may also further comprise elements such as enhancers, framework or matrix binding regions (SARS or MARS), and introns. Various intronic sequences such as maize adh1 intron 1 have been shown to enhance expression when included in the 5 'untranslated region of genes and optionally used in the chimeric genes of the present invention.

Plants that have been transformed according to the invention to display the desired female / male sterility characteristics may also have been transformed with a polynucleotide comprising protein-encoding regions capable of conferring on plant material containing it at least one of the following agronomically desirable traits: resistance to insects, fungi, viruses, bacteria, nematodes, tension, desiccation and herbicides.

Genes that confer resistance to herbicides can, for example, be selected from the group encoding the following proteins: glyphosate oxidase (GOX), EPSP synthase, phosphinothricin acetyl transferase (PAT), hydroxyphenyl pyruvate dioxygenase (HPPD), glutathione S-transferase (GST) ), cytochrome P450, Acetyl-CoA carboxylase (ACCase), Acetolactate synthase (ALS), protoporphyrinogen oxidase (PPO), dihydropteroate synthase, polyamine transport proteins, superoxide dismutase (SOD), bromoxynyl nitrilase, phytoene desaturase (PDS) product of the tfdA gene that can be obtained from Alcaligenes eutrophus, and otherwise known mutated or modified variants of said proteins. The person skilled in the art will recognize the need to choose such genes and the promoters that direct their expression, carefully, taking into account the nature of the enzyme that it uses to convert the non-phytotoxin substance. In the event that the polynucleotide provides resistance to multiple herbicides, such herbicides can be selected from the group consisting of a dinitroaniline, triazolo-pyrimidine herbicide, a uracil, phenylurea, tricetone, isoxazole, acetanilide, oxadiazole, triazinone, sulfonanil, herbicide amide, anilide, isoxaflutol, flurochloridone, norflurazon and triazolinone and the post-emergence herbicide is selected from the group consisting of glyphosate and salts thereof, glufosinate, asulam, bentazon, bialaphos, bromacil, setoxidim or other cyclohexanodione, dicamba, fos flupoxam, phenoxy propionate, quizalofop, or other aryloxy-phenoxypropanoate, picloram, fluormetron, butafenacil, atrazine, and other triazine, metribuzine, chlorimuron, chlorsulfuron, flumetsulam, belosulfuron, sulfometron, imazaquine, imazetapir, isoxapirur, isoxapirur , nicosulfuron, fomesaphene, fluroglycophen, KIH9201, ET751, carfentrazone, mesotrione, sulco trione, paraquat, diquat, bromoxynil and fenoxaprop.

In the event that the polynucleotide comprises sequences encoding insecticidal proteins, these proteins can be selected from the group consisting of crystalline toxins derived from Bt, including secreted Bt toxins such as those known as "VIP"; Xenhorabdus / Photorhabdus protease, lectin and toxin inhibitors. Genes that confer resistance to fungi can be selected from the group consisting of those that encode known AFPs, defensins, chitinases, glucanases, and Avr-Cf9. Particularly preferred insecticidal proteins are cryIAc, cryIAb, cry3A, Vip 1A, Vip 1B, Vip3A, Vip3B, cysteine protease inhibitors and snowdrop lectin. In the event that the polynucleotide comprises genes that confer bacterial resistance, these can be selected from the group consisting of those that encode cecropins and tequiplesin and analogs thereof. Genes that confer virus resistance can be selected from the group consisting of those that encode virus coat proteins, movement proteins, viral replicases, and antisense and ribozyme sequences known to provide virus resistance; while genes conferring stress, salt and drought resistance can be selected from those encoding Glutathione-S-transferase and peroxidase, the sequence constituting the known CBF1 regulatory sequence, and genes known to provide accumulation of trehalose.

The polynucleotides used in accordance with the present invention may have been "modified" to enhance the expression of the protein coding sequences comprised therein, because the motifs of mRNA instability and / or the random splice regions may have been removed or Preferred codons may have been used for culture so that expression of the thus modified polynucleotide in a plant produces substantially similar protein having activity / function substantially similar to that obtained by expression of the protein coding regions of the unmodified polynucleotide in the organism in which such regions of the unmodified polynucleotide are endogenous. The degree of identity between the modified polynucleotide and a polynucleotide endogenously contained within said plant and encoding substantially the same protein may be such as to prevent cosuppression between the modified and endogenous sequences. In this case the degree of identity between the sequences should preferably be less than about 70%. Furthermore the sequence around a translation start position can be modified so that it is "Kozack preferred". The person skilled in the art knows well what this means.

The invention further includes morphologically normal conditionally fertile whole plants resulting from the crossing of plants that have regenerated from material that has been transformed with nucleic acid according to the present invention and therefore provides such a trait. The invention also includes progeny of the resulting plants, their seeds and parts.

Plants of the invention can be selected from the group consisting of large crops, fruits and vegetables such as rape, sunflower, tobacco, sugar beet, cotton, corn, wheat, barley, rice, sorghum, beet, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, soybean spp., sugar cane, pea, beans, poplar, grape, citrus, alfalfa, rye, oats, grass and forage herbs, flax and rapeseed and nut-producing plants to the extent that their descendants, seeds and parts have not been specifically mentioned already.

Particularly preferred such plants include wheat, barley, oats, rice, corn, millet, and sorghum.

A preferred procedure for producing hybrid wheat seeds comprises the steps of

(i)

 transforming plant material with a polynucleotide or vector comprising a gene that confers conditional male sterility after exogenous application of a proherbicide or other non-phytotoxic substance;

(ii)

 select the transformed material in this way; and

(iii) regenerate the material selected in this way in morphologically normal male conditionally sterile whole plants.

(iv)

cultivate a homozygous male conditionally sterile female parental line

(v)

 transforming plant material with a polynucleotide or vector comprising a gene that confers conditional female sterility after exogenous application of the same proherbicide or non-phytotoxic substance as in (i);

(saw)

 select the transformed material in this way; and

(vii) regenerate the material selected in this way in morphologically normal female conditionally sterile whole plants.

(viii) cultivate a homozygous female conditionally sterile male parental line

(ix)

interspersed planting of such conditionally sterile male and female parental lines in such a relationship as to ensure effective pollination

(x)

 Apply this proherbicidal or other non-phytotoxic substance to the planted parental lines interleaved at a dose and at a stage of development that minimizes self-fertilization.

(xi)

 Collect hybrid wheat seeds from the intercrop parent plants.

Also disclosed is a diagnostic kit comprising means for detecting the proteins or DNA sequences that encode them, which are present in plants produced according to the present method of the invention and therefore suitable for identifying tissues or samples containing them. . DNA sequences can be detected by PCR amplification as known to the person skilled in the art, based on primers that can be easily derived from the enzyme coding sequences disclosed or mentioned in the present application. Enzymes by themselves can be detected by, for example, the use of antibodies that have been raised against them to diagnostically distinguish the antigenic regions they contain.

Enantiomerically pure D-Phosphinothricin (D-PPT) can be produced by a process comprising the steps of:

(to)

 Providing cells that contain an enzyme capable of selectively N-acylating PPT;

(b)

 Let these cells grow in a medium containing D-L PTT to produce conditioned medium;

(c)

 Separate cells from conditioned medium from (b);

(d)

 Optionally extracting the conditioned medium with a non-aqueous, non-miscible solvent at various pHs, so that the fraction containing PPT is separated from the fraction containing molecules more soluble in water than PPT;

(and)

 Optionally mix with the extracted conditioned or PPT-containing medium from step (d) a cation exchange resin in its protonated form, in an amount, and at a pH, sufficient to absorb a substantial proportion of the cations, other than PTT, from the means, medium;

(F)

 Mix with the conditioned medium, extracted medium or medium resulting from step (e) a cation exchange resin in its protonated form, in an amount, and at a pH, sufficient to bind the volume of the PTT in the medium;

(g)

 Collect the cationic ion exchange resin from step (f) to which the PPT is bound and selectively elute PPT from it using an elution medium having sufficient pH and ionic strength, provided that the pH of said elution medium is not so low as to cause racemization of the PPT thus eluted.

With respect to transformation of plant material, those of skill in the art will recognize that although particular types of target material are specified (eg, embryogenic cell suspension culture or undifferentiated immature embryos) and particular transformation procedures (eg, using Agrobacterium or bombardment of Particles) In the Examples below, the present invention is not limited to these particular embodiments and such target materials and processes can be used interchangeably. Furthermore, the term "plant cells" as used throughout the present description of the invention can refer to isolated cells, including suspension cultures as well as cells in intact or partially intact tissue such as embryo, gusset, microspore, embryo derived from microspore or somatic cells of plant organs. Similarly, although the specific examples are limited to corn and wheat, the invention is equally applicable to a wide range of agricultural crops that can be transformed using suitable plant cell transformation procedures.

Mutant forms of D-amino acid oxidase enzymes and genes encoding them are disclosed in which a lysine is present at the position corresponding to phenylalanine at position 58 of wild type Rhodotorula gracilis D-amino acid oxidase. A double mutant form of Rhodotorula gracilis D-amino acid oxidase is disclosed having a lysine at position 58 (F58K) and a serine at position 213 (M213S). In a preferred embodiment the present invention provides a double mutant form of Rhodotorula gracilis D-amino acid oxidase having a lysine at position 58 (F58K) and a threonine at position 213 (M213T). These enzymes are capable of efficiently oxidizing D-phosphinothricin and other similar negatively charged D amino acids such as aspartate and glutamate.

These enzymes and the genes that encode them are used in more applications than the generation of hybrid cultures and, for example,

1) Enzymes can be used in Detection devices for D amino acids such as D-phosphinothricin

(for example as a means of detecting pesticide residues). For example, oxygen reduction and peroxide ion generation can be coupled to a series of chemical or electrochemical detection procedures and used in a sensor device.

2) Enzymes can be used in biocatalytic procedures for enantiorresolution of DL mixtures of acidic amino acids. For example, the herbicide, phosphinothricin is normally manufactured as the DL racemate while only Form L is the active herbicide. It would be desirable to convert all form D to L to achieve a more pure and twice as active herbicide formulation by weight of the chemical agent. The genes and enzymes of the present invention provide a method to achieve this. For example, racemic DL phosphinothricin is added to the growth medium of a host cell such as E. coli or yeast, etc. transformed to express the R. gracilis F58K, M213S D-amino acid oxidase or the F58K, M213T. Optionally, the host cells are also genetically engineered to express high levels of L-glutamate amino acid transferase. The growth medium preferably contains a source of glutamine so that cells can still grow despite inhibition of glutamine synthase by the L-component of phosphinothricin. After a suitable time it is discovered that the racemic phosphinothricin in the medium has been converted to substantially all of the L-form. The medium is then taken to provide substantially pure L-phosphinothricin. Analogous procedures that will be apparent to the person skilled in the art can, optionally, use isolated enzymes instead of cell culture procedures and, optionally, can use chromatography to isolate the residual 2-keto product of residual L-phosphinothricin. These procedures can also be applied to the enantiorresolution of a series of acidic amino acids.

The present invention will be further apparent from the following non-limiting examples taken in conjunction with the associated Sequence List and Drawings.

SEQ ID NO: 1 and 2 represent the PCR primers used to obtain the TA29 promoter region.

SEQ ID NO: 3 represents a DNA sequence, isolated from Rhodotorula gracilis, that encodes an enzyme that has the activity of a D-amino acid oxidase.

SEQ ID NOS: 4 and 5 represent degenerate oligos used to provide variant D-amino oxidase.

SEQ ID NOs: 6 and 7 represent motifs in which alternative amino acids can be substituted to provide variant Daminoacid oxidases.

Figure 1 is a schematic representation of a Rhodotorula Daminoacid oxidase construct for tobacco transformation under operational control of the stig 1 promoter region. Components indicated are LB (far left sequence), AOPR1 (AoPR1 promoter), PSTIG1 (EMBL access number X77823), RGDAO (OPT) (SEQ ID NO: 7), PROMOTOR PC (EMBL access number X16082), PAT (EMBL access number A02774), NOS (terminator we obtained from number of EMBL access ATU237588) and RB (far right sequence).

Figure 2 is a schematic representation of a tobacco transformation construct in which the Rhodotorula D-amino acid oxidase coding sequence is truncated by 3 codons at the 3 'end and at the 5' end (RGDAO (OPT) - SKL), fuses with a region encoding an optimized transit peptide (FR2673643).

General molecular biology procedures are carried out according to well established procedures.

For the most part the following examples each comprise multiple exemplifications of the present invention. When the term "promoter region of a gene" is used, it is understood that this means DNA sequences comprising the promoter, upstream sequences of the promoter and also, optionally, all or part of the DNA sequence encoding the 5 'free region. untranslated from the mRNA.

Example 1. Tobacco plants that are conditionally female sterile depending on the exogenous application of D-phosphinothricin, D-aspartate or D-glutamate.

The DNA sequence that encodes the protein sequence of D-amino acid oxidase P80324 (Swissprot) within the sequence of EMBL A56901 is obtained by RT-PCR of Rhodosporidium toruloides (Rhodotorula gracilis) mRNA.

or a similar one is obtained synthetically (which makes it easier to control which internal restriction enzyme sites are present and to create flanking sites to facilitate cloning), such as, for example, SEQ ID NO: 3 which is designed to explain codon use of the plant (in this case wheat) and minimize the DNA elements potentially adverse to expression. The DNA sequence is altered by PCR mutagenesis so that it encodes a mutant form of D-amino acid oxidase that has a lysine at position 58 instead of a phenylalanine (F58K) and, optionally, a serine or threonine at position 213 instead of a methionine (M213S or M213T). The flanking synthetic DNA and PCR primer sequences are designed to locate unique restriction sites useful for subcloning. Preferably, in the case where the oxidase coding sequence does not contain internal confusion sites, an Nco1 or Nde1 site is placed at the 5 'end to facilitate the

cloning of phase fusions with added 5 'sequences of the ORF such as the coding sequences of the chloroplast transit peptide. In some variants of the example the D-amino acid oxidase gene is cloned in such a way that the three terminal amino acids are truncated and the encoded enzyme is therefore no longer a peroxisomal target. In a further series of method variants the gene is genetically engineered by PCR such that it encodes Rhodotorula gracilis D-amino acid oxidase with alternative amino acids at positions 213, 223 and 238, and in particular when at position 213, wild-type methionine is replaced by His, Thr, Gly, Pro, Gln, Ser, Cys, Asn, or Ala, wild-type tyrosine at position 223 is replaced by His, Thr, Gly, Pro, Gln, Ser, Cys, Asn or Ala and / or the wild type tyrosine at position 238 is replaced by His, Thr, Gly, Pro, Gln, Ser, Cys, Asn or Ala. Methionine at position "213" is identified as M in the native RCTMDSS protein sequence motif. Tyrosine at position 238 is identified as the "Y" within the GGTYGVG native protein sequence motif.

Optionally, restriction sites are placed upstream of the intermediate sequences of the ATG translational initiation site to conform to plant translational consensus sequences such as according to Kozack.

The S 13 delta promoter is a promoter region useful for obtaining preferential expression in parts of female flowers. This comprises a -339 to -79 region of the SLG13 promoter region fused with -46 to +8 of the CMV 35S core promoter (Dzelkalns et al (1993) Plant Cell, 5, 833-863). This S13 promoter region is cloned into bluescript sk by further restricting said plasmid and ligating with restriction fragments comprising the 3 'transcription terminator region of Nos and one or the other of the amino acid oxidase coding sequences to create an expression cassette " delta S13-D-amino acid oxidase-terminator Nos ”within a bluescript sk plasmid. This is then appropriately restricted as, for example, an EcoR1 fragment and, as such, ligated back into a suitable site in a vector such as pBIN19 (Bevan (1984) Nucleic Acids Res.) Or pCIB200 or pCIB2001 (document WO 98/39462) for use for transformation using Agrobacterium. As described in WO 98/39462 pCIB200 contains the following unique polylinker restriction sites: EcoR1, Sst1, Kpn1, BglII, Xba1 and SalI. PCIB2001 contains an insert in the polylinker that adds additional unique restriction sites including MluI, BclI, AvrII, ApaI, HpaI and StuI. pCIB200 and pCIB2001 also provide selectable marker genes for plant and bacterial selection in kanamycin, left and right T-DNA ends, RK2-derived trfA function for mobilization between E. coli and other hosts, and the oriT and oriV functions of RK2. As an alternative, the binary vector pCIB 10 incorporating sequences from the wide host series plasmid, pRK252 (Rothstein et al (1987) Gene 53, 153-161), or one of its derivatives that incorporates both kanamycin resistance genes, is used such as the hygromycin phosphotransferase gene, such as pCIB715 (Gritz et al (1983) Gene 25, 179-188).

As an alternative the ∼ 1.6 kb Stig 1 promoter region (access derivative of EMBL X77823) is used. For example the coding region of the GUS gene in the stig1-GUS construct described by Goldman et al (1994) in EMBO J., 13, 2976-2984, is replaced with the DNA sequence encoding the P80324 or Q99042 coding sequences using enzymes restriction restriction and the resulting D-amino acid oxidase-stig1 expression construct is cloned into a suitable vector such as pCIB200 at a position upstream of a 3 'terminator sequence adjacent to a suitable marker gene and between sequences of DNA ends T.

In a further particular example the T-DNA insert within the binary vector is constructed according to Figure 1. A construct comprising the synthetic DNA sequence (SEQ ID NO: 3) encoding Rhodotorula gracilis D-amino acid oxidase altered by PCR mutagenesis to encode a mutant form of Daminoacid oxidase that has a lysine at position 58 instead of a phenylalanine (F58K) and a serine or threonine at position 213 instead of a low methionine (M213S or M213T) operational control of the stig1 promoter region and also the DNA sequence (A02774) encoding L-phosphinothricin N-acetyl transferase (PAT) under operational control of the pea plastocyanin promoter region is cloned at a site between the gene LB / npt II and the RB of the T DNA of the binary vector. In brief, the altered SEQ ID NO: 3 encoding the double mutant (F58K, M213S or F58K, M213T) is cloned into plasmid pFse4-Stiglnos (described in WO 99/42598) behind the Stig1 promoter and in front of the region terminator Nos (comprised within EMBL: ATU237588) as an NcoI / PstI fragment. The pea plastocyanin promoter region (derived from Accession number EMBL X16082) is obtained from pea genomic DNA by PCR and cloned in front of the nos / PAT gene terminator. The resulting PC-PAT-nos cassette is cloned behind Stig1-RGDAMOX-nos as a NotI fragment and this entire two-gene construct is transferred to a binary vector (pVB6, a derivative of Bin19) as an FseI fragment.

In a further variant of the procedure the construction used is in accordance with the schematic representation of Figure 2. The coding sequence for D-amino acid oxidase of Rhodotorula, SEQ ID NO: 3, again mutated in a targeted manner to encode the double form F58K, M213S or F58K, M213T mutant of the enzyme, is truncated by three codons at the 3 'end and, at the 5' end, is cloned to immediately place it upstream of a region encoding a chloroplast transit peptide. encoding a D-amino acid oxidase / chloroplast transit peptide fusion protein. The sequence encoding the chloroplast transit peptide is derived from the Arabidopsis gene encoding the small EPSP synthase subunit (Klee et al 1987 in Mol.Gen.Genet., 210, 437). Optionally this is modified to include the Sph1 site at the CTP processing site, thereby replacing the Glu-Lys at this location with Cys-Met (SEQ in Figure 9 of WO 92/044490). Consequently, an SPh 1 site can be genetically engineered in the

N-terminus of the D-amino acid oxidase coding sequence (converting the amino acid after methionine to a leu). Alternatively, the coding sequence for the chloroplast transit peptide is derived from the petunia gene encoding EPSP synthase (Figure 11 of WO 92/044490). As an alternative, the chloroplast coding sequence is any one of a wide variety of possibilities including those derived from genes encoding the Rubisco small subunit and including the so-called "optimized" chimeric transit peptide sequence (FR 2673643). In all cases, instead of relying on subcloning, the entire desired DNA sequence encoding the double mutant Rhodotorula D-amino acid oxidase / chloroplast transit peptide fusion polypeptide can simply be synthetically obtained. This sequence is cloned at a site downstream of the stig1 promoter region and upstream of a terminator sequence (for example nos) into a suitable vector (for example by replacing the GUS coding sequence in the vector containing the stig1-GUS construct. described by Goldman et al (1994) in EMBO J., 13, 2976-2984). The complete gene expression construct is then cloned into a suitable site between the left and right ends of the T-DNA of a PVB6 vector.

Tobacco leaf discs are transformed with the recombinant binary vectors using procedures similar to those described in Horsch et al (1985) Science, 227, 1229-1231. Many variations of the procedure can be used. The binary vector can be transformed into, for example Agrobacterium tumefaciens strain LBA 4404 using the freeze-thaw transformation procedure. Tobacco transformation and whole plant regeneration is performed using Nicotiana tabacum variant Samsun according to the protocols described by Draper et al (Plant Genetic Transformation, Blackwell Sci. Pub. 1989). Transformation events are selected in MS medium containing kanamycin or another suitable antibiotic. The presence of integrated transgenes is confirmed by PCR. Plants regenerate and are allowed to reach maturity and self-sow to produce seeds. Northern and / or Western analysis is used to confirm tissue-specific expression of the D-amino acid oxidase genes. The selected plants are self-fertile but have the condition of conditional female sterility. Seeds of the T1 generation are sown. Once seedlings have grown to a sufficient size they are assayed by PCR for the presence of the transgene. The PCR positive plants are transferred to the greenhouse. These plants are completely fertile in the absence of exogenously applied proherbicide. A subset of these conditionally sterile plants are (potentially) treated with D-phosphinothricin, D-aspartate, or D-glutamate in varying amounts and at various stages of growth. Such treatments are carried out on T1 plants confirmed as positive by PCR for the D-amino acid oxidase gene or, likewise, three treatments are carried out directly on plants of the T0 generation (which are vegetatively cloned so that Untreated clones can be separated from each event for seed production.) The observed fertility is then used as the basis for selecting suitable plant lines showing the most obvious conditional sterility phenotype. For example these amino acids are pure Denantiomers or alternatively they are DL racemates. For example, they are applied as a foliar spray, before or during the early stages of flower formation, at rates usually between 0.25 and 20 kg / ha. Amino acids that can crystallize out of solution on the leaves can be redissolved after foliar application and mobilized again for uptake by the leaves by additional applications of water such as spray mist. Amino acids are also applied, for example, as a root potion and optionally additionally applied as 50 µl of a 10-200 mM solution spilled directly onto the emerging flower buds. Pollen is collected from the treated plants and viability is tested. Plants are obtained that produce relatively little or no seed after treatment with D-phosphinothricin, D-aspartate or D-glutamate but that nevertheless, under the same treatment conditions, produce almost normal levels of viable pollen. Controls include both transgenic and non-transgenic plants and are grown under identical conditions and in an identical regimen of physical treatments except that the treatment solutions are water or an equivalent concentration of pure L-amino acid.

In a variant of the procedure, the applied amino acid is racemic DL phosphinothricin. In this case, the DNA construct used for transformation comprises, in addition to the DNA sequence encoding a Daminoacid oxidase under operative expression control of a tissue-specific female floral promoter region such as "stig 1", also a DNA sequence (EMBL: A02774) a "PAT" gene under operational control of a promoter region such as the 5 'region of the plastocyanin gene translation start from the Pisto sativum plastocyanin gene (EMBL accession number X16082). For example, the construction is the same as that shown in Figure 1.

The plastocyanin promoter region enables preferential expression in the green tissues of the plant. It is unexpectedly discovered that a promoter such that, unlike, for example, the 35S promoter region, is expressed substantially only in certain plant tissues and most notably in green tissues, however, when used in combination with the PAT gene enables substantially complete reproductive tolerance to herbicide DL PPT even at rates above 2 kg / ha. Furthermore, in the absence of the heterologous D-amino acid oxidase that is co-expressed in flower tissues, the PAT gene / plastocyanin combination provides essentially complete reproductive tolerance without significant loss of performance even though the level of PAT expression is low or not. existing in many of the critical floral tissues when expressed low in control of this promoter region. Therefore, in this variant of the example, the non-phytotoxic substance D-phosphinothricin is applied in its least expensive and most readily available form as the commercial herbicide racemate DL phosphinothricin. At appropriate spraying times and at rates between 250 g / ha and 5 kg / ha of DL phosphinothricin, the treated plants did not

they are visibly damaged but become conditionally female sterile remaining with normal or near normal male fertility.

Example 2. Tobacco plants that are conditionally male sterile depending on the exogenous application of D-phosphinothricin, D-aspartate or D-glutamate

The mutant D-amino acid oxidase protein sequences and the DNA sequences encoding them are as in the preceding example, Example 1.

The TA29 promoter region (Kriete et al (1996) Plant J., 9, 808-818) is cloned from tobacco genomic DNA by PCR using the 5'-AACTGCAGCTTTTTGGTTAGCGAATGC-3 'primers (SEQ ID NO: 1) and 5' -CAGACTAGTTT-TAGCTAATTTCTTTAAGTAAAAAC-3 '(SEQ ID NO: 2). Through a series of restriction and subcloning steps, the PCR fragment obtained in this way is placed upstream of the D-amino acid oxidase coding sequence and a transcriptional terminator is not added 3 ’of the coding region. The resulting nos-D-amino acid oxidase-TA29 terminator expression cassette is then cloned, obtained as a suitable restriction fragment and cloned into a binary vector as in Example 1.

Alternatively, any of the above D-amino acid oxidase coding sequence regions are cloned as a suitable restriction fragment (eg BamH1, Bgl / II in which synthetic variants of coding sequences are designed to remove internal restriction sites) and they fuse with the CaMV 35S promoter and the nopaline synthase terminator regions by insertion into (eg) the BamH1 site of the binary vector pROK1 (Baulcombe et al (1986) Nature, 321, 446-449) in a sense configuration. The EcoR1-BamH1 fragment carrying the 35S promoter region is then excised and replaced with an EcoR1-BamH1 fragment from pAP30 (Kriete et al (1996) The Plant Journal 9, 809-818) carrying the TA29s promoter region fragment ( -810 to + 54). The resulting vectors can be named pGKTA29_Q99042, pGKTA29_P80324, pGKTA29_Q9HGY3, and pGKTA29_P24552 etc. according to the encoded protein sequence.

Tobacco plant material is transformed by Agrobacterium with vector and transgenic plants are regenerated in a similar way as described in the previous example. The plants produced are self-fertile but are conditionally male sterile. The T1 generation seeds are planted but in soil. Once the seedlings have grown to a sufficient size they are assayed by PCR for the presence of the transgene. PCR positive plants are transferred to the greenhouse. These plants are completely fertile in the absence of exogenously applied proherbicide. A subset of these potentially conditionally sterile T1 plants or, alternatively, T0 "event" seedlings (direct regenerative transformation) are treated with D-phosphinothricin, D-aspartate or D-glutamate in various amounts and at various stages of growth. When T0 plants are treated they are vegetatively cloned so that untreated sisters are separated from the events for seed production. The observed fertility is then used as the basis for selecting suitable plant lines showing the most obvious conditional sterility phenotype. For example these amino acids are pure D enantiomers or alternatively they are DL racemates. For example, they are applied as a foliar spray, before or during the early stages of flower formation, at rates usually between 0.25 and 20 kg / ha. Amino acids that can crystallize from solution on the leaves after foliar application can be redissolved and re-mobilized for uptake by the leaves by additional applications of water such as spray mist. Amino acids are also applied, for example, as a root potion, and optionally additionally applied as a 10-200 mM solution directly to the emerging flower buds.

Pollen is collected from the treated plants and their viability is tested. Plants are obtained that give off relatively little or no pollen and / or pollen that is not viable. Pollen collected from some of the treated plants is tested and found to be poorly formed and not viable. However, such infertile male plants remain fertile female and produce (hybrid) seed when pollinated with pollen collected from other untreated or conditionally sterile female non-GM tobacco plants. Controls include both transgenic and non-transgenic plants and are allowed to grow under identical conditions and in an identical regimen of physical treatments except that the treatment solutions are water or an equivalent concentration of pure amino acid L.

In an alternative embodiment the promoter region used is a 2.2 kb region (reference EMBO X57295) upstream of the Antirrhinum tap 1 gene (Spena et al (1992), Theor. Appl. Genet., 84, 520-527 ).

Analogously to Example 1, in a variant of the example, the applied amino acid is racemic DL phosphinothricin. In this case, the DNA construct used for transformation comprises, in addition to the DNA sequence encoding a D-amino acid oxidase under the control of operational expression of a tissue-specific male floral promoter region such as "TAP 1" or "TA 29 ", Also a DNA sequence encoding a phosphinothricin N-acetyl transferase gene such as the" PAT "gene under operational control of a promoter region such as that of the plastocyanin gene (in this case the plastocyanin gene region of Pisum sativum). At appropriate times of spraying and rates between 250 g / ha and 5 kg / ha of DL phosphinothricin the treated plants are not visibly damaged but become conditionally male sterile while remaining normal or near normal female fertility.

Example 3. Chimeric genes capable of preferentially expressing themselves in the male reproductive structures of wheat and encoding enzymes capable of oxidizing D-phosphinothricin, D-glutamate or D-aspartate.

The mutant D-amino acid oxidase protein sequences and the DNA sequences encoding them are as in Example 1. Plasmid pGK73 carries the EcoR1-BamH1 fragment of promoter region TA29s from -810 to +54 (Kriete et al (1996) , 9, 809-818). This restriction fragment or a similar suitable PCR-generated fragment is cloned, preferably as a phase fusion, at a position upstream of the DNA sequence encoding, for example, the double mutant R. gracilis D-amino acid oxidase ( F58K, M213S or F58K, M213T) in bluescript sk. Using a suitable series of restriction, ligation and subcloning steps, a terminator of the 3 'transcription of the coding region is added to generate, according to the coding sequence, alternative expression cassettes of the type TA29-carboxylesterase-nos in Bluescript plasmids sk, pBLTA_RGF58KM213T, pBLTA_RGF58KM213S etc.

In a further example, the anther-specific SGB6 promoter region SEQ ID NO: 1 of USP 5470359 is used. For example, pSGBNE1 containing a 3 kb genomic EcoR1-Nhe1 subcloned fragment of pSGB6g1 (USP 5470359) is further subcloned. to place a blunt cloned 1558 bp ApaII / Xba1 fragment in bluescript ks at the SmaI site. As before, through additional restriction and cloning steps this fragment is fused upstream of a mutant D-amino acid oxidase DNA coding sequence. Again a terminator nos 3 ’is added from the coding region to create, alternatively, Bluescript sk plasmids, pBLB6_RGF58K etc. comprising the alternative SGB6-DAMOX-nos expression cassettes.

In a similar set of examples the rice RA8 anther specific promoter region (genbank access / EMBL AF042275; Jeon et al (1999) PMB, 39, 35-44; WO 00/26389) is also similarly fused into an in-phase and upstream site of one or the other of the DNA sequences encoding the mutant Daminoacid oxidase F58K and a 3 'terminator to comprise alternative RA8-DAMOX-nos expression cassettes in a series of bluescript sk vectors, pBLRA8_RGF58K, M213S etc.

Example 4. Chimeric genes capable of preferentially expressing themselves in the female reproductive structures of wheat and encoding enzymes capable of oxidizing D-phosphinothricin, D-aspartate and / or D-glutamate

DNA sequences encoding D-amino acid oxidase protein sequences are obtained as described in Example 1.

The genomic clone pSH64 was deposited under the terms of the Budapest Treaty on 02/27/1998 at NRRL and assigned NRRL B-21920. It was detected as a genomic clone that hybridizes with the silk-specific cDNA clone B20014-2 (WO 98/39462). Chimeric genes that are preferentially expressed in female reproductive structures are constructed as follows. A bluescript ks-derived plasmid similar to pSH70 is constructed having an "empty" expression cassette comprising, from 5 'to 3', the 5 'promoter region B200i consisting of nucleotides 1-3790 of SEQ ID NO: 11 from WO 98/39462, a BamH1 site and the B200i 3 'untranslated terminator region comprising nucleotides 4427-6397 of SEQ ID NO: 11 from WO 98/39462 as described in WO 98/39462. Using partial BamH1 digestion or, alternatively by further subcloning, alternative D-amino acid oxidase coding sequences are ligated into PCR and ligation steps at or adjacent to the BamH1 site so that they are immediately 3 'from the promoter region B200i and 5 'from the B200i terminator region. Consequently, a series of bluescript vectors pBLB200_RGF58K, pBLB200_RGF58KM213T, pBLB200_RGF58KM213S etc. are created. encoding alternative mutant B200i-D-amino acid oxidase expression cassettes.

As an alternative, as described in WO 98/39462, a Pst I / Nco I fragment of the 5 'promoter region of the P19 gene from the X2-1 genomic clone was cleaved which was deposited under the terms of the Budapest Treaty on 27 / 02/1998 in NRRL and assigned the access number B-21919. The Nco I site at nucleotide 1088 of SEQ ID NO: 14 of WO 98/39462 corresponds to the start of the ATG translation of the P 19 gene. Using appropriate subcloning, restriction, ligation and PCR steps this fragment is ligated to form a phase fusion with one or the other of the DNA sequences encoding D-amino acid oxidase and a terminator sequence is added to us 3 'of the coding sequence. Consequently, a series of bluescript vectors pBLP19_RGF58KM213S, pBLP19_RGF58KM213T etc. is created. encoding the alternative nos-D-amino acid oxidase-P19 expression cassettes. Alternatively, using similar standard procedures, similar plasmids having the 5 'promoter region (comprising some or all of nucleotides 1-3987 of SEQ ID No. 2 of WO 98/39462) of the P26 gene are obtained instead of the region promoter P19. Genomic clone P26-A4, pCIB10302 deposited under the terms of the Budapest Treaty on January 21, 1997 in the Agricultural Research Service (NRRL) patent culture collection, accession number NRRL B-21655 is subcloned as described in WO 98/39462. Consequently, a series of bluescript vectors pBLP26_RGF58KM213T, pBLP26_RGF58KM213S etc. are created. encoding alternative nos-D-amino acid oxidase-P19 expression cassettes.

Example 5. A pair of complementary constructs useful in a method of providing (a) a female inbred parental line that is conditionally sterile male depending on the application of DL phosphinothricin and (b) a complementary male inbred parental line that is conditionally sterile female depending of the application of DL fosfinotricina.

The first suitable DNA construct to provide a female inbred parental cereal or rice plant line that is conditionally sterile male depending on the application of DL phosphinothricin comprises three genes A), B) and C). A) consists of a DNA sequence encoding a PAT enzyme capable of Nacetyl L-phosphinothricin under operational control of the ∼ 1 kb promoter region of the barley plastocyanin gene (EMBL: Z28347) and a suitable terminator region such as of the 35S gene or nos, B) consists of a PAT coding sequence similar to the first but in this case under the operative control of a tissue-specific female floral promoter region (such as P19 or P26 as described above) plus a suitable terminator and C) consists of a suitable DAMOX coding sequence as described in Examples 1, 10 and 11, encoding, for example, a double or triple mutant of the F58K mutant form of D-amino acid oxidase of Rhodotorula gracilis which has changes at positions 213, 223 and 238 and, in particular, where, at position 213, the wild-type methionine is replaced by His, Thr, Gly, Pro, Gln, Ser, Cys, Asn or Ala , the wild-type tyrosine in the posi tion 223 is replaced by His, Thr, Gly, Pro, Gln, Ser, Cys, Asn or Ala and / or the wild type tyrosine at position 238 is replaced by His, Thr, Gly, Pro, Gln, Ser, Cys , Asn or Ala under the operational control of a tissue specific male floral promoter region (such as SGB6 or RA8 as described above) and a suitable terminator region. In preferred examples the encoded R. gracilis DAMOX enzyme is a double mutant form F58K, M213S or F58K, M213T. This construction is assembled using procedures that are conventional in the art and reported by the previous examples.

The second DNA construct suitable for providing a male inbred parental cereal or rice plant line that is conditionally female sterile depending on the application of DL phosphinothricin comprises three genes A), D) and F). A) consists of a DNA sequence encoding a PAT enzyme capable of Nacetylar L-phosphinothricin under operational control of the promoter region of the barley plastocyanin gene and a suitable terminator region such as that of the 35S or nos gene, D) consists in a PAT sequence similar to the first but in this case under the operational control of the same tissue-specific male floral promoter region (such as SGB6 or RA8P19) that is used in construct 1 plus a suitable terminator and F) consists of a DAMOX gene suitable as, for example, that used in Construction 1 and under the operational control of the same tissue-specific female floral promoter region (such as P 19 or P26) used in Construction 1 and a suitable terminator region. This construction is assembled using procedures that are conventional in the art and reported by the previous examples.

A pair of DNA constructs in this example contain, for example, the following elements

Construction 1

A = Barley plastocyanin promoter region - PAT coding sequence, terminator Nos; B = P26 promoter region - PAT coding sequence, 35S terminator; C = RA8 promoter region - Rhodotorula D-amino acid oxidase coding sequence (mutant F58K, M213T) terminator Nos

Construction 2

A = Barley plastocyanin promoter region - PAT coding sequence, terminator Nos; D = RA8 promoter region - PAT coding sequence, 35S terminator; E = P26 promoter region - Rhodotorula D-amino acid oxidase coding sequence (mutant F58K, M213T) terminator Nos

Example 6. Polynucleotide Vectors for Wheat Transformation

Examples 3, 4 and 5 describe the construction of various chimeric genes in expression cassettes that are usually cloned in bluescript sk. Optionally these vectors are prepared in bulk for direct DNA transformation for use with a co-bombarded selectable marker such as pSOG35 (DHFR / methotrexate) or pUbi-Hyg (hygromycin phosphotransferase / hygromycin) as described in WO 98/39462. Preferably, after bulk preparation, the vectors are linearized using a suitable restriction enzyme to remove the bluescript ampicillin resistance gene.

Optionally, instead of using co-bombardment, said bluescript vectors are further engineered by conventional procedures such that they further comprise a plant selectable marker gene such as kanamycin resistance, hygromycin resistance, methotrexate resistance, or glyphosate resistance and they are used directly. In some of the above examples a PAT gene is integral to vector design and, in these cases, DL phosphinothricin can optionally be used for selection at some stage after transformation.

Alternatively, expression cassettes are cleaved within a suitable restriction fragment and cloned into vectors derived from pIGPD9 (described in Figure 12 of WO 00/66748). The use of this vector for transformation avoids the transfer of antibiotic marker genes to the plant since its maintenance in bacteria is based on complementation of an autotrophic mutant of E. coli. The vector comprises a gene that expresses IGPD (the product of HisB) and is further engineered to comprise a plant selectable marker gene such as an EPSPS gene cloned at the Xma I site such as, for example, pZEN16i and

pZEN18i of WO 00/66748. As an alternative, a marker gene that provides positive selection for mannose or xylose is used (USP 5767378).

In particular examples of use of pIGPD9 vectors, plasmids are constructed for wheat transformation. Illustrative examples are pZEN18_ BLB200_Q99042 and pZEN18_ BLRAB_Q01470. These are vectors derived from pIGPD9 that comprise the EPSPS gene from pZEN18 (WO 00/66748) and, in this case, the expression cassettes B200i-) D-amino acid oxidase-B200i or RA8-D-amino acid oxidase-nos, respectively .

Large-scale DNA preparations for use in plant transformation are obtained using the Maxiprep procedure (Qiagen) using protocols provided by the manufacturer.

Example 7. Transformation / regeneration of wheat with polynucleotides that comprise chimeric genes preferentially expressed in male or female reproductive structures and that encode enzymes capable of oxidizing D-phosphinothricin, D-aspartate and / or D-glutamate.

In one example, immature embryos (0.75-1.0 mm long) of the UC703 genotype are plated in MS medium containing 3 mg / l 2,4-D and 3% sucrose. After approximately 4 hours the embryos are plated in MS medium containing 15% maltose, 3% sucrose and 3 mg / l 2,4-D superimposed with a filter paper supported agarose block containing the same components. Embryos are allowed to plasmolyse for 2-3 hours prior to bombardment.

DNA prepared as described in Example 6 and in the previous examples is precipitated into micron-sized gold particles using conventional procedures. Four target plates with 16 embryos per target are fired twice with a DuPont Biolistics helium device using a burst pressure of 7.58 MPa. The plates were shot with an 80 mesh screen located between the vehicle cover and the target. After bombardment targets were placed in the dark at 25 ° C for 24 hours before the blocks with the embryos were placed on MS medium plates containing 3% sucrose and 3-mg 2,4-D. The individual embryos are removed from the blocks and placed directly in fresh medium of the same composition after another 48 hours. Approximately 6 weeks after gene delivery the tissue is placed in MS medium with 2,4-D 3 mg / l, sucrose 3% and methotrexate 0.2 mg / l over a period of 3 weeks. The tissue is then placed in regeneration medium comprised of MS medium containing 1 mg / l zeatin riboside and 1 mg / l methotrexate. After 2 weeks, the regenerating seedlings are placed in sterile containers with half concentration MS medium containing 2% sucrose, 1 mg / l naphthalinic acid and 4 mg / l methotrexate.

In particular variants of the example, vectors comprising chimeric genes preferably expressed in male reproductive structures are bombarded in conjunction with alternative selectable marker genes. Therefore, for example, plasmid DNA is prepared and gold particles coated with it along with pUbiHyg (a plasmid encoding hygromycin phosphotransferase under the operational control of the corn polyubiquitin promoter). In this case transformation and regeneration is carried out as described above except that, after bombardment, the regeneration medium contains increasing concentrations of hygromycin between 2 and 20 mg / l.

In a further example wheat is transformed with pZEN18_BLB200_RGF58KM213S (D-amino acid oxidase), selected using glyphosate and regenerated as described in Example 15 of WO 00/66748.

DNA is extracted from tissues of transformation derived plant leaves and PCR is run for the presence of the selectable marker gene and the gene encoding D-amino acid oxidase. PCR positive plants are propagated. During flowering pistils and anthers are collected and RNA is prepared. DNA expression is confirmed by Northern analysis. Furthermore, D-amino acid oxidase genes are expressed using pET vectors in E. coli and are partially purified. Protein bands of the expressed protein are cut from an SDS gel and used to generate polyclonal antibodies. These antibodies are used to detect expression in flower and other tissues by Western analysis.

Example 8. A method for efficiently producing hybrid cereal crops in which DL phosphinothricin is applied both for weed control and at the same time as the chemical hybridization agent and in which the generation of F1 hybrid from plants resulting from the hybrid seed produced in this way is substantially tolerant both vegetatively and reproductively to the application of DL phosphinothricin.

Hybridization chemicals are expensive. It would be desirable to use a relatively cheap substance such as a commercial herbicide as a chemical hybridization agent. This would further achieve additional efficacy since weed control could be combined with chemical hybridization. However, there are several problems to overcome in order for this proposition to be realized. First, one would need to establish male and female parental lines that are tolerant to the herbicide in question. Furthermore, to achieve the desired "conditional" fertility in response to herbicide application the two lines would need to be engineered so that tolerance to the herbicide did not extend to all tissues but was expressed in a tissue-specific manner. so that each of the required floral tissues remained selectively susceptible. Therefore, in one line (the female parent line), the volume of the plant plus the female tissue

they must be made tolerant while some critical part of the male floral tissue must remain susceptible to the application while in the other (the male parental line), the reverse is needed while only a certain critical part of the female gamete-forming tissue is susceptible. Even granting that this can be achieved there is still an additional problem to overcome with respect to hybrid seed and F1 generation. Since this generation of the crop would necessarily contain at least two genes capable of conferring resistance to the herbicide, it would be desirable that this same herbicide could also be used for weed control in the crop. However, it is very difficult to conceive of a combination of herbicides, tissue-specific promoter regions and tolerance genes that would allow this use of the same herbicide in the F1 generation. The hybrid crop would likely have vegetative tolerance but little or no grain yield after application of the herbicide to the F1 generation. For example, for the glyphosate herbicide the usual mechanism of resistance is the expression of a resistant form of EPSP synthase. It is difficult to identify a promoter region or combination of promoter regions that would allow sufficient expression of an R-EPSPS in all tissues and at all times other than, for example, at a critical stage in the development of stamens or stigmas. The easiest way to avoid this would be to use an antisense or similar approach in which the expression of R-EPSPS is driven by a constitutive / tissue-specific promoter and is only locally and transiently suppressed in, for example, stamens due to expression of an antisense EPSPS gene (see for example WO 99/46396). However, in that case the suppression of expression in the stamen (or stigma) would be directed by a dominant gene. It is evident that, for any such mechanism, application of the herbicide to the F1 generation would result in a non-producing sterile culture due to the additive effects of the dominant male and female conditional sterility genes.

The present invention provides a method of overcoming the problem of allowing the use of a cheap commercial herbicide, DL phosphinothricin, both as a weed control and as a hybridizing agent in the production of hybrid cereals, and by providing said method, in addition, to hybrid cereals or rice. resulting in that DL phosphinothricin (or L-phosphinothricin) can be safely used for weed control without substantial loss of performance. As a further added benefit, F1 generation self-sown seeds that may emerge later as volunteers in subsequent crops will be easier to control since they, themselves, will generally be sterile if sprayed with control amounts of DL phosphinothricin. The same holds true for the cross-pollinated offspring of pollen from F1 plants to weeds (eg red rice) or other cereals. The present invention provides genes and enzymes that convert a non-phytotoxic component, D-phosphinothricin, from a commercially available herbicide formulation DL phosphinothricin to the active L-form. The PAT gene that converts L phosphinothricin to N-acetyl L-phosphinothricin is already known and is used commercially to avoid tolerance to DL phosphinothricin in cultures. A further critical observation regarding the present example is that, surprisingly, wheat containing a PAT gene under the control of the operational expression of the barley plastocyanin promoter region has been found to be substantially reproductively tolerant to the application of DL phosphinothricin at rates of up to at least 2kg / ha. Therefore a critical feature of the constructs described in Example 6 that are used to provide the plants of the present example is that the PAT gene that provides the resistance trait is expressed under the operational control of a promoter region that provides substantially expression only in green fabrics. A feature of such a useful promoter region is that it should express PAT in such a way that it adequately protects all non-green flower tissues from foliar applied DL phosphinothricin while, at the same time, providing only a minimal level of PAT expression in the floral fabric itself and especially low on the target parts for conditional sterility. With PAT expressed under the operational control of the barley plastocyanin promoter region this condition appears to be met since substantially all of the sprayed Lphosphinothricin enters through the leaves and is intercepted and converted to non-phytotoxic N-acetyl-L-phosphinothricin before of it translocating to developing floral tissues. Therefore, in the present invention, the Lfosfinotricina that causes the effects of selective tissue sterility in the parental lines is generated only transiently and locally from phloem-mobile non-phytotoxic D-phosphinothricin by D-amino acid oxidase. Exactly matching the floral control elements that direct the expression of PAT with the elements that direct the expression of D-amino acid oxidase in the complementary pair of constructs (Example 5) ensures that, in the F1 hybrid, the transient explosion of L-phosphinothricin in the target floral tissue will be rapidly neutralized by a corresponding explosion of PAT expression at the same time and in the same local tissue. Therefore the application of the herbicide does not induce a sterility effect in the hybrid. However, in later generations, the floral-corresponding corresponding D-amino acid oxidase and PAT from the hybrid will segregate and therefore, once again, the resulting plants will be sterile male or female upon application of control amounts of DL phosphinothricin.

Using the procedures described in Examples 6 and 7, the constructs described in Example 5 are transformed into yeast or (using conventional superbinary vector procedures) into rice that is selected and regenerated into seedlings. Transforming T0 events are selected (using clonal propagation of shoots to maintain untreated lines) and suitable events to continue cultivation as, alternatively, male inbred parental lines that are conditionally female sterile depending on the application of DL phosphinothricin or female inbred lines that conditionally sterile male depending on the application of DL phosphinothricin are selected using procedures essentially as described in Examples 1 and 2. The best lines show the best herbicide tolerance, minimal loss of performance, most evident conditional sterility phenotype etc. Parental lines

Alternative male and female parentals are selected and optionally back-crossed into elite lines suitable for several generations. The genetic inserts in these finally selected events are fully characterized as well as the inheritance genetics of conditional fertility and herbicide resistance traits and the characteristics of expressed gene products.

The female and male parental lines selected in this way are then interleaved together in suitable ratios in a field and sprayed with DL phosphinothricin at a suitable rate of between 0.05 and 5 kg / ha and selecting time to period of early flowering to optimize hybrid seed production. Seeds produced in this way have the advantage that they will give rise to plants that not only benefit from hybrid vigor but are also tolerant to herbicide formulations containing DL phosphinothricin which can therefore be used for selective weed control in cultivation. Hybrid seeds also have the advantage that the herbicide tolerance trait they express will be passed only incompletely to future self-sown generations or by cross-pollination to related weeds. Therefore, for example, the resulting hybrid rice of the present invention can be grown using DL phosphinothricin as a weed control agent without significant yield loss. However future generations of red rice plants to emerge as the pollen offspring of hybrid cross-pollinated rice with female parents of red rice will be vegetatively tolerant to DL phosphinothricin treatment but will have reduced self-fertility (due to D-amino acid expression oxidase in the floral tissue) and thus produce little grain. Therefore using the hybrid rice of the present invention DL phosphinothricin can be used for weed control with a greatly reduced future risk of grain contamination with red rice as a result of the herbicide resistance trait being cross-pollinated to rice closely related red. Similarly, spontaneous second-generation rice or wheat plants arising from the hybrid crop will, for the most part, not produce grain after spraying with DL phosphinothricin.

Example 9. Transformation / Regeneration of corn with a polynucleotide comprising a chimeric gene preferentially expressed in male reproductive tissue and encoding an enzyme capable of oxidizing Dphosphinotricin.

RA8-D-amino acid oxidase expression cassettes are cloned into a series of bluescript sk vectors, pBLRA8_RGF58KM213T, pBLRA8_RGF58KM213S etc. as described above. Optionally, these are bombarded in conjunction with DNA comprising selection markers such as pUbiHyg or pSOG35; selected and regenerated using hygromycin or methotrexate as described, for example, in Example 11 of WO 98/39462.

As an alternative, pZEN18_BLRA8_RGF58KM213S etc. they are directly bombarded or transferred in silicon carbide whiskers to corn cells and corn plants are selected and regenerated in glyphosate as, for example, described in Examples 12 and 13 of WO 00/66748.

Alternatively, transformation of corn was carried out using Agrobacterium tumefaciens containing a superbinary vector. For example, the pZEN18 expression cassette and the BLRA8_F58K D-amino acid oxidase chimeric gene is cleaved from pZEN18_ BLRA8_RGF58K and cloned into positions between the left and right T-DNA ends of a superbinary vector derived from pSB1 through a series of subcloning and homologous recombination in a series of steps similar to those described in WO 00/66748. Plant matter derived from immature embryos is infected with Agrobacterium containing superbinary vector comprising the glyphosate marker gene and the chimeric gene of the present invention. Plants are selected and regenerated using glyphosate as described in WO 00/66748.

DNA is extracted from leaf tissues of transformation-derived plants and a PCR is run for the presence of the selectable marker gene and the gene encoding mutant D-amino acid oxidase. PCR positive plants are propagated. During flowering pistils and anthers are collected and RNA is prepared. DNA expression is confirmed by Northern analysis. Furthermore, mutant D-amino acid oxidase genes are expressed using pET vectors in E. coli and partially purified. The protein band of the expressed protein is cut from an SDS gel and used to generate polyclonal antibodies. These antibodies are used to detect expression in floral and other tissues by Western analysis.

Example 10 Targeted Mutagenesis to Generate Additional Mutants Derived from R. gracilis D-Amino Acid Oxidase Mutant Form with Additional Enhanced Abilities to Oxidize D-Phosphinothricin and / or D-Aspartate etc.

This example relates to the production of genes encoding R. gracilis D-amino oxidase variants that have enhanced ability to oxidize D-phosphinothricin and / or other D-amino acids. These genes are used in preferred embodiments of the invention, described in the other examples, when sterility becomes conditional upon application of D-phosphinothricin or D-aspartate. In the present particular example these genes encode enzymes that have, in addition to the F58K mutation, a single amino acid change at position "213" and / or at position "238". One of skill in the art will recognize that completely analogous procedures are used to make a similar series of mutations to replace tyrosine at equally preferred position 223 with the same set of alternative amino acids. Methionine at position "213" is identified as M in the sequence motif

native protein RCTMDSS (SEQ ID NO: 6). Tyrosine at position 238 is identified as the "Y" within the native protein sequence motif GGTYGVG (SEQ ID NO: 7). There are many approaches known in the art to provide a series of genes encoding a series of D-amino acid oxidase variants with amino acid changes at one or both of these positions. Selection of DNA template for mutagenesis also depends on the intended use. Thus, for example, when the intended use of the mutant gene is for expression in plants then a plant optimized synthetic DNA encoding a R. gracilis D-amino acid oxidase such as the F58K mutant encoding the mutant form of SEQ ID. No. 3 is a suitable starting point. On the other hand, when the intended immediate use of the mutant gene is as a starting point for additional cycles of random mutagenesis and improvement in a yeast or E. coli based selection system (as in Example 11) then the F58K mutant from the native DNA sequence or a synthetic sequence optimized for expression in S. cerevisiae is more suitable.

A preferred procedure to provide suitable variants of R. gracilis D-amino acid oxidase is through the use of degenerate oligonucleotides using Strategenes Quickchange mutagenesis kit. The procedures used are in accordance with the manufacturer's instructions.

For example, in case the mutant F58K encoding the mutant form of the native R. gracilis DNA sequence encoding D-amino acid oxidase is the template DNA for mutagenesis, the "superior" degenerate oligonucleotide pairs (RGMUTTOP) e "Bottom" (RGMUTBOT) may suitably be 50-250 nucleotides in length and designed to comprise, within them, sequence regions as follows.

RGMUTTOP comprises within it a sequence (SEQ ID NO: 4)

RGMUTBOT comprises within it a sequence (SEQ ID NO: 5)

Furthermore, these two oligonucleotides, RGMUTTOP and RGMUTBOT comprise at each end, sequences that, once the two oligonucleotides have hybridized to each other, will constitute the 5 'and 3' ends that will exactly coincide with the ends created when the template DNA is cut in a suitable pair of unique restriction sites (ie designed so that the hybridized oligonucleotides can replace a single restriction fragment cut from the template DNA encoding the D-amino acid oxidase.

0.5 to 1.0 ug of each oligonucleotide is transferred to a 0.5 ml Eppendorf centrifuge tube and heated to a suitable temperature (for example 94 ° C, depending on calculated melting points) for 5 minutes and it hybridizes slowly by cooling to room temperature. The template DNA (eg yeast shuttle vector pYES6 / CT) is then cut with two restriction enzymes (according to the two unique restriction sites in template DNA that span the region that includes the two codons to be replaced and that characterizes the ends of the hybridized DNA), gel purified, ligated with the hybridized oligonucleotide, and transformed into yeast so that the alternative D-amino acid oxidases created by mutagenesis are expressed. Then, as described, yeast clones that produce the best growth in D phosphinothricin analogs (such as D-homocysteic acid) or in D-phosphinothricin (when the PAT gene is co-expressed) are selected as the sole source of nitrogen such as those containing the coding sequences of D-amino acid oxidase variants with the desired properties. Alternatively, D-amino acid oxidase expression is carried out in some microorganism other than yeast and, for example, under the control of the expression of the T7 promoter of a pET vector in an E. coli lysogen. In this case, after transformation, individual colonies can be selected, plated, cultured, induced, lysed and screened for the desired substrate activity against D-phosphinothricin using procedures known in the art (eg, a scan fluorometric for peroxide generation or a colorimetric assay for ammonia generation or for 2-keto acid using well established assay procedures. Transforming lines of test organism can be grown appropriately in 2 ml wells of microtiter plates, lysate in situ and colorimetrically assayed for D-amino acid oxidase activity using, alternatively, phosphinothricin or D-aspartate as substrate (depending on optimization of activity being sought) Lines providing levels are selected higher activity. Alternatively, transgenic E. coli lines are transformed ad optionally such that they express the PAT gene (eg, constitutively) and are induced with IPTG so that they express the "test" mutant Daminoacid oxidase. Induced lines showing the best growth in minimal medium provided with phosphinothricin (or, optionally, a phosphinothricin analog) are selected as the primary source of N (optionally a small amount of ammonium ions are included).

Optionally, lines are selected not only based on maximizing the ability to use Dphosphinothricin or other acidic side chain D alpha amino acids but also to improve thermal stability

(eg, extracts or cell lines are subjected to a short heat treatment prior to the enzyme assay or cells are grown at elevated or reduced temperatures) or other properties.

Yeast or other selected microbial clones are grown, DNA and full-length D-amino acid oxidase DNA sequence cloned are prepared by revision PCR and cloned into pCRBlunt using the Invitrogen zero Blunt TOPO kit. The D-amino acid oxidase coding sequences that characterize the selected clones are determined. These D-amino acid oxidase coding sequences are further subcloned for expression in a pET vector (eg pET 24a from Novagen) and transformed into E. coli BL21 DE3. The cells are grown in a fermenter in LCM50 medium containing 100 ug / ml kanamycin, induced with IPTG, collected, disrupted and partially purified the extract and assayed for Daminoacid oxidase activity (as detailed below). D-amino acid oxidase genes that encode D-amino acid oxidase enzymes that produce acceptable stability and the highest activity (kcat / Km) per mg of pure protein against D-phosphinothricin at pH 7.0 are selected.

Additionally, a series of particular DNA sequences are generated that particularly encode Daminoacid oxidase target enzymes. In particular, genes encoding the mutant form F58K of D-amino acid oxidase of Rhodotorula gracilis are generated with additional mutational changes at positions 213, 223 and 238 and, in particular, when at position 213, wild-type methionine is replaced with His, Lys, Arg, Thr, Gly, Pro, Gln, Ser, Cys, Asn or Ala, the wild type tyrosine at position 223 is replaced by His, Lys, Arg, Thr, Gly, Pro, Gln, Ser , Cys, Asn or Ala and / or the wild type tyrosine at position 238 is replaced by His, Lys Arg, Thr, Gly, Pro, Gln, Ser, Cys, Asn or Ala. The procedures used are the same as described above except that, instead of a mixture of oligonucleotides, individual pairs of oligonucleotides are designed and used to effect each single or double amino acid change. Each resulting mutant D-amino acid oxidase coding sequence is cloned for expression (unlabelled) behind the T7 promoter in pET 24A from Novagen and transformed into E. coli BL21 DE3. Cells are grown in a 1.01 fermenter in LCM50 medium supplemented with 100 ug / ml kanamycin, induced for expression with 1mM IPTG and harvested by low speed centrifugation.

LCM50 medium contains (in 1 liter)

KH2PO4 (3g), Na2HPO4 (6g), NaCl (0.5g), Casein Hydrolyzate (Oxoid) (2g), (NH4) 2SO4 (10g), Yeast Extract (Difco) (10g) , Glycerol (35 g) (these ingredients are made up in solution and autoclaved). The following additional ingredients are filter-sterilized as solutions and added to the medium: MgSO4 (2.5 ml of 246.5 mg / ml solution), Thiamine.HCl (1 ml of 8 mg / ml solution) CaCl2.2H2O (0 , 2 ml of solution 147 g / l), * reserve of Fe SO4.7H2O / citric acid (2 ml), ** Solution of trace elements (5 ml) and make up to 1 liter.

* The reserve of Fe SO4.7H2O / citric acid per 100 ml consists of Fe SO4.7H2O (0.415 mg), citric acid (0.202 mg).

** The composition of trace element solution per 1 ml is AlCl3.6H2O (20 mg), CoCl2.6 H2O (8 mg), KCo (SO4) 2.12 H2O (2 mg), CuCl2.H2O (2 mg), H3BO3 (1 mg), KI (20 mg), MnSO4.H2O (0.8 mg), Na2MoO4.2H2O (4 mg), ZnSO4.7H2O (4 mg).

Approximately 7 g of wet weight of cells are washed in water. The cells are resuspended in an equal volume of 50mM Mops / KOH buffer at pH 7.0 containing 2mM EDTA, 2mM DTT and 0.01mM FAD. Cells are suspended uniformly using a glass homogenizer and then disrupted using a one shot head on the Basic Z Cell Disruptor from Constant Systems (BudBrooke Rd, Warwick UK) at 93.05 MPa. The crude extract is kept cold (∼4 ºC), it is centrifuged at 30,000 g on average for 1 h and the sediment is discarded. Some of the extract protein is run on a Coomassie Blue stained SDS PAGE gel and, through lateral comparison with similarly prepared extracts of cells containing only "empty" pET vector, it is estimated that 2-50% of the total soluble protein in the extract is D-amino acid oxidase. Part of the extract protein is exchanged in 50 mM Mops / KOH buffer at pH 7.0 containing 0.01 mM FAD. This is diluted with the same buffer in a conventional oxygen electrode cell (calibrated at 25 ° C between zero and a saturated oxygen concentration). Optionally, the D-amino acid oxidase is further purified using ion exchange, phenyl sepharose, fractional ammonium sulfate precipitation, and gel filtration. Assays at 25 ° C are initiated by adding a 200mM solution of the DL phosphinothricin ammonium salt to the diluted enzyme or by adding 25mM D-aspartate. For the measurement of the Vmax and Km values, the substrate concentrations are varied in the normal way. Vmax values are estimated based on total protein and estimated purity of D-amino acid oxidase. Based on SDS PAGE, the mutant D-amino acid oxidase normally constitutes 15-35% of the soluble protein in crude protein extracts (or more when D-amino acid oxidase is further purified). The final reaction volume in the oxygen electrode cell is 2 ml. Final amounts of protein in the cell vary up to 5 mg depending on the level of activity being measured. Oxygen uptake rates are measured (after subtracting any drift at baseline).

The following are exemplary results obtained under the conditions described above. Wild-type R. gracilis D-amino acid oxidase shows no detectable ability to oxidize D-phosphinothricin and only low activity (∼30 nmol / min / mg) when D-aspartate is used as substrate (compared to control rates of > 40 umol / min / mg observed when using 25 mM D-alanine as substrate). The F58K mutant form shows

some low activity against phosphinothricin (> ∼15 nmol / min / mg) and moderate activity (∼ 1.8 umol / min / mg) with D-aspartate. The F58KM213S double mutant form shows very high activity against D-aspartate of ∼40 umol / min / mg and, against DL phosphinothricin, a high level of activity of ∼3.2 µmol / min / mg. The Km for D-phosphinothricin of the double mutant F58KM213S is estimated to be ∼12 mM. The triple mutant F58K, M213S, Y223H shows moderate activity of ∼0.4 umol / min / mg against D-phosphinothricin and ∼0.7 µmol / min / mg against D-aspartate.

In control experiments the pure L-form does not oxidize to detectable levels and, depending on the concentration, the pure D-form oxidizes up to twice the rate at which the DL racemate is oxidized.

Additional results are also obtained using a higher yield assay procedure based on the Konno procedure in 'Methods for the Detection of D-Amino-Acid Oxidase'. Biol. Proced. On-line. (1998) May 14; 1: 27-31. This is an especially useful procedure for initial mutant selection prior to more precise analysis using the oxygen electrode assay. Targeted / random mutants of the D-amino acid oxidase gene obtained as described above are cloned into vector PET24 or PET21 (Novagen) as NdeI / EcoRI fragments and transformed by heat shock into Competent Cells (DE3) -RP BL21-CodonPlus® (Stratagene). After selection and plating, individual colonies are selected and grown in 1 ml of L (+ Kanamycin or Ampicillin + Chloramphenicol) broth at 30 ° C overnight in a 96-well plate containing large 2-wells. ml. The plate is then subjected to low speed centrifugation, the cells are centrifuged to the bottom of the plate, and the supernatant is carefully removed. The cells are resuspended by vortexing in 0.5 ml of fresh L-Culture Broth (without antibiotic) and allowed to incubate and shake for an additional 2 hours at 30 ° C. An additional 0.5 ml of IPTG L + 4 ul Culture Broth is added and the plate is placed back in a shaking incubator (30 ° C) for 3 hours. The plate is centrifuged once more so that the cells are pelleted by centrifugation, the supernatant is removed and the plate is frozen at -80 ° C for 10 minutes. The plate is then returned to room temperature and the cell pellets are lysed prior to the enzyme assay. 0.4 ml of CelLyticB (Sigma) containing 1 mg / ml lysozyme is added to each well and left for 10 minutes. A glass bead is added to each well and then the wells are sealed as a bead mill grinding block for 2 minutes. The plate is then centrifuged to separate the residues from the cycle. 10 ul of the resulting supernatant extract are then assayed by adding 30 ul of test D-amino acid (eg, potassium or ammonium salts of D-glutamate, D-aspartate, or DL glufosinate to, eg, 5, 10, 25, 50 / 100 or 200 mM in water) together with 30 ul of 0.133 M pyrophosphate buffer at pH ∼ 8.3 (including beta mercaptoethanol 1 ul / ml and Catalase 5 mg / ml), 20 ul of 0.1 mM FAD and 10 ul 70% methanol. The plate is then incubated to allow the assay to run at room temperature for 10-60 minutes and then the reaction is stopped in each well with the addition of 100 ul of 10% TCA. 50 ul are then removed from each well into the corresponding well of a new plate in which each well contains 50 ul of 5M KOH. 50 ul of 0.5M HCl containing "Purpald" (4-amino-5) is then added. -hydrazino-1,2,4-triazol-3-thiol) 0.5% to each well and the plate is left for 15 minutes at room temperature. After this time 50 µl of 0.2 M KOH containing 0.75% potassium periodate is added to each well and finally 5 ul of isopropanol is added to each well to avoid formation. The optical density of each well of the last plate is then measured at 550 nM. High levels of D-amino acid oxidase activity correspond to high DO readings. The assay can be quantified using standard keto acid additions and specific activities are calculated based on protein concentrations measured using standard procedures such as the Bradford or Lowry procedures. DAMOX specific activities are estimated based on the percentage of the total protein in extracts that is D-amino acid oxidase (as estimated by Coomassie stained SDS PAGE). Using the above assay, it is shown, for example, at a concentration of 25 mM D-phosphinothricin substrate, that the R. gracilis D-amino acid oxidase double mutant form F58K, M213T is approximately 2-5 times more active than form F58K, M213S (see Table 1).

Table 1

Exemplary results obtained from a plate-based assay comparing the activities of mutant forms of D-amino acid oxidase with D-phosphinothricin as a substrate. The assay is run under the conditions described above using 25 mM phosphinothricin DL as the substrate. Extracts containing the wild-type form of the enzyme do not produce any detectable color under these conditions. The optical density at 550 nm obtained after a 30 minute test of the extract of the R. gracilis DAMOX mutant form F58K, M213T is more than double that obtained from a similar extract of the F58K mutant form, M213S. Since the assay is non-linear at high optical densities, the F58K, M213T mutant form is estimated to be any one of 2 to 5 times more active than the F58K, M213S form under the assay conditions.

Mutant

Experiment Absorbance at 550 nm

F58K, M213T

one 2,2019

F58K, M213T

2 2.2966

F58K, M213T

3 1.5633

F58K, M213T

4 1,4484

F58K, M213T

Half 1.8775

F58K, M213S

one 0.9843

F58K, M213S

2 0.8374

F58K, M213S

Half 0.9109

F58H, M213S

one 0.5982

F58H, M213S

2 0.6030

F58H, M213S

Half 0.6006

Example 11. Random Mutagenesis and Selection to Generate Additional Mutants of Daminoacid Oxidase Genes F58K Encoding Enzymes with Enhanced Specificity (kcat / Km) for D-phosphinothricin Oxidation

A DNA sequence is cloned, with codons optimized for expression in yeast and encoding the mutant form F58K or the double mutant form F58K, M213S or F58K, M213T of the Rhodotorula gracilis D-amino acid oxidase in the shuttle vector pYES6 / CT from Invitrogen as a HindIII / PmeI fragment downstream of the GAL1 promoter. Similarly, these DNA sequences are cloned into the protein expression shuttle vector pAUR123 (Panvera) as an XbaI fragment downstream of the constitutive promoter ADHI. The construction of these vectors

10 is performed in E. coli followed by transformation into Saccharomyces cerevisiae S288C. When appropriate, the PAT gene is used to replace blasticidine or aureobasidine antibiotic resistance genes in vectors pYES6 / CT / pAUR123 respectively and DL phosphinothricin is used in place of the antibiotic to maintain selection. Additional mutant variants of D-amino acid oxidase are created using various mutagenesis procedures. For example, multiple variants of the D-amino acid oxidase coding sequence are generated by PCR.

Poisoned with Mn2 +, the mixed population is cloned in front of the GAL1 or ADH1 promoters of the two shuttle vectors, transformed into yeast, the yeast is further transformed with a PAT gene, and selection is made based on the ability of the new sequence to confer on yeast the ability to grow more rapidly in a minimal medium with phosphinothricin as a major source of nitrogen. Alternatively, mutation and selection is carried out directly on the transformed yeast. For example, yeast is allowed to grow

Transformed with the above plasmids in a fermenter in the presence of a chemical mutagen such as EMS in a nitrogen limited culture medium containing 20-100 mM DL phosphinothricin and induced for expression of D-amino acid oxidase (eg cultured in galactose as a carbon source). Following successive subcultures, the fastest growing subcultures in phosphinothricin are identified as the primary N source, plated, and sequences encoding D-amino acid oxidase are subcloned, sequenced, and expressed

25 in E. coli for further characterization.

In a further, preferred procedure, mutagenesis is carried out on the two shuttle vectors using an amplification and passage through the E. coli XL1-red strain. This strain is deficient in three DNA repair pathways

primaries, mut S, mut D and mut T. This results in a 5000-fold increase in mutation rates during DNA replication. The protocol used is in accordance with Stratagene. For example, 10 ng of shuttle vector is transformed into E. coli XL1-red strain, cells are grown and then plated on Ampicillin-containing L-Culture Broth agar plates for 24 hours. From each plate> 200 batches of colony transformants are pooled by scraping the colonies from the plate in L broth and then 1 in 100 and 1 in 1000 dilutions are grown and successively subcultured in L / ampicillin broth at 37 ° C. for 1-2 weeks so that a large number of cell divisions occur. A similar procedure is carried out starting from several plates. Shuttle vector DNA minipreps are prepared from cells grown overnight and transformed back to yeast. The transformed yeast is allowed to grow and the improved D-amino acid oxidase containing colonies are selected as described above.

Alternatively, expression and selection of D-amino acid oxidase is carried out in some microorganism other than yeast and, for example, under the control of the expression of the t7 promoter of a pET vector in an E. coli lysogen. In this case, the D-amino acid oxidase coding sequence (optionally mutated by Mn2 + poisoned PCR) is cloned into a pET vector, transformed into E. coli XL1 red, and after passage for several generations, transformed back to a E. coli lysogen such as E. coli BL212 DE3. Individual colonies can then be selected, plated, cultured, IPTG-induced, lysed, and screened for desired substrate activity against D-phosphinothricin using procedures known in the art (eg, a fluorometric scan for peroxide generation or a colorimetric assay for generation of 2-keto acid or ammonia). Alternatively, the transgenic E. coli lines are further transformed to express the PAT gene (eg, constitutively) and IPTG-induced to express the "test" mutant D-amino acid oxidase. Induced lines showing the best growth in minimal medium provided with phosphinothricin (or, optionally, a phosphinothricin analog) are selected as the primary source of N (optionally a small amount of ammonium ions are included).

Optionally, the medium used for yeast selection contains a low concentration of solvent (for example 0.1% DMSO).

Example 12. Production of D-phosphinothricin in an enantiomerically pure form

E. coli BL21 DE3 codon plus RIL is transformed with pET 24A from Novagen having the coding sequence for PAT (A02774) cloned for expression (not labeled) behind the T7 promoter. These cells are grown to a OD600nm density of ∼40 in a 10 liter fermenter of kanamycin-containing LCM50 medium, induced with 0.2mM IPTG, harvested by low speed centrifugation and rapidly transferred to minimal medium Containing 9.91 g of the ammonium salt of D / L phosphinothricin (PPT).

Minimum medium (in 1 liter) is.

Na2HPO4 (6g), KH2PO4 (3g), NaCl (1g), NH4Cl (1g) were dissolved in water and autoclaved and the following solutions were added after filter sterilization:

CaCl2 (1 ml of 14.7 g / l), MgSO4 (1 ml of 246.5 g / l), Thiamine HCl (5 ml of 1 mg / ml)

Glucose (30 ml of 20% solution autoclaved separately), DMSO 0.5 ml.

Fermentation details are as follows. LCM 50 medium is inoculated in a 10-liter fermenter with inoculum grown in LB culture broth (200 ml) of E. coli BL21 DE3 codon plus RIL containing the PAT gene and maintained at 30 ° C, stirring rate 200 rpm, pH 6.5, 50% oxygen concentration saturated in air. After approximately 12 hours the culture grows to an OD 600 nm of ∼30. The culture is then induced for PAT expression by the addition of 0.2 mM IPTG. After 1.5 hours, the culture typically grows further to a 600nm OD of ∼40, before cells are harvested by centrifugation and washed in 8 liters of minimal medium. Cells are centrifuged once more and resuspended in a final volume of 10 liters in the fermenter in minimal medium containing 9.91 g of the commercially available ammonium salt of D / L-phosphinothricin and 0.2mM IPTG additional. The temperature is raised to 37 ° C and the samples from the fermenting medium are monitored by NMR of phosphorus and protons to determine a) when glucose levels have dropped substantially and need to be supplied and b) the extent of conversion of phosphinothricin to N-acetyl phosphinothricin . Over the course of ∼ 12 hours, ∼ 500 g of glucose are added to the fermenter. It is observed that the formation of n-acetyl phosphinothricin begins after a few hours and at ∼ 20 hours it reaches> 93% conversion of L-PPT (46.5% by volume of D / L) to N-acetyl- L-PP. The fermentation medium is collected shortly thereafter by removing the cells by low speed centrifugation.

D-PPT is purified from fermentation medium using ion exchange chromatography. The fermentation medium (∼ 9.5 l) is stored at 4 ºC. It is mixed with 900 ml of Dowex 5000-X8 200400 mesh cation exchange resin (previously prepared with HCl) as H + so that the pH of the supernatant above the resin drops to ∼ pH 3.2. The Dowex resin is allowed to settle by gravity and the supernatant is decanted along with a water rinse 21 of the Dowex resin and then centrifuged for clarification. The washed Dowex is discarded (to be recycled over time). The clarified supernatant is then extracted using a

decantation funnel with ethyl acetate (1/4 of the volume of the supernatant) and the aqueous fraction (∼ 121) is preserved. Then an additional 2.31 of H + form of Dowex 50W-X8 resin is added and stirred with -121. The resin is then allowed to settle. The pH of the supernatant above the resin is ∼ pH 1.6 at this stage. The supernatant is decanted off and discarded, and the resin is washed with ∼12 liters of water and, again, allowed to settle. Again, the supernatant is discarded and the resin is poured into a sintered Buchner funnel filter and rinsed with an additional ∼ 4.51 of water (to remove most of the residual N-acetyl-phosphinothricin). The main fraction containing D-phosphinothricin is eluted from the resin with 151 of 0.4M ammonium hydroxide, followed by a rinse with 1.41 of water from the resin. The pH of this D-phosphinothricin containing reaction is ∼ 11.4. Optionally, this is reduced to ∼ pH 10 by the addition of, for example, ∼ 0.13 mol of acetic acid and ml 600 ml of cation exchange resin in H + form. If added, the resin is allowed to settle. The D-phosphinothricin fraction (supernatant) is then loaded onto a 565 ml (5 x 28 cm) column of Dowex 1X8 400 mesh anion exchange resin in OH form (pre-equilibrated with NaOH and washed with water). A gradient of 0M - 0.32M ammonium acetate is applied to the column over 17 column bed volumes. 55 ml fractions are collected throughout the gradient. Fractions are monitored by UV at 215nm and also by 31P NMR and protons. This analysis indicates that highly pure phosphinothricin is eluted between fractions 39 and 78. N-acetylphosphinothricin is eluted as unbound material early in the gradient and some glutamate subsequently elutes fractions 79-90.

Fractions 63 to 78 (corresponding to 6-7.6 bed volumes) constitute the volume of highly pure phosphinothricin. The phosphinothricin fractions are lyophilized and found to be pure by NMR of phosphorus and protons (no peaks other than acetate are visible,> 95% of the organic material is phosphinothricin), although, based on discrepancies between calculated and observed dry weights it is typically found that some residue of inorganic salts (eg ammonium chloride) remains in the phosphinothricin samples. For practical purposes, when D-phosphinothricin is used (for example for spraying in plants) inorganic agents can be considered to be inert and need only be taken into account to adjust the calculated concentrations when D-phosphinothricin solutions are composed of samples weighed in dry.

The isolated phosphinothricin according to the above procedure is expected to be substantially enantiomerically pure Dphosphinothricin. This is verified according to the fluorescent HPLC assay procedure of Hori et al. (2002) J. Chrom. B 776, 191-198. For example, 50 ul of commercial DL phosphinothricin (0.01-10 ug / ml) or sample is dissolved in 0.1M Borate buffer pH 8.5 and mixed with 200 ul of the same Borate buffer. Then 50 ul of 18 mM FLEC ((+) - 1- (9-fluorenyl) ethyl chloroformate) are added and the mixtures are further incubated for 30 minutes at 40 ° C. Excess FLEC is removed by shaking for 3 minutes with 500 ul of ethyl acetate. 100 µl of the aqueous layer is removed from the bottom for HPLC analysis.

A practical 5 µM Inertsil ODS2 (15 x 4.6) column is equilibrated with 10 mM 77% aqueous ammonium acetate (pH 5.0): 23% acetonitrile at a flow rate of 0.8 ml / minute. A 2 ul sample is injected into the column and run isocratically for 60 minutes and monitored using fluorescence detection with excitation at 260nm and emission wavelength at 305nm. It is observed that the D and L isomers of phosphinothricin are clearly separated and elute at 12.4 and 13.4 minutes respectively. A sample of isolated D phosphinothricin is run according to the current procedure and it is estimated that it is in an enantiomeric excess greater than 99%. This is estimated based on the addition of known amounts of commercial DL phosphinothricin and observing how small a detectable increase in the right side 13.4 minute peak is against the bottom of the apparently simple 12.4 minute peak produced by the sample. .

Furthermore, the HPLC procedure is used to estimate the amount of phosphinothricin based on peak integration and comparison with a standard curve. Additionally, the total amounts of phosphinothricin are estimated by integration of NMR signals.

Overall, it is estimated that from ∼ 9.91 g of starting DL racemate, fos 1.9 g (38% yield) of pure D-phosphinothricin ammonium salt is produced in an enantiomeric excess of> 99% . 50-70% of the dry weight of the sample comprises inorganic salts that are maintained. Optionally these are removed by additional steps of ion exchange and lyophilization (after exchange to volatile salts).

SEQUENCE LISTING

<110> Syngenta Limited

<120> Procedure for selectively producing male or female sterile plants

<130> PPD70629

<160> 7

<170> PatentIn version 3.1

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40 <400> 7

Claims (10)

one.

A male or female sterile plant production method comprising the steps of transforming plant material with a polynucleotide encoding at least one enzyme that reacts with a non-phytotoxic substance to produce a cytotoxic and regenerate the material thus transformed into a plant, in which said non-phytotoxic substance is applied to the plant until the formation and / or maturation of male or female gametes, so that the non-phytotoxic substance enables the production of a phytotoxic that selectively prevents the formation of or acts as a otherwise to said non-functional gametes, in which the enzyme is preferentially expressed in male or female reproductive structures and the non-phytotoxic substance is a D-alpha amino acid, characterized in that the enzyme is a D-amino acid oxidase obtained from Rhodotorula gracilis and encoded by the nucleic acid sequence as represented in SEQ ID NO: 3, said enzyme being a mutated version of the protein encoded by SEQ ID NO: 3 and comprising a lysine at position 58 and at position 213 an amino acid selected from the group consisting of Thr, Gln and Gly; at position 238 an amino acid selected from the group consisting of Thr, Gln and Gly and / or at position 223 an amino acid selected from the group consisting of Thr, Cys, Gly, Gln and / or Asn.

2.

 A method according to claim 1, wherein said non-phytotoxic substance is applied in mixture together with at least one additional substance which is selected from the group consisting of healers, gametocides, glutathione-S-transferase inducers, cytochrome inducers P450, Herbicides, Fertilizers, Nematocides, Synergists, Insecticides, Fungicides, Hormones, Plant Growth Regulators, and P450 Cytochrome Inhibitors.

3.

 A process according to claim 1 or 2, in which the non-phytotoxic substance is applied foliarly and is a mobile oxidizable substrate in the phloem and metabolically stable of the enzyme, in which the enzyme provides the phytotoxic product, as a direct one or indirect of the non-phytotoxic substance.

Four.

 A process according to the preceding claim, in which the phytotoxic product is an indirect one produced in the form of peroxide and / or a superoxide anion.

5.

 A method according to one of claims 3 or 4, in which the non-phytotoxic substance is Daspartate or D-glutamate and the enzyme oxidizes said amino acid to a 2-keto acid with joint reduction of oxygen to a peroxide anion.

6.

 A method according to the preceding claim in which the amino acid at position 213 is Thr.

7.

 A method according to any one of claims 3-6, wherein the enzyme has a target other than the peroxisome.

8.

 A method according to claim 1 or 2, wherein the non-phytotoxic substance is the D-enantiomer of phosphinothricin or a D-enantiomer of bialaphos.

9.

 A process according to one of claims 1 or 2, in which the non-phytotoxic substance is comprised within a mixture, which contains a phytotoxic substance and in which the enzyme oxidizes an amino acid to a 2-keto acid with joint reduction oxygen to a peroxide anion.

10.

 A method according to any one of claims 8 or 9, wherein the mixture comprises both D and L phosphinothricin and the plant material expresses a PAT gene substantially only in green tissues and / or in floral tissue that produces gametes that are other than those that become non-functional.