ES2360611T3 - METHOD FOR SELECTIVELY PRODUCING MALE OR FEMALE STERILE PLANTS. - Google Patents

Abstract

Male- or female-sterile plant production method, comprising the step of transforming plant material with a polynucleotide that encodes 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 formation and / or maturation of the male or female gamete, so that the non-phytotoxic substance causes the production of a phytotoxic that selectively prevents the formation of gametes , or renders them nonfunctional, in which the enzyme is expressed in male or female reproductive structures, characterized in that: (i) the non-phytotoxic substance is a D-alpha-amino acid and (ii) the enzyme is a D- amino acid oxidase.

Description

FIELD OF THE INVENTION

Heterosis in cultivated plants has a noticeable effect on improving performance. In general, hybrids have increased yields compared to non-hybrid varieties. Hybrids typically provide a higher return unit for growth factors like water and fertilizer. Hybrids often provide superior stress tolerance, product uniformity and maturation, and also provide a simple opportunity for genetic improvement 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 widely used in many crops, including corn, sorghum, sugar beet, sunflower, and rape. However, mainly due to the lack of economic methods of hybrid seed production, wheat, barley and rice are still mainly cultivated as inbred.

Traditionally, hybrid seed production involves planting separate blocks of male and female parental lines, harvesting only the seeds from the female parent. To ensure that this seed is hybrid, the self-pollination of the female parental line must be minimized, making the line male-sterile. Methods for making the male-sterile female parent line include mechanical, chemical, and genetic methods. In monoecious plants such as maize, male sterility can be easily achieved mechanically by despendonation of the male inflorescence. However, most of the cultures are dioecious and the presence of male and female organs in the same flower makes such castration unfeasible. Therefore, genetic approaches have sometimes been used.

The features of male genetic sterility that appear are normally controlled by nuclear genes in which alleles associated with the sterile phenotype are generally expressed recessively with respect to alleles associated with fertility. When genetic male sterility occurs it is normally associated with a single recessive gene that has to be homozygous for male sterility to be expressed. In order to use such genetic male sterility traits in a practical way, breeders normally develop a phenotypically uniform female line that segregates into male-sterile and female-fertile plants. Male-sterile plants, once identified, have to be destroyed, which is laborious. There is always a problem with maintaining the parental line, as fertile male plants cannot be removed from the population because they are essential for population maintenance. Rather than relying on the existence of natural male sterility alleles, it is also possible to use molecular biology methods. Plants that express, for example, ribozyme or antisense genes that reduce or eliminate the expression of key genes necessary for the formation of viable pollen can be obtained by genetic engineering. Such transgenic plant lines are male-sterile and are used for hybrid seed production by crossing, using male-fertile plant pollen. The main problem with such lines is that they can only be maintained in an hererozygous 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, male-fertile plants can be selectively destroyed, this still requires plants to be initially planted at extra-high densities.

The use of cytoplasmic male sterility for the production of commercial hybrids requires a stable male-sterile cytoplasm and a source of pollen. The cytoplasmic-genetic system of male sterility requires the existence of three types of line for hybrid production: line A (male-sterile cytoplasmic), line B (male-fertile maintenance), and line R (male- fertile with restorative genes). Crosses in the three senses produced with this system involve the maintenance and production of four lines, and one line A and B of one consanguineous and consanguineous masculine-fertile of the other two. Relying on a single source of sterile male cytoplasm can minimize the flexibility of genetic improvement and lead to offspring with high overall susceptibility to particular diseases.

Hybrid seeds can also be produced through the use of chemicals that inhibit the formation of viable pollen. These chemicals, called gametocides, are used to produce transient male sterility. However, the cost, registrability and reliability of gametocides has limited their use.

A deficiency of traditional hybrid seed production systems is the need to plant separate rows or blocks of the male and female parent lines. In this regard, low pollination efficiency is a particularly serious problem in cultivated species, such as wheat, which release small amounts of pollen that does not travel far in the wind. In such crops up to two thirds of the hybrid's production land has to be dedicated to male pollen donor plants and consequently, the production of hybrid seeds becomes, therefore, not profitable.

In order to achieve more profitable seed production in wheat and other crops, it is necessary to move male and female plants closer to each other for more efficient pollen transfer; with maximum efficiency by interplanting males and females in the same rows at a distance of centimeters between them. In such a system it would be unfeasible to harvest only the seed of the female (male-sterile) parents. Contamination with non-hybrid seed from the female parent can be minimized by using as low a percentage as possible of such male parent plants in the potting mix and / or by using male plants that are female-sterile. A method for obtaining a dominant female-sterile line has been described (EP-412.006-A1 (1991); Goldman et al., (1994) EMBO, J., 13, 2976-2984) but, as for the male- sterile, the line has to remain heterozygous.

Consequently, the need for simple economic methods of hybrid seed production persists. In particular, in order to efficiently produce hybrid seed, there remains a need to provide both male-sterile female parent lines and male-sterile male parent lines that can be easily maintained as pure homozygous lines and are useful for efficient seed production. hybrids. The methods described in the state of the art to achieve this include methods in which the 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 returns to the conditionally sterile line, depending on the exogenous application of a non-phytotoxic substance, which can be converted locally and specifically to a phytotoxin, by means of an enzyme encoded by the heterologous chimeric gene and which is preferably expressed, either in male or female reproductive structures . The non-phytotoxic substance can be a herbicide precursor. 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 altered only after exogenous application of the non-phytotoxic substance. In one such example of a conditional male sterility system, a gene encoding the deacetylase enzyme is preferentially expressed in the male flower tissue mat cells, where it converts the applied herbicide precursor to N-acetyl-L-phosphinothricin herbicide exogenously, in phytotoxin Lfosfinotricina and, therefore, prevents the formation of viable pollen. In other similar examples: (i) the preferential expression of the bacterial cytochrome P450 mat catalyzes the conversion of the herbicide precursor R7402 into a sulfonylurea phytotoxin that prevents the production of viable pollen; and (iii) the preferred expression on the mat of a phosphonate monoester hydrolase catalyzes the conversion of the glyceryl glyphosate herbicide precursor to the phytotoxin glyphosate, which likewise prevents the production of viable pollen. WO 98/03838 describes examples of a conditional female sterility system, in which enzymes capable of converting herbicide precursors to phytotoxins are preferably expressed in female reproductive structures.

Despite the existence of these methods of obtaining male and female parental lines that are conditionally sterile, the production of hybrid seeds is still far from being routine in crops such as wheat. The present application relates, inter alia, to improvements in the art with respect to the production of female parental lines that are conditionally male-sterile and female parental lines that are conditionally female-sterile.

The present application relates to improvements in methods for the production of hybrid seed for cultivation. In particular, the application refers to a method of producing hybrid seed from female and male parental lines, at least one of which is conditionally female or male-sterile, depending on the exogenous application of a substance that is not phytotoxic to the culture and which includes herbicide precursors. The application also relates to a method in which said non-phytotoxic substance is applied at once and in a sufficient quantity so that self-fertilization is minimized or avoided in the conditionally sterile parental line or lines. The present application also relates to a method of producing conditionally male- or female-sterile plants, by (i) transforming plant material with one or more chimeric genes that, in isolation or together, encode one or more enzymes capable of react with a non-phytotoxic substance, preferably as a herbicide precursor, to produce a phytotoxic substance. Enzymes are expressed under the modulable control of one or more promoters that, in the case of conditionally male-sterile plants, causes the enzyme or enzymes to be expressed preferentially in male reproductive structures or which, in the case of conditionally female plants -Sterile, causes said enzyme or enzymes to be preferentially expressed in female reproductive structures. Plant material is regenerated into morphologically normal fertile plants, which are conditionally male- or female-sterile. The application also includes the use of conditionally male-sterile plants in combination with conditionally female-sterile plants to produce hybrid seed more efficiently, the use, as non-phytotoxic substances, of certain herbicide precursors and the use of chimeric genes to produce more efficiently Hybrid seeds, chimeric genes and enzymes useful for the invention. The application also provides conditionally male-sterile plants, conditionally female-sterile plants, seeds of these plants, and hybrid seeds produced by said method. In preferred embodiments of the application, cultivated plants to which the method of obtaining hybrid seed 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 formation and / or maturation of the female or male gamete, so that the non-phytotoxic substance allows the production of a phytotoxic that prevents selectively the formation of gametes, or, if not, transforms it becomes nonfunctional gametes, in which the enzyme is preferably expressed either in male or female reproductive structures. The method is characterized in that

(i) the non-phytotoxic substance is a D-alpha-amino acid and (ii) the enzyme is a D-amino acid-oxidase.

Because a desirable goal is to maximize hybrid seed yield, and therefore minimize any crop damage, in preferred embodiments, the non-phytotoxic substance is a herbicide precursor selected from compounds that are relatively non-phytotoxic to the crop. In order for them to be able to produce an effect against floral tissues, it is also desirable that herbicide precursors be phytotoxin precursors that are effective on "non-green" tissues. Thus, in preferred embodiments of the invention, herbicide precursors are selected from those that are phytotoxin precursors that are directly phytotoxic to non-green tissues, rather than those that have a major site of action in photosynthesis or pigment production. photosynthetics. It is also a desirable goal to minimize the costs of hybrid seed production. Thus, in preferred embodiments, herbicide precursors are selected from those chemicals for which approval for use in crops has already been granted or is being processed by the appropriate regulatory authorities.

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 comprising a polyadenylation site.

"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 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 other sites that facilitate precise excision from a DNA locus and its insertion in other.

"Non-phytotoxic substances" are, in the context of the present invention, substances that are relatively non-phytotoxic to the plants, cells or tissues of any particular crop to which the method of the invention is applied. Non-phytotoxic substances must be non-phytotoxic in all plant tissues of all plants. Non-phytotoxic substances include herbicide precursors that are substances with a negligible direct toxic effect on plant tissues, but that are precursors of active phytotoxins. In susceptible plant species, such herbicide precursors act indirectly as herbicides through the action of endogenous enzymes that convert them into a phytotoxin within 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 to which the method of the invention is applied. Such phytotoxins do not have to be phytotoxic to all tissues of all plant species.

"Female reproductive structure" means female gametes and those portions of the plant that are specialized in the production, maturation, and viability of female gametes. Typically, these comprise those portions of a plant that comprise the carpel or gynoecium ("pistil"). Carpel of a plant includes, but is not limited to, a stigma, style, ovary, and cells or tissues within the stigma, style, and ovary.

"Male reproductive structure" means male gametes and those portions of the plant that are specialized in the production, maturation, and viability of male gametes. These comprise those portions of a plant that comprise, for example, microspores, stamens, rugs, anthers, and pollen.

"Female-sterile plant", as used here, 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 genetic selection.

or the presence of a transgene. A "conditionally female-sterile plant" refers to a plant that, under normal growth conditions, is female-fertile and can become female-sterile under specific conditions. In the context of the present invention, said conditions comprise the exogenous application of a herbicide precursor or other non-phytotoxic substance. In the context of the present invention, said "female-sterile plant" or "conditionally female-sterile plant" remains male-fertile and capable of producing viable pollen.

"Male-sterile plant", as used here, is a plant that is unable to withstand the formation of viable pollen. Such male sterility may be the result of genetic selection or the presence of a transgene. A "conditionally male-sterile plant" refers to a plant that, under normal growth conditions, is male-fertile and can become male-sterile under specific conditions. For example, conditions may include physical exoneration or the application of a specific chemical gametocide. In the context of the present invention, said conditions particularly comprise the exogenous application of a herbicide precursor or other non-phytotoxic substance. In the context of the present invention, such a "male-sterile plant" or "conditionally male-sterile plant" remains female-fertile and capable of producing viable seeds when pollinated with functional or viable pollen.

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

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

In the method according to the invention, said non-phytotoxic substance can be applied mixed with at least one additional substance, which can be selected from the group consisting of amino acids, protectors, gemetocides, 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 mixed with piperonyl butoxide or malathion. In particular embodiments, said non-phytotoxic substance can be applied mixed with the same phytotoxic substance of which said non-phytotoxic substance is a precursor.

Said enzyme can be a D-amino acid oxidase and the non-phytotoxic substance can then be a D-amino acid and, in particular, it can be the D enantiomer of phosphinothricin, the D enantiomer of bialaphos or be selected from the group consisting of D-alanine, D -serine, D-isoleucine, D-methionine, D-leucine or D-valine. As used herein "D-amino acid oxidase" means any enzyme capable of oxidizing a D-amino acid to produce a 2-keto-acid, and includes enzymes with specificity for aspartate, known as "D-aspartate oxidases".

Chimeric genes that encode enzymes capable, alone or in combination with others, of reacting with a non-phytotoxic substance to produce a phytotoxic can be selected from genes comprising DNA coding sequences that encode one or more of the following enzymes:

D-amino acid oxidases capable of catalysing oxidation:

D-amino acid + O2 + H2O → NH3 + H2O2 + 2-oxoacid

and, in certain embodiments, particularly the reaction:

D-phosphinothricin + O2 + H2O → NH3 + H2O2 + 2-oxo-4-methyl-phosphinobutyrate.

The enzyme D-amino acid oxidase (DAMOX) can be selected from those produced by Rhodosporidium sp. (Rhodotorula sp.), Trigonopsis sp. , pig. Fusarium sp. Candida sp., Shizosaccharomyces sp. and Verticillium sp., and can be selected from proteins having sequences corresponding to Swissprot accession numbers P80324, Q99042, P00371, P24552 or SPTREMBL numbers Q9HGY3 and Q9Y7N4. DNA sequences encoding Daminoacid oxidase can be selected from sequences within EMBL Accession Numbers A56901, RGU60066, Z50019, SSDA04, D00809, AB042032, RCDAAOX, A81420, and SPCC1450. D-amino acid oxidases are ubiquitous flavoenzymes.

When the non-phytotoxic substance is D-phosphinothricin or D-bialaphos or D-aspartate or D-gitamate, then the particularly preferred Daminoacid oxidases are obtained from Rhodotorula gracilis mutants or it is a D-aspartate oxidase. Such mutants, whatever the phytotoxic substance, may comprise single or double amino acid substitutions at positions 213 and 238, when compared to the wild-type sequence. Preferably, at position 213 the wild methionine is replaced by Arg, Lys, Ser, Cys, Asn or Ala, and the wild type Tyr at position 238 is replaced by His, Ser, Gys, Asn or Ala.

However, the enzyme can comprise substitutions in addition to, or in other positions than those mentioned in the previous paragraph. In particular, Phe at position 58 in the wild sequence can be replaced by a residue selected from the group consisting of His, Ser, Lys, Ala, Arg, and Asp, and is preferably His, Ser, or Ala. In addition, or alternatively, the Met at position 213 in the wild sequence can be replaced by a residue selected from the group consisting of His, Ser, Lys, Ala, Arg and Asp and preferably is Ser or Ala. In addition, or alternatively, the Tyr at position 238 in the wild sequence can be replaced by a residue selected from the group consisting of His, Ser, Lys, Ala, Arg and Asp.

A particularly preferred mutant form of the enzyme comprises at least two of the aforementioned mutations. A first embodiment of such a double mutant has a His at position 58 (better than Phe in the wild sequence) and a Ser at position 213 (better than Met). A second embodiment of such a double mutant has Ser at position 58 (better than wild-type Phe) and Arg at position 213 (better than wild-type Met).

When the non-phytotoxic substance is a D-amino acid other than D-phosphinothricin or D-bialaphos, then the enzyme is a D-amino acid oxidase. The D-amino acid is preferably not an endogenous metabolite of the plant, and is selected and selected to be a mobile one in the phloem, metabolically stable in the plant (preferably having a t ½ in the plant greater than ~ 1 week) and an efficient substrate for said oxidase. The oxidation of Daminoacid by the enzyme is concomitant with the reduction of oxygen to peroxide and / or phytotoxic superoxide anions.

In a preferred embodiment, the enzyme oxidase is directed to a subcellular location other than the peroxisome. This is achieved, for example, by gene modification, such that three C-terminal amino acids are deleted or modified (eg, SKL in the case of Rhodotorula gracilis D-amino acid oxidase) and / or by addition of a sequence for adding a mitochondrial or chloroplastic transit peptide to the N-terminus.

Other suitable D-amino acid oxidases can be obtained preferably by fungal sources, by mutational and selective procedures known to the person skilled in the art and increased by the present description.

Other D-amino acid oxidase enzymes (or likewise phosphinothricin-racemase) and DNA coding sequences suitable for practicing the invention are selected from those organisms, optionally subjected to mutagenesis, in which growth in a medium with Limited N, under conditions in which D-amino acid oxidase (or phosphinothricin-racemase) is induced (for example growth on D-alanine), said enzyme is selectively inhibited in the presence of D-phosphinothricin. The D-amino acid oxidase genes suitable for the invention are then obtained, for example, by testing libraries for such organisms with suitable degenerate DNA probes (eg based on the establishment of consensus D-amino acid oxidase sequences, such as PROSITE, PS00677) and subcloning. Alternatively, genes encoding suitable enzymes are obtained by screening gene expression libraries in a suitable host cell such as E. coli or a yeast (suitable host strains lack endogenous oxidase or dehydrogenase activity against Dphosphinotricin) for transformation into a phenotype with increased sensitivity to growth inhibition by D-phosphinothricin in minimal medium. This method 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 heterologous 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 methods to directly test the activities of D-amino acid oxidases, such as those based on peroxide detection (Enzyme Microb. Technol., (2000), 27 (8), 605-611), oxygen depletion using a oxygen electrode or based on direct detection of ammonia.

In one embodiment of the invention, a DAMOX gene, preferably derived from fungi, is cloned into a transporter vector under the modulable control of a promoter (eg, a GAL promoter) capable of being expressed in the host organism in which it is will make the selection (preferably yeast). This gene is then subjected to mutagenesis, for example by Mn2 + poisoned PCR; plasmid DNA replication in a strain that is defective in DNA repair / editing processes such as the E. coli XL1 red strain; or by replication of the plasmid DNA in a host strain that undergoes mutagenesis using, for example, X-rays, ultraviolet light, addition of chemical mutagen, and transformation into a host organism (preferably yeast). The desired DNA encoding a DAMOX with the desired property of an increased ability to oxidize D-PPT is selected (following an optional initial selection step for transformants based on selectable markers present in the transporter vector, allowing, for example, selection via restoration of prototrophy or growth in the presence of hygromycin, etc.), via, for example:

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

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b) Selection of transformed cells capable of using D-PPT itself as the sole source of N. For this selection to work, the host cell must also transform itself with a gene capable of reversing the inhibitory effect of L-phosphinothricin on the glutamine synthetase. For example, the transporter vector may also comprise a gene encoding an enzyme such as PAT, which inactivates L-PPT.

Mutation and selection cycles can be repeated. D-amino acid oxidases can be cloned, expressed, partially purified, and kinetically characterized in order to identify genes and DAMOX with the most suitable properties (eg, enzyme stability, high kcat / km value for D-PPT oxidation, minimal oxidation of any endogenous plant substrate, 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 background level of N-acetyl-PPT-deacetylase activity) transformed and induced to express a PAT gene (encoding an enzyme that transfers an acetyl group from acetyl CoA to L-PPT), at high levels. After allowing adequate time for substantially all of the L- component to be N-acetylated (assayed, for example, by monitoring the conversion using 31-P NMR), D-PPT is recovered and purified from the cell-free medium , using successive steps, for example, high and low pH solvent extraction, cation and anion exchange chromatography, selective crystallization with chiral cations such as quincocin, or other procedures known in the art, such as liquid / liquid extractions with two aqueous phases non-miscible, such as the phase system (eg methods in US Patent 5,153,355). Typically, a final step is cation exchange chromatography, from which D-PPT is recovered as the ammonium salt.

Alternatively, D-PPT can be obtained by an enzymatic method in which DL PPT + 2-ketoglutarate is converted first to a mixture of D PP5, 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 obtained from the reaction mixture using methods known in the state of the art, as outlined above.

D-PPT can also be obtained using an enzymatic method in which DL-PPT + 2-ketoglutarate + NAD is first converted to 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 further method of obtaining D-PPT, DL-PPT is treated with an L-amino acid oxidase, so that the remaining amino acid is in the desired D-form. This D-PPT is recovered after the reaction mixture.

Still another method involves (I) converting DL-PPT to N-acetyl-DL-PPT / using acetic anhydride or other acetylating reagents and methods known in the art) and (II) treating N-acetyl DL PPT with D-amino -acylase, so that only N-acetyl D-PPT is deacetylated. The resulting D-PPT is purified from the reaction mixture. For example, D-PPT is recovered from N-acetyl-L-PPT, by binding to a 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.

Still another method involves treating DL-PPT with L-aminoacylase and an acylate in a nonaqueous solvent, such that only the desired D-PPT is left in non-acetylated form.

Yet another method of preparing pure D-PPT involves enantioselective crystallization of DL PPT using a chiral base, such as quincocin, and addition of a chiral base seed crystal with pure D-PPT.

Still another method of preparing pure D-PPT from DL-PPT is by direct chiral chromatography, using a chiral base column.

In one of the examples below, a detailed method for the production of pure D-PPT is provided.

The DNA sequences encoding the enzymes used in the present invention can optionally be further mutated and screened, in order to generate more useful enzymes, with improved utility. Many enzyme characteristics can therefore be improved, including catalytic activity (kcat / km) against the desired substrate, stability against temperature and optimal pH. The methods for generating, screening and selecting such improved variants are well known. For example, suitable DNA variant sequences are generated by a mutagenesis procedure (eg, passing DNA through bacterial or yeast strains with error-prone DNA replication, such as E. coli XL1 red, by mutagenesis. chemistry or oligonucleotide-directed PCR). In particular, such genes are produced by any alternative DNA mixing or "sexual PCR" method as outlined, for example in WO-0061740, pages 28-41, all of which are included herein by reference. Many methods are suitable for selecting such improved genes. Genes can be adequately expressed in a suitable host cell, such as E. coli or yeast, and are selected for improvement using suitable assays, such as those described herein.

Chimeric genes encoding enzymes for use in the invention that are capable, alone or in combination with one another, of converting a non-phytotoxic substance to a phytotoxic, may each comprise a DNA sequence encoding one of said enzymes operably linked to a 5 'promoter region, which, preferably, directs expression to either male or female reproductive structures. This specificity of expression ensures that the effect of the enzyme or enzymes expressed will be exerted only in the location of the tissues and cells necessary for the formation of viable seed or pollen, and will not be deleterious for the plant, beyond its effect on the fertility in the presence of a suitable non-phytotoxic substance, perhaps a herbicide precursor. In addition to promoter regions, the chimeric genes according to the present invention also comprise an en3 'transcriptional terminator sequence. This is responsible for the termination of the transcription and the correct polyadenylation of the mRNA. Many of these 3 'transcriptional 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 'transcriptional terminator sequence is selected from the CMV 35S terminator, the tm1 terminator, the nopaline synthase (nos) terminator, and the pea rbcS EO terminator.

The 5 'promoter regions, suitable for use in certain embodiments of such chimeric genes, include 5' regions of genes that are preferably 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 (Dzellcalns 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, other suitable promoter regions are obtained from regions upstream of the coding sequences of genomic DNA, corresponding to cDNA sequences, which are known in the art to be preferably expressed in female reproductive structures In certain embodiments, such cDNA probes are selected from the group consisting of the Arabidopsis genes Fbp7 and Fbp11 (Angenent et al., (1995) Plant Cell, 7, 1569-1582) and the orchid-specific cDNAs O40, O108, O39, O126 and O141 (Nadeau et al., (1996) Plant Cell, 8, 213-239). Preferred ations, the 5 'promoter regions comprising genomic DNA associated with preferential expression in female reproductive structures, are selected from DNA regions comprised in the group consisting of the genomic DNA clone pSH64, with accession number NRRL B21920, clone genomic pCIB10302 that hybridizes with the P26-A4 cDNA with accession number NRRL B 21655 and the genomic DNA clone X2-1, which hybridizes with the cDNA clone P19-QA, with accession number NRRL B-21919. In additional particular embodiments, these promoter regions comprise 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, other 5 'promoter regions, suitable for use in the chimeric genes of the invention, are isolated and cloned by methods that are customary to one skilled in the art. For example, new transcripts expressed in female reproductive structures are identified by isolating RNA from tissues such as corn, silk genera, or wheat pistils, followed by differential screening using techniques such as differential visualization, subtraction of selected cDNA by PCR, and construction of subtractive cDNA libraries. . CDNA clones that are preferentially expressed in female tissues and not in other parts of the plant, such as leaves, roots, and banners, are isolated. Tissue specificity of expression is optionally confirmed by Northern blotting. The cDNA clones are used as probes for screening genomic libraries. 5 'promoter regions and, optionally 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 female reproductive structures.

The 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 structures. These include promoter regions for expression in pollen, mat, or other anther structures. In certain embodiments, these 5 'promoter regions are selected from the group consisting of the LAT52 promoter (Twell et al. (1989) Dev., 190, 705-713), the tomato A127 promoter (Dotson et al., ( 1996) Plant J., 10, 383-393), the Zmg maize promoter (Hamilton et al., (1989) Sex. Plant Reprod. 2, 208-212), the maize CDPK promoter (Guerro et al., (1990) Mol. Gen. Genet., 224, 161168) and the anther-specific promoters ant32 and ant43D, described in US 5477002, incorporated herein by reference in their entirety. In certain further embodiments, the 5 'promoter region is selected from the group consisting of the corn CA55 specific mat promoter ("Pea55" described in WO 92/13956), the rice E1 mat specific promoter (described in US 5,639,948), the rice-specific anther RA8 promoter (EMBL / Genbank accession number AF042275; Jean Js et al., (1999) PMB, 39, 35-44), the TapI anther-specific promoter (Spena et al (1992) Thero. Appl. Genet. 84, 520-527) and the maize ZmC5 pollen specific promoter (EMBL / Genebank accession number Y13285; Wakeley et al, (1998) PMB, 37, 187-192 ). Optionally, suitable additional promoter regions are obtained from upstream regions relative to genomic DNA coding sequences corresponding to prior art cDNA sequences that are preferentially expressed in male reproductive structures. In certain embodiments, such cDNA probes are selected from the group consisting of the cytochrome P450 gene, specific to the orchid pollen tube (Nadeau et al., (1996) Plant Cell, 8, 213-239), the gene Arabidopsis bcp1 (Xu et al (1995) PNAS, 92, 2106-2110) and the male corn flower specific MFS14 gene (Wright SY et al., (1993) Plant J 3, 41-49). In further embodiments, other 5 'promoter regions, suitable for use in the chimeric genes of the invention, are isolated and cloned by methods that are known to one skilled in the art. For example, new transcripts expressed in male reproductive structures are identified, isolating RNA from tissues such as banners, pollen tubes, anthers, or mat, followed by differential screening using techniques such as differential visualization, subtraction of cDNA selected by PCR, and construction of cDNA libraries. subtractive. CDNA clones are isolated which are preferentially expressed in male tissues and not in other parts of the plant, such as leaves, roots and stigma. The tissue specificity of the expression is optionally confirmed by Northern blot. The cDNA clones are used as probes for screening genomic libraries. 5 'promoter regions and, optionally, 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.

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

The chimeric genes may additionally comprise a region, which immediately precedes the DNA sequence encoding the enzyme involved in the conversion of the non-phytotoxic substance to phytotoxin, which encodes a peptide sequence capable of directing said enzyme to subcellular organelles such as chloroplast, peroxisome (in a case other than that in which the phytotoxin is a peroxide or superoxide anion) or mitochondria, and said targeting protein can have the sequence of (i) a chloroplastic transit peptide or (ii) an N-terminal portion of a chloroplastic transit peptide of a chloroplastic protein or chloroplastic transit peptide. This may be especially advantageous when, for example, such a DNA sequence encodes a D-amino acid dehydrogenase enzyme, which would be expected to work best in a compartment such as the mitochondria or chloroplast comprising a membrane electron transport chain or when, for example, For example, the DNA sequence encodes an enzyme that catalyzes only a partial step in the desired overall transformation and where the total reaction requires combination with compartmentalized metabolites and endogenous activities. In particular, to target the mitochondria, said DNA region immediately preceding the enzyme-encoding DNA sequence encodes a mitochondrial transit peptide sequence. In certain embodiments, the sequence of the transit peptide can be selected from the group consisting of the endogenous transit peptide sequences of the beta subunit of the mitochondrial ATP synthase of Nicotinia plumbanginifolia, NADP-dependent mitochondrial isocitrate dehydrogenase, a subunit that is binds NADPH of respiratory chain I complex and yeast mitochondrial tryptophanyl-tRNA synthetase (WO 6121513).

The polynucleotides for use in the present inventive method may comprise one or more chimeric genes encoding enzymes that catalyze reactions involved in the generation of phytotoxins from non-phytotoxic substances. Optionally, said polynucleotides comprise additional chimeric genes and 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. The marker DNA encodes an RNA, protein, or polypeptide, which, 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 such 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 render plant cells resistant to antibiotic selection, lethal. in other circumstances (eg, the aac (6 ') gene encoding resistance to gentamicin, WO 94/01560, or hygromycin transferase genes, which provide resistance to hygromycin), or herbicides such as glyphosate (eg. , EPSPS genes, such as in US 5510471 or WO 00/66748), fenmedifam (eg pmph gene US 5347047; US 5543306), bromoxynil (eg genes described in US 4810648), sulfonylureas (p 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 synthase inhibitors, such as L- phosphinothricin (such as, for example, the N-acetyl transferase genes described in EP 0242246, EP 0242246, and EP 0257542). In a preferred embodiment of the polynucleotide of the present invention, which comprises a herbicide resistance gene as a marker gene, said herbicide is a herbicide that is useful for the control of weeds in the crop and, additionally, the resistance gene to Herbicide is sufficiently expressed to provide a strong tolerance to the proportions of said herbicide in the field. In a further preferred embodiment of the invention, 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 a positive metabolic advantage to transformed plant cells, in the context of a particular medium. US 5767378 describes various suitable positive selection genes and systems.

When the polynucleotide of the present invention comprises a herbicide resistance gene, the herbicide is applied exogenously to cultivated plants, which are interleaved at a density sufficient to eliminate non-hybrid seed production originating from self-fertile parent plants. In a preferred embodiment, the herbicide is glyphosate or one of its agronomically useful salts, and said herbicide resistance marker gene is selected from those genes that confer herbicide resistance described in WO 00/66748.

When a marker gene is present, means can 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 eliminate herbicide resistance genes that could interfere with the operation of the herbicide precursor dependent conditional fertility mechanism of the present invention. For example, it might be desirable to remove an N-acetyl transferase (PAT) herbicide resistance marker gene from a polynucleotide that also comprises a chimeric gene, useful for providing conditional male or female sterility, dependent on exogenous application of herbicide precursor D -phosphinothricin. The presence of the PAT gene could potentially interfere with successful conditional sterility, inactivating the L-phosphinothricin phytotoxin. Thus, polynucleotides comprising marker genes may optionally comprise specific recognition sites for specific recombinases at positions flanking the marker gene and allowing the sequence to be removed. Crossing a plant carrying the marker gene thus flanked with a plant bearing a marker gene encoding the corresponding specific recombinase produces a progeny of plants from which the marker is specifically cleaved. Examples of such 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 inventive method may optionally comprise one or more translation activators, located in the 5 'untranslated regions of the protein coding regions. The person skilled in the art is aware of the identity of such suitable translation activators, such as the Omega and Omega prime primer sequences derived from TMV and those derived from the tobacco engraving virus, and how such translation activators can be introduced into the polynucleotide, to provide the desired result of increased protein expression.

Additional examples include translational activators 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 protein expression from chimeric genes and chimeric marker genes, said polynucleotides may further comprise elements such as activators, framework binding regions (SAR or MARS), and introns. Various intron sequences, such as the maize adh 1 intron, have been shown to activate 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 exhibit the desired male / female sterility characteristics may also have been transformed with a polynucleotide comprising protein-coding regions capable of conferring on the plant material containing them, at least one of the following agronomically desirable characteristics: resistance to insects, fungi, viruses, bacteria, nematodes, stress, desiccation and herbicides.

Genes that confer resistance to herbicides can be selected, for example, from the group encoding the following proteins: glyphosate oxidase (GOX), EPSP synthase, phosphinothricin acetyltransferase (PAT), hydroxyphenyl-pyruvatodioxygenase (HPPD), glutathione -S-transferase (GST), cytochrome P450, acetyl-CoA-carboxylase (ACCase), acetolactatosintase (ALS), protoporphyrinogen oxidase (PPG), dihydropteroate synthase, polyamine transport proteins, superoxide dismutase (SOD), bromoxynil -nitrilase, phytoene desaturase (PDS), the product of the tfdA gene obtainable from Alcaligenes eutrophus, and known variants, mutagenized or modified in other ways, 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 used to convert the non-phytotoxic substance. In case the polynucleotide provides resistance to multiple herbicides, such herbicides can be selected from the group consisting of a dinitroaniline, triazolo-pyrimidine, herbicide, uracil, phenylurea, tricetone, an ixoxazole, acetanilide, oxadiazole, triazinone, sulfonanilide, , anilide, isoxaflutol, fluorochloridone, norflurazon and triazolinone, and the post-emergence herbicide is selected from the group consisting of glyphosate and its salts, glufosinate, asulam, bentazon, bialaphos, bromacil, setoxidim, or other cyclohexanedione, dicamba, fosamine, flupoxy -propionate, quizalofop or other aryloxy-phenoxy-propanoate, picloram, fluormetron, butafenacil, atrazine or other triazine, metribuzin, chlorimuron, chlorosulfuron, flumetsulam, halosulfuron, sulfometron, imazaquine, imazetrapir, isoxaben, imazam, , nicsulfuron, fomesaphene, fluroglycophen, KIH9201, ET751, carfentrazone, mesotrione, sulcotrione, paraquat, diquat, b romoxynil and fenoxaprop.

In the event that the polynucleotide comprises sequences encoding insecticidal proteins, these proteins can be selected from the group consisting of crystalline Bt-derived toxins, including secreted Bt toxins, such as those known as "VIPs", protease inhibitors, lectins, and toxins from Xenhorabdus / Photorhabdus. Genes that confer resistance to fungi can be selected from the group consisting of those encoding known AFPs, defensins, chitinases, glucases, and Avr-Cf9. CryIAc, cryIAb, cry3A, Vip 1a, Vip 1B, Vip 3a, Vip 3B, cysteine protease inhibitors and snowflake lectins are particularly preferred insecticidal proteins. In the event that the polynucleotide comprises genes that confer resistance to bacteria, these can be selected from the group consisting of those that encode cecropins and tequiplesin and their analogues. Genes that confer virus resistance can be selected from the group consisting of those that encode envelope proteins, motion proteins, viral replicases, and viral antisense and ribozyme sequences, which are known to provide resistance to viruses, while genes that confer resistance to stress, salt and drought can be selected from those that encode Glutathione-S-transferase and peroxidase, the sequence that constitutes the regulatory sequence CBF1 and genes known to allow the accumulation of trehalose.

The polynucleotides used according to the present invention may have been modified to activate the expression of the protein coding sequences comprised by them, so that the motifs and / or fortuitous cleavage regions of mRNA instability may have been removed, or the codons Culture preferred may have been used such that expression of the thus modified polynucleotide in a plant produces a substantially similar protein having activity / function substantially similar to that obtained by expression of the coding regions of the unmodified polynucleotide protein. , 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 in said plant and encoding substantially the same protein, may be such as to avoid 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 to be "Kozack preferred". What this means is well known to the person skilled in the art.

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

The plants of the invention can be selected from the group consisting of crop products, fruits and vegetables, such as rape, sunflower, tobacco, sugar beet, cotton, corn, wheat, barley, rice, sorghum, fodder beet, tomato, mango , peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, soybean, sugarcane, sugarcane, pea, small bean, poplar, grape, citrus, alfalfa, rye, oats, grass and forage grasses, flax oil and nuts-producing seeds and plants, provided that their progeny, seeds and parts are not specifically mentioned.

Such specifically preferred plants include wheat, barley, oats, rice, corn, millet, and sorghum.

The invention also provides a preferred method of producing hybrid wheat seed, comprising the steps of:

(i)

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

(ii)

 select the material thus transformed; and

(iii) regenerate the material so selected in conditionally male-sterile, morphologically normal complete plants;

(iv)

 obtain a feminine, conditionally masculine-sterile, homozygous parental line;

(v)

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

(saw)

 select the material thus transferred; and

(vii) regenerate the material thus selected in conditionally female-sterile, morphologically normal complete plants

(viii) obtain a male parental line, conditionally female-sterile, homozygous;

(ix)

intercropping said male and female parental lines, conditionally sterile in such a proportion as to ensure efficient pollination;

(x)

 applying said herbicide precursor or other non-phytotoxic substance to the intercropped planted parent lines, in a dose and at a stage of development such that self-fertilization is minimized;

(xi)

 Harvest hybrid wheat seed from intercropped parent plants.

The present invention likewise provides variants of the above method, in which the male parent is female-sterile by any method, the female parent is male-sterile by any method, the male and female parent lines are conditionally sterile, depending on the application of two different herbicide precursors, both being applied, and the crop is different from wheat.

Regarding the transformation of plant material, those skilled in the art will recognize that, although specific types of target material (eg suspension culture of embryogenic cells) are specified in the examples below.

or immature embryos that are being dedifferentiated) and particular transformation methods (eg using Agrobacterium or particle bombardment), the present invention is not limited to these particular embodiments and such target materials, and the methods can be used interchangeably . Furthermore, the term "plant cells", as used throughout the description of the invention, can refer to isolated cells, including suspension cultures, as well as to cells in intact or partially intact tissues, such as embryos, escutello, microspore, embryo derived from the 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 methods.

The present invention will become clearer from the following non-limiting examples, taken in conjunction with the sequence listing and associated drawings.

SEQ ID NOS: 5 and 6 represent the PCR primers used to obtain the TA29 promoter region

SEQ ID NO: 7 represents a DNA sequence, isolated from Rhodotorula gracilis, that encodes an enzyme with D-amino acid oxidase activity.

SEQ ID NOS: 8 and 9 represent degenerate oligos used to provide a variant of D-amino oxidase.

SEQ ID NOS: 10 and 11 represent motifs in which alternative amino acids can be substituted to provide D-amino acid oxidase variants.

Figure 1 is a schematic representation of a construct for tobacco transformation, which has Rhodotorula D-amino acid oxidase under the modulable control of the stig 1 promoter region. The components indicated are LB (left border sequence), AOPRE1 (promoter AoPR1, PSTIG1 (EMBL access number X77823), RGDAO (OPT) (SEQ ID No.: 7), PC PROMOTER (EMBL access number X16072), PAT (EMBL access number A02774), NOS (terminator we obtained EMBL access number atu237588) and RB (right limit sequence).

Figure 2 is a schematic representation of a construct for tobacco transformation, in which a 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 to a region encoding an optimized transit peptide (FR2673643).

General molecular biology methods are performed according to well-established methods.

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, this is considered to refer to DNA sequences comprising the promoter, sequences upstream of the promoter, and also, optionally, all or part of the DNA sequence encoding the leader region. 5'-untranslated mRNA.

Example 1. Tobacco plants that are conditionally feminine-sterile, dependent on the exogenous application of D-phosphinothricin or D-alanine or D-leucine or D-methionine or D-asparagine or D-aspartate or D-glutamate

The DNA sequence encoding the D99 amino acid oxidase protein sequence Q99042 (Swissprot) comprised in the EMBL Z50019 sequence is obtained either by RT-PCR from Trigonopsis variabilis mRNA, or obtained synthetically. Alternatively, the DNA sequence encoding the P80324 D-amino acid oxidase protein sequence (Swissprot) included in the EMBL A56901 sequence, is obtained either by RT-PCR from Rhodosporidium toruloides (Rhodotorula gracilis) mRNA or synthetically obtained ( which makes it easier to control which internal restriction enzyme sites are present and to create flanking sites to facilitate cloning) such as SEQ ID No. 7, which is designed with the use of the plant codon in mind (in this case wheat ), and to minimize DNA characteristics potentially unfavorable for expression. Alternatively, the DNA sequence (eg derived from EMBL accession number X95310) encodes a "D-aspartate oxidase" as P31228 (Swiss Prot) and, again, is synthesized taking into account the use of the codon of the plant and to minimize the unfavorable characteristics for expression. Alternatively, the D-amino acid oxidase coding sequences obtained are the same as in Example 2. The flanking sequences of the PCR primer and the synthetic DNA sequences are designed to establish unique restriction sites useful for cloning. Preferably, in the case where the oxidase coding sequence does not contain confusing internal sites, an Nco1 or Nde 1 site is placed at the 5 'end, to facilitate the cloning of internal fusions to the reading frame with sequences added to the ORF in 5 ', as coding sequences for the chloroplastic transit peptide. In some variants of the example, the D-amino acid oxidase gene (called in some variants "D-aspartate oxidase") is cloned in such a way that the 3'-terminal amino acids are truncated and the enzyme, therefore, already it is not directed at the peroxisome. In a further series of variants of the method, the gene is obtained by genetic engineering by PCR, to encode Rhodotorula gracilis D-amino acid oxidase with alternative amino acids at positions 213 and 238 and, in particular, with an arginine, serine, cysteine, lysine , asparagine or alanine replacing methionine at position 213 and / or a histidine, serine, cysteine, asparagine or alanine replacing tyrosine at position 238. Methionine at position "213" is identified as M in the protein sequence motif native RCTMDSS. Tyrosine at position 238 is identified as "Y" in the motif of the native GGTYGVG protein sequence. These variants of Rhodotorula D-amino acid oxidase or "D-aspartate oxidase" are used when female sterility has to be made conditional upon application of D-aspartate, D-glutamate or D-phosphinothricin.

Restriction sites can be placed upstream of the introns of the ATG translational initiation site, consistent with the consensus translation sequences of the plant, such as those according to Kozak.

The "delta S13" promoter is a promoter region useful for obtaining preferential expression in female flower parts. It comprises a -339 to -79 region from the SLG13 promoter region, fused with that ranging from -46 to + 8 of the promoter nuclear CMV 35S (Dzelkains et al (1993) Plant Cell, 5, 833-863). This S13 promoter region is cloned into the bluescript sk plasmid, which is again subjected to restriction and ligated with restriction fragments that comprise the 3 'transcriptional terminator region and one or the other of the amino acid oxidase coding sequences, so that create an "delta S13-D-amino acid oxidase-terminator Nos" expression cassette within a bluescript sk plasmid. This is then suitably removed as, for example, in the form of an EcoR1 fragment and, as such, ligated back into a suitable site of a vector such as Pbin19 (Bevan (1984) Nucleic Acids Res.) Or pCIB200

or pCIB2001 (WO 98/39462) for use for transformation using Agrobacterium. As described in WO 98/39462, pCIB200 contains the following unique polyconnector restriction sites: EcoR1, Sst1, Kpn1, BgIII, Xaba1, and SaII. PCIB 2001 contains an insert in the polyconnector, which adds additional unique restriction sites, including MluI, BcII, AvrII, ApaI, HpaI and StuI. PCIB200 and pCIB2001 also provide selectable marker genes for plant and bacterial selection on kanamycin, left and right T-DNA boundaries, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the oriT and oriV functions of RK2. Alternatively, the binary vector Pcib10 is used, which incorporates sequences of plasmid Prk252 from a wide range of hosts (Rothstein et al (1987) Gene 53, 153-161) or one of its derivatives, which incorporates kanamycin resistance genes and the gene hygromycin-phosphotransferase, such as Pcib715 (Gritz et al (1983) Gene 25, 179-188).

Alternatively, the Stig promoter region 1 ~ 1.6 kb (derived from EMBL accession number X77823) is used. For example, the coding region of the GUS gene in the stg1-GUS construct, described by Goldman et al (1994) in EMBO J., 13, 2976-2984, is replaced with the DNA sequence that encodes the P30324 or Q99042 coding sequences. , using suitable restriction enzymes, and the resulting stig1-D-amino acid oxidase 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 T-DNA border sequences.

In a further particular example, the T-DNA insert comprised in the binary vector is constructed according to Figure 1. A construct comprising the synthetic DNA sequence (SEQ ID No. 7) encoding Rhodotorula gracilis D-amino acid oxidase is cloned. , under the modulable control of the stig1 promoter region and also the DNA sequence (A02774), which encodes L-phosphinothricin-N-acetyltransferase (PAT), under the modulable control of the pea plastocyanin promoter region, in a site between the LB / upt II gene and the RB of the T-DNA of the binary vector. In summary, sequence ID No. 7 is cloned into plasmid pFse4-Stig1nos (described in WO9942598) behind the Stig1 promoter and against the nos terminator region (comprised in EMBL ATU237588), as an NcoI / PstI fragment. The pea plastocyanin promoter region (derived from EMBL accession number X16082) is obtained from the pea genomic DNA by PCR and cloned against the PAT / terminator nos gene. The resulting PC-PAT-nos cassette is cloned behind the Stig1RGDAMOX-nos, as a NotI fragment, and this entire two-gene construct is transferred to a binary vector (Pvb6, a derivative of Bin19), as an Fse1 fragment.

In a further variant of the method, the construct used is in accordance with the schematic representation of Figure 2. The coding sequence for Rhodotorula D-amino acid oxidase, SEQ ID No. 7, optionally mutated by targeted mutation to encode the M213R form, it is truncated by 3 codons at the 3 'end, and at the 5' end it is cloned to place it immediately downstream of a region encoding a chloroplastic transit peptide, so that a chloroplastic transit peptide fusion protein is encoded / D-amino acid oxidase. The coding sequence for the chloroplastic 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, it is modified to include an Sph1 site at the CTP processing site, thereby replacing Glu-Lys at this site with Cys-Met (SEQ EN Fig. 9 of WO 92044490). Correspondingly, a site at the N-terminus of the D-amino acid oxidase coding sequence can be genetically engineered (converting the amino acid following methionine to a Leu). Alternatively, the coding sequence for the chloroplastic transit peptide is derived from the Petunia gene encoding EPSP-synthase (Fig. 11 of WO 92044490). Alternatively, the coding sequence for the chloroplastic transit peptide is one of a large number 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, rather than relying on subcloning, the total desired DNA sequence encoding the Rhodotorula chloroplastic transit peptide / Daminoacid oxidase fusion polypeptide can be synthetically obtained in a straightforward manner. This sequence is cloned at a site downstream of the stig1 promoter region and upstream of a terminator sequence (eg, nos), into a suitable vector (eg,. Replacing the GUS coding sequence in the vector containing the stig1 construct

→ GUS, described by Goldman et al. (1994) in EMBO J., 13, 2976-2984). The total gene expression construct is then cloned into a suitable site between the left and right limits of the T-DNA of a PVB6 vector.

Tobacco leaf discs are transformed with the recombinant binary vectors, using methods similar to those described in Horsch et al (1985) Science, 227, 1229-1231. Many variations of the method can be used. The binary vector can be transformed, for example, into the Agrobacterium tumefaciens LBA 4404 strain using the freeze-thaw transformation method. The transformation of the tobacco and the regeneration of the total plant is carried out using Nicotiana tabacum var. Samsum, according to the protocols described by Draper et al. (Plant Genetic Transformation, Blackwell Sci. Pub. 1989). Transformation events are selected on MS medium containing kanamycin or other suitable antibiotic. The presence of integrated transgenes is confirmed by PCR. Plants regenerate and are allowed to reach maturity and allowed to produce seeds on their own. Northern and / or Western analysis is used to confirm the specific tissue expression of the D-amino acid oxidase genes. The plants are self-fertile but have the characteristic of conditional female sterility. The seeds of the T1 generation are planted. 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 fully fertile in the absence of an exogenously applied herbicide precursor. A subset of these conditionally sterile (putatively) plants are treated with D-phosphinothricin or D-alanine or D-leucine or D-asparagine or D-methionine or Daspartate or D-glutamate, in varying amounts and in varying stages of growth. Such treatments are carried out on T1 plants confirmed as PCR positive for the D-amino acid oxidase gene or, similarly, such treatments are carried out directly on plants of the T0 generation (which are cloned vegetatively so that the untreated clones of each event can be set aside for seed production). The observed fertility is then used as the basis for selecting suitable plant lines that display the clearest conditional sterility phenotype. For example, those amino acids are pure D enantiomers or, alternatively, DL racemates. For example, they are applied as a foliar spray, before or during the early stages of flower formation, in proportions usually between 0.25 and 20 kg / ha. Amino acids that can be removed by crystallization from the solution on the leaves, after foliar application, can be re-dissolved and mobilized again for absorption by the leaves by new applications of water in the form of a spray mist. Amino acids are also applied, for example, as root drench and optionally can also be applied as ~ 50 ml of a 10-200 mM solution that directly floods the buds of the emerging florets. Pollen is collected from the treated plant and its viability is tested. Plants are obtained that produce relatively little or no seed after treatment with Dphosphinothricin or D-alanine or D-leucine or D-asparagine or D-methionine or D-aspartate or D-glutamate, but which, however, under the same conditions of treatment, produce close to normal levels of viable pollen. Control plants are transgenic and non-transgenic, and are grown under identical conditions and under an identical regimen of physical treatments, except that the treatment solutions are water or an equivalent concentration of pure Lamino Acid.

In a variant of the method, 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 modulable expression of a specific female florar promoter region as "stig 1", also a sequence DNA (embl a02774) a "PAT" gene under the modulable control of a promoter region as the 5 'region of the start of translation of the Pisto sativum plastocyanin gene (accession number EMBL X16082). For example, the construction is the same as that shown in Fig. 1, except that the DNA sequence encoding Daminoacid oxidase is mutated in a targeted manner, so that the M213R form of the enzyme is encoded. The plastocyanin promoter region provides the preferred expression in the green tissues of the plant. It is unexpectedly discovered that such a promoter, which, unlike for example the 35S promoter region, is expressed substantially only in certain plant tissues and more markedly in green tissues, provides, however, when used in combination with the gene PAT, a complete reproductive tolerance to the herbicide DLPPT, even in proportions in excess of 2 kg / ha. Furthermore, in the absence of any suitable heterologous D-amino acid oxidase, or similar D-to-L-converting activity co-expressed in flower tissues, the plastocyanin / PAT gene combination provides essentially complete reproductive tolerance, without any significant performance loss, despite that the PAT expression level is low or non-existent in many of the critical floral tissues, when it is expressed under the control of this promoter region. Therefore, in this variant of the example, the non-phytotoxic substance Dphosphinothricin is applied in its least expensive and most readily available form, as the commercial herbicide racemate DL-phosphinothricin. At suitable proportions and spraying time intervals between 250 g / ha and 5 kg / ha of DLphosphinothricin, the treated plants are not visibly damaged but become conditionally female-sterile, while remaining normal or close to normal for male fertility .

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

The DNA sequence encoding the sequence of the Q9HGY3 D-amino acid-oxidized protein (Sptrembl) included in the EMBL sequence AB042032 is obtained either by RT-PCR of Candida boidini mRNA, or obtained synthetically. Alternatively, the DNA sequence encoding the P24552 D-amino acid oxidase protein sequence (Swissprot), included in the EMBL D00809 sequence, is obtained either by RT-PCR of the Fusarium solani mRNA, or obtained synthetically. The synthetic or PCR primer flanking sequences are designed to locate unique restriction sites useful for cloning. Preferably, and in the case where the oxidase coding sequence does not contain confusing internal sites, an Nco1 or Nde1 site is placed at the 5 'end, to facilitate cloning of fusions within the reading frame with sequences added to the ORF in 5'. Alternatively, when the restriction sites are placed upstream of the ATG translation start site, the introns are designed to be in accordance with consensus plant translation sequences according to Kozak. Alternatively, the sequences encoding D-amino acid oxidase obtained are the same as in Example 1. Again, as in the previous example, when sterility has to be made dependent on the application of D-aspartate, D-glutamate or D-phosphinothricin, then, preferably, D-amino acid oxidases are variants at amino acid positions 23 and / or 238.

The TA29 promoter region (Kriete et al (1996) Plant J., 9, 808-818) is cloned from tobacco genomic DNA by PCR, using primers 5'- AACTGCAGCTTTTTGGTTAGCGAATGC-3 '(SEQ ID No. 5) and 5'CAGACTAGTTTTAGCTAATTTCTTTAAGTAAAAAC-3 '(SEQ ID No. 6). Through a series of restriction and subcloning steps, the PCR fragment thus obtained is placed upstream of the D-amino acid oxidase coding sequence and a transcriptional terminator nos is added 3 'to the coding sequence. The resulting TA29-D-amino acid oxidase-terminator expression cassette is then cloned, obtained as a suitable restriction fragment, and cloned into a binary vector as in Example 1. As in Example 1, when sterility has rather than conditional upon application of D-aspartate, D-glutamate, or D-phosphinothricin, the use of D-amino acid oxidase variants of Rhodotorula gracilis with mutations at positions 213 and / or 238 is preferred.

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

The tobacco plant material is transformed via Agrobacterium with the vector and transgenic plants are regenerated in a similar way to that described in the previous example. The plants produced are self-fertile but are conditionally male-sterile. Seeds of the T1 generation are planted in the field. Once the 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 fully fertile in the absence of an exogenously applied herbicide precursor. A subset of these T1 plants, which are, putatively, conditionally sterile, or, alternatively, seedlings of the T0 (direct regenerative transformation) events are treated with D-phosphinothricin, D-alanine, D-leucine, D-methionine, D -asparragin, D-aspartate or D-glutamate in various amounts and in varying stages of growth. When T0 plants are treated, they are cloned vegetatively, so that untreated siblings of the events are set aside for seed production. The observed fertility is then used as the basis for selecting suitable plant lines that display the clearest conditional sterility phenotype. For example, these amino acids are D-enantiomers or, alternatively, DL racemates. For example, they are applied as a foliar spray, before or during the early stages of flower formation, in proportions usually between 0.25 and 20 kg / ha. Amino acids that can be removed by crystallization from the solution on the leaves, after foliar application, can be re-dissolved and mobilized again for absorption by the leaves by new applications of water in the form of a spray mist. Amino acids are also applied, for example, as root drench and optionally can also be applied as ~ 50 ml of a 10-200 mM solution that directly floods the buds of the emerging florets.

Pollen is collected from the treated plants and its viability is tested. Plants are obtained that produce 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 malformed or not viable. However, such male-sterile plants remain female-fertile and produce (hybrid) seed when pollinated with pollen collected from other non-GM or conditionally female-sterile untreated tobacco plants. Control plants are transgenic and non-transgenic, and are grown under conditions identical to physical treatments, except that the treatment solutions are water,

or an equivalent concentration of pure L-amino acid.

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 the D-amino acid oxidase under modulable expression control of a tissue-specific male floral promoter region, such as "TA29", also a DNA sequence encoding a phosphinothricin-N-acetyltransferase gene, such as the "PAT" gene, under the modulable control of a promoter region, such as that of the plastocyanin gene (in this case the Pisto sativum plastocyanin gene region) . At appropriate spraying times and ratios 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 close to normal as to to female fertility.

Example 3. Chimeric genes preferentially expressed in female reproductive structures and encoding enzymes capable of oxidizing D-phosphinothricin and / or D-alanine and / or D-leucine and / or D-methionine and / or Dasparragin and / or D-aspartate and / or D-glutamate

DNA sequences encoding the D-amino acid oxidase protein are obtained, as described in Examples 1 and

2.

The genomic clone pSH64 was deposited under the terms of the Budapest Treaty on 02/27/1998 with NRRL and was assigned NRRL B-21920. It was detected as a genomic clone that hybridized to the silk-specific cDNA clone B200i4-2 (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 was 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 of WO 98/39462, a BamH1 site and the 3 'B200i untranslated terminator region, comprising nucleotides 4427-6397 of SEQ ID No. 11 of WO 98/39462, as described in WO 98/39462 . Using partial digestion with BamH1 or, alternatively, through new steps of subcloning, PCR and ligation, alternative D-amino acid oxidase coding sequences are ligated at or adjacent to the BamH1 site position, so that they are immediately positioned 3 'with respect to the B200i promoter region and 5' from the B200i terminator region. Consequently, a series of Bluescript vectors pBLB200_Q99042, pBLB200_P80324, pBLB200_Q9HGY3 and pBLB200_P24552 are created, encoding the alternative expression cassettes B200iD-amino acid oxidase-B200i.

Alternatively, as described in WO 98/39462, a Pst I / Neo I fragment from the 5 'promoter region of the P19 gene is cleaved from the X2-1 genomic clone, which was deposited under the terms of the Budapest Treaty on 27 / 02/1998 in NRRL and to which the access number B-21919 was assigned. 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 P19 gene. Using appropriate subcloning, restriction, ligation and PCR steps, this fragment is ligated to form an in-frame fusion with one or the other of the DNA sequences encoding D-amino acid oxidase and a terminator sequence nos is added at position 3 'with respect to the coding sequence. Consequently, a series of Bluescript vectors pBLP19_Q99042, pBLP19_P80324, pBLP19_Q9HGY3 and pBLP19_P24552, etc. are created, encoding the alternative P19D-amino acid oxidase expression cassettes. Alternatively, using alternative standard methods, similar plasmids having the 5 'promoter region (comprising some or all nucleotides 1-3987 of SEQ ID No. 2 of WO 98/39462) of the P26 gene are obtained instead of the promoter region from P19. The genomic clone P26-A4, pCIB10302, deposited under the terms of the Budapest Treaty on January 21, 1997 with accession number NRRL B-21655 from the Agricultural Research Service patent culture collection, is subcloned as described in WO 98/39462. Consequently, a series of Bluescript vectors pBLP26_Q99042, pBLP26_P80324, pBLP26_Q9HGY3 and pBLP26_P24552 are created, encoding the alternative expression cassettes P19-D-amino acid-oxidase-us.

Example 4. Chimeric genes preferentially expressed in male reproductive structures and encoding enzymes capable of oxidizing D-phosphinothricin and / or D-alanine and / or D-leucine and / or D-methionine and / or Dasparragin and / or D-aspartate and / or D-glutamate

D-amino acid oxidase protein sequences are obtained as described in Examples 1 and 2.

Plasmid pGK73 carries the promoter region TA29s EcoR1-fragment BamI-II from ~ 810 to +54 (Kriete et al. (1996), 9, 809-818). This restriction fragment or a suitable similar fragment generated by PCR is cloned, preferably as an in-frame fusion, at a position upstream of the DNA sequence encoding Daminoacid oxidase, in Bluescript sk. Using a suitable series of restriction, ligation and subcloning steps, a transcriptional terminator nos is added in position 3 'with respect to the coding region to generate, according to the coding sequence, alternative expression cassettes of the type TA29-D-amino acid oxidase- nos, in Bluescript sk plasmids.

In a further example, the anther-specific SGB6 promoter region, SEQ ID No. 1 of US 5470359 is used. For example, pSGBNE1, which contains a subcloned genomic EcoR1-Nhe1 fragment from pSGB6g1 (US 5470359) is subcloned again to place a 1558 bp ApaII / Xba1 blunt-ended fragment in Bluescript ks at the SmaI site. As before, by new restriction and cloning steps, this fragment is fused in-frame, upstream of the D-amino acid oxidase coding sequence. Again, a nos terminator is added 3 'to the coding sequence to create alternative Bluescript sk plasmids, comprising the alternative SGB6-D-amino acid oxidase-nos expression cassettes.

In a similar group of examples, the rice anther specific promoter region, RAS (EMBL / Genbank Accession No. AP042275; Jean Js et al. (1999) PMB, 39, 35-44) is also similarly fused into an in-frame and upstream site of one or another of the DNA sequences encoding D-amino acid oxidase and a 3 'nos terminator, to comprise alternative RA8-D-amino acid oxidase-nos expression cassettes in a series of Bluescript sk vectors.

Example 5. A pair of complementary constructs useful in a method of providing (a) a female inbred parental line, which is conditionally male-sterile, depending on the application of DL-phosphinothricin and (b) a complementary male inbred parent line, which it is conditionally feminine-sterile, depending on the application of DL-phosphinothricin.

The first suitable DNA construct to provide a female inbred parental line of rice or cereal plants, which is conditionally male-sterile, 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 N-acetylating Lphosphinothricin under the modulable control of the ~ 1 kb promoter region of the barley plastocyanin gene (EMBL Z28347) and a suitable terminator region such as that of gene nos or 35S, B) consists of a PAT coding sequence similar to the first, but this time under the modulable control of a tissue-specific female floral promoter region (such as P19 or P26, as described in Example 4), plus a suitable terminator; and C) consists of a suitable DAMOX coding sequence, as described in Examples 1, 2, 9 and 10, for example, a Rhodotorula gracilis D-amino acid oxidase mutant having an arginine, serine, cysteine, lysine, asparagine, or alanine replacing methionine at position 213 and / or a histidine, serine, cysteine, asparagine, or alanine replacing tyrosine at position 238, under the modulable control of a tissue-specific male floral promoter region (such as SGB6 or RA8, as described in Example 5), and a suitable terminator region. This construction is assembled using methods that are standard in the art and described in the previous examples.

The second DNA construct suitable for providing a male inbred parental line of cereal or rice plants, which 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 Nacetyl L-phosphinothricin under the modulable control of the promoter region of the barley plastocyanin gene and a suitable terminator region, such as that of the nos or 35S gene; D) consists of a PAT sequence, similar to the first, but this time under the modulable control of the same tissue-specific male floral promoter region (such as SGB6 or RA8, as used in Example 4), as used in construction 1, plus a suitable terminator; and F) consists of a suitable DAMOX gene, as described in Examples 1, 2, 9 and 10, and, for example, encoding a mutant form of Rhodotorula gracilis D-amino acid oxidase having an arginine, serine , cysteine, lysine, asparagine or alanine replacing methionine at position 213 and / or a histidine, serine, cysteine, asparagine or alanine replacing tyrosine at position 238, under the modulable control of a female floral promoter region (such as P19 or P26, as described in Example 3), as used in Construction 1, and a suitable terminator region. This construction is assembled using methods that are standard in the art and described in 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 = P19 promoter region → PAT coding sequence, 35S terminator;

C = RA8 promoter region → Rhodotorula amino acid oxidase (mutant M213R), terminator Nos.

Construction 2

A = barley plastocyanin promoter region → PAT coding region, terminator Nos

B = RA8 promoter region → PAT coding region, 35S terminator;

E = P19 promoter region → Rhodotorula D-amino acid oxidase coding region (M213R mutant), 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 (for example, pBLRA8_Q01470, pBLRA8_P37967, pBLRA8_P40363, pBLB200_Q99042, pBLB200_P80324, pBLB200_Q9H200_P, etc.). 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, rather than using co-bombardment, said Bluescript vectors are further engineered by standard methods, further comprising a selectable marker gene such as the kanamycin resistance, hygromycin resistance, methotrexate resistance, or glyphosate resistance gene, and are used directly. In some of the previous examples, a PAT gene is integral to the vector design and, in these cases, DL-phosphinothricin can optionally be used for selection at some stage after transformation.

Alternatively, the expression cassettes are removed into suitable restriction fragments 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 the bacterium is based on the complementation of a mutant of E. coli his B auxotrophic. The vector comprises a gene that expresses IGPD (the HisB product) and is genetically engineered to comprise a plant-selectable marker gene, such as an EPSPS gene, cloned into the Xma I site, such as pZEN16i and pZEN18i of WO 00/66748. Alternatively, a marker gene that provides positive selection for mannose or xylose is used (US 5767378).

In particular examples of use of pIGPD9 vectors, plasmids are constructed for wheat transformation. Illustrative examples are pZEN18_BLB200_Q99042 and pZEN18_BLRA8_Q01470. These are vectors derived from pIGPD9 that comprise the pZEN18 EPSPS gene (WO 00/66748) and, in this case, the B200i- (Q99042) Daminoacid oxidase-B200i or RA8- (Q01470) carboxylesterase-nos expression cassettes, respectively.

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

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

In one example, immature embryos (0.75-1.0 mm long) of the UC703 genotype are plated on MS medium containing 3 mg / L 2,4-D and 3% sucrose. After approximately 4 h, the embryos are plated in MS medium containing 15% maltose, 3% sucrose and 3 mg / l 2,4-D, covered with an agarose layer supported on filter paper, which contains the same components. The embryos are left to plasmolyze for 2-3 h before bombardment.

DNA prepared as described in Example 6 and subsequent examples, is precipitated onto micron-sized gold particles, using standard procedures. Four target plates with 16 embryos per target are bombarded twice with a DuPont Biolistics helium device, using a burst pressure of 1100 psi. The plates are bombarded with a screen 80 screen placed between the carrier phase and the target. After bombardment, the targets are placed in the dark at 25 ° C for 24 h before the layers with the embryos are deposited on plates of MS medium containing 3% sucrose and 3 mg / l 2,4-D. The individual embryos are removed from the slides and placed directly in freshly prepared medium of the same composition after another 48 h. Approximately 6 weeks after gene delivery, the tissue is placed on MS medium with 3 mg / l 2,4-D, 3% sucrose and 0.2 mg / l methotrexate, over a period of 3 weeks. The tissue is then placed in regeneration medium comprising MS medium containing 1 mg / l zeatin riboside and 1 mg / l methotrexate. After 2 weeks of regeneration, the seedlings are placed in sterile containers with medium strength MS medium containing 2% sucrose, 1 mg / l naphthyl acetic acid and 4 mg / l methotrexate.

In particular, example variants of the vectors comprising chimeric genes expressed preferentially in male reproductive structures are co-bombarded with alternative selectable marker genes. Thus, for example, plasmid DNA such as pBLRA8_P24552 (expressing a D-amino acid oxidase) is prepared under the modulable control of the RA8 promoter region and coated on gold particles together with pUbiHyg (a plasmid encoding hygromycin phosphotransferase under control modulable from the corn polyubiquitin promoter). In this case, transformation and regeneration is carried out as described above except that, following bombardment, the regeneration medium contains increasing concentrations of hygromycin between 2 and 20 mg / l.

In a further example, the wheat is transformed with pZEN18_BLB200_Q99042, selected using glyphosate and regenerated as described in Example 15 of WO 00/66748.

DNA is extracted from tissues of plant leaves derived from transformation and PCR is performed for the presence of the selectable marker gene and the gene encoding D-amino acid oxidase. PCR positive plants are propagated. During flowering, the 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 obtained by cutting 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 8. An efficient production method of hybrid cereal crops is applied in which DLphosphinothricin is applied for weed control and, at the same time, as a hybridizing chemical, and in which the hybrid F1 generation of plants resulting from seed hybrid thus produced is substantially tolerant, both vegetatively and reproductively, to the application of DL-phosphinothricin.

Chemical hybridizing substances are expensive. It would be desirable to use a relatively cheap substance, such as a commercial herbicide, as a chemical hybridizer. This would also achieve greater efficiency, since weed control could be combined with chemical hybridization. However, there are several problems to solve in order to achieve this purpose. Firstly, it would be necessary to establish male and female parental lines that are tolerant to the herbicide in question. Furthermore, in order to achieve the desired “conditional” fertility in response to herbicide application, both lines would need to be engineered so that tolerance to the herbicide does not extend to all tissues, but express in a tissue specifically, so that each of the required floral tissues remains selectively susceptible. Therefore, in one line (the female parental line), the thickness of the plant plus the female tissue must become tolerant, while some critical part of the female floral tissue must remain susceptible to application, while in the other (the male parental line), the opposite is required, with only a critical part of the female gamete-forming tissue remaining susceptible. Even if this could be achieved, there is an additional problem to solve regarding hybrid seed and F1 production. Since this generation of the crop would necessarily contain at least two genes capable of conferring resistance to the herbicide, it would be desirable if this same herbicide could be used for the control of weeds in the crop. However, it is very difficult to conceive of a combination of herbicides, tissue-specific promoter regions and tolerance genes that allows this use of the same herbicide in the F1 generation. It would be probable that the hybrid culture presented vegetative tolerance, but little or no grain yield, after the application of 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 allows sufficient expression of an R-EPSPS in all tissues and at times other than, say, a critical stage in the development of stamens or stigmas. The most direct way in this regard would be to use an antisense or similar approach, in which the expression of R-EPSPS is driven by a tissue / constitutive non-specific promoter and only locally and transiently suppressed, for example in stamens, due to expression of an antisense EPSPS gene (see eg WO 9946396). However, in this case the suppression of expression in the stamen (or stigma) would be directed by a dominant gene. It is clear that, for any such mechanism, application of the herbicide to the F1 generation would produce a sterile, non-productive culture, due to the additive effects of the dominant male and female conditional sterility genes.

The present invention provides a method of solving the problem of allowing the use of a cheap commercial herbicide, DL-phosphinothricin, as a weed control and hybridizing substance in the production of hybrid cereals and, providing such a method in addition, resulting hybrid cereals or rice. , in which DL-phosphinothricin (or L-phosphinothricin) can be used safely for weed control without substantial loss of performance. As an added bonus, homogeneous F1 generation seed, which can later grow as a naturally regenerating plant in subsequent crops, will be easier to handle since it will itself be generally sterile if sprayed with controlled amounts of DL-phosphinothricin . The same will be true of the cross pollen progeny of F1 plants for weeds (eg red rice) or other cereals. The present invention provides genes and enzymes that convert a non-phytotoxic component, D-phosphinothricin, to a commercial herbicide DL-phosphinothricin formulation into the active L-form. The PAT gene, which converts L-phosphinothricin to N-acetyl-L-phosphinothricin, is known and is used commercially to provide tolerance to DL-phosphinothricin in cultures. A further critical observation relating to the present example is that, surprisingly, wheat containing the PAT gene under the control of modulatory expression of the barley plastocyanin promoter region was found to be substantially reproductively tolerant to the application of DL -phosphinothricin in proportions of at least 2 kg / ha. Therefore, a critical feature of the constructs described in Example 5, which are used to provide the plants of the present example, is that the PAT gene, which provides the resistance characteristic, is expressed under the modulable control of a promoter region that it provides expression substantially only in green tissues. A feature of such a useful promoter region is that it should express PAT in a way that adequately protects all non-green flower tissues from foliar applied DL-phosphinothricin, while providing, at the same time, only a minimal level of PAT expression in the floral fabric itself, and especially low in those parts to which conditional sterility is directed. With PAT expressed under the control of the barley plastocyanin promoter region, this condition appears to be met, since substantially all of the L-phosphinothricin that is sprayed enters via the leaves, is intercepted, and is converted to non-N-acetyl-L-phosphinothricin. toxic, before being translocated to developing floral tissues. Therefore, in the present invention, the L-phosphinothricin that produces the effects of selective tissue sterility in the parental lines, is generated only temporarily and locally by mobile non-phytotoxic D-phosphinothricin through the phloem, via D-amino acid oxidase . By associating exactly the floral control elements that direct the expression of PAT with those elements that direct the expression of D-amino acid oxidase in the complementary pair of constructs (Example 5), it is ensured that, in the F1 hybrid, the explosion Transient L-phosphinothricin in the target floral tissue is 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 induces a non-sterility effect in the hybrid. However, in subsequent generations, the floral-corresponding PAT and D-amino acid oxidase of the hybrid will segregate and therefore, once again, the resulting plants will be male- or female-sterile after application of controlling amounts of DL -phosphinothricin.

Using the methods described in Examples 6 and 7, the constructs described in Example 5 are transformed into wheat or (using standard superbinary vector methods) rice, which is selected and regenerated into seedlings. Using methods essentially as described in Examples 1 and 2, we select the transforming events of T0 (using clonal propagation of shoots to keep the lines untreated) and suitable events to alternatively reproduce male inbred parental lines that are conditionally female- sterile, dependent on the application of DL-fofinotricina or female inbred lines that are conditionally masculine-sterile, dependent on the application of DL-fosfinotricina. The best lines have the best herbicide tolerance, minimal loss of performance, the clearest phenotype of conditional sterility, etc. Alternate male and female parental lines are selected and optionally backcrossed into suitable elite lines for several generations. The genetic inserts in these selected events are finally fully characterized as are the inheritance genetics of the characteristics of conditional fertility and herbicide resistance, and the characteristics of the expressed gene products.

The female and male parental lines thus selected are then planted interleaved together, in suitable proportions in a field, and sprayed with DL-phosphinothricin in a suitable proportion between 0.05 and 5 kg / ha and at a rate up to the period Early flowering, selected to optimize hybrid seed production. The seed thus produced has the advantage that it will originate plants that not only benefit from the vigor of the hybrids, but are also tolerant to herbicidal formulations containing DL-phosphinothricin, which they can therefore use to control weeds. herbs. Hybrid seed also has the advantage that the herbicide tolerance trait it expresses will only be incompletely transmitted to future homogeneous or exocruced generations of related weeds. Thus, for example, the hybrid rice resulting from this invention can be grown using DL-phosphinothricin as a weed control substance, without significant yield loss. However, future generations of red rice plants that are generated as progeny from hybrid rice exocrossing with female red rice generators will be vegetatively tolerant to DL-phinothricin treatment, but have reduced self-fertility (due to expression of a D-amino acid oxidase in the floral tissue) and therefore produce little grain. Therefore, using the hybrid rice of the present invention, DLphosphinothricin can be used for weed control with a greatly reduced future risk of contamination of the grain with red rice, as a result of the herbicide resistance trait being exocruciated towards red rice, closely related. Similarly, second generation natural regeneration plants, which are generated from the hybrid crop, will not produce grain, for the most part, after spraying with DL-phosphinothricin.

Example 9. Targeted mutagenesis to generate genes encoding D-amino acid oxidases that oxidize Dphosphinothricin.

This example relates to the production of genes encoding R. gracilis D-amino acid oxidase variants that have an enhanced ability to oxidize D-phosphinothricin. These genes are used in preferred embodiments of the invention, described in the other examples, where sterility is made conditional upon application of Dphosphinothricin. In the present example, these genes encode enzymes that have a single amino acid change at position "213" and / or at position "238". Methionine at position "213" is identified as M in the sequence motif of the native protein RCTMDSS. Tyrosine at position 238 is identified as "Y" in the motif of the native GGTYGVG protein sequence. 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. The choice of template DNA for mutagenesis also depends on the use. Thus, for example, when the intended use of the mutant gene is for expression in plants, then a suitable starting point is synthetic DNA encoding a R. gracilis D-amino acid oxidase, such as SEQ ID No. 7. On the other hand, when the immediate use of the mutant gene is for subsequent rounds of mutagenesis and enhancement in a yeast-based selection system (as in Example 10), then the native DNA sequence (optionally enhanced for expression in S. cerevisiae) it is more suitable.

A preferred method of providing suitable variants of R. gracilis D-amino acid oxidase is through the use of degenerate oligonucleotides, using the Stratagene Quickchange Mutagenesis Kit. The methods are used according to the manufacturer's instructions.

For example, (in the case where the native R. gracilis DNA sequence encoding D-amino acid oxidase is the template DNA for mutagenesis), pairs of degenerate "start" (RGMUTTOP) and "end" oligonucleotides ( RGMUTBOT) may suitably be 50-250 nucleotides in length and designed to comprise within it sequence regions as follows:

RGMUTTOP comprises a sequence (SEQ ID No. 8)

tccccatgcaagcgatgcacgNNNgactcgtccgaccccgcttctcccgcctacatcattccccgaccaggiggcgaagtcatctgcggcgggacgNNNggcgtgg gagactgggacttg.

RGMUTBOT comprises a sequence (SEQ ID No. 9)

caagtcccagtctcccacgccNNNcgtcccgccgcagatgacttcgccacctggtcggggaatgatgtaggcgggagaagcggggtcggacgagtcNNNcgtgca tcgcttgcatgggga.

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

0.5 to 1.0 µg of each oligonucleotide is transferred to a 0.5 ml Eppendorf centrifuge tube and heated to a suitable temperature (eg, 94 ° C, depending on the calculated melting points), for 5 minutes, and hybridize slowly, cooling to room temperature. The template DNA (for example the yeast transporter vector pYES6 / CT) is then cut with two restriction enzymes (according to the only two restriction sites in the template DNA that span the region that includes the two codons to be replaced and that characterize the ends of the hybridized DNA), gel purified, ligated with the hybridized oligonucleotide, and transformed into yeast as described, for example, in Example 19, so that alternative D-amino acid oxidases, created by mutagenesis, were express in yeast. Then, as described, the yeast clones that produce the best yield are selected in D-phosphinothricin analogs (such as D-homocysteic acid) or in D-phosphinothricin (when the PAT gene is co-expressed) as the sole source of nitrogen, such as those that contain the sequences that encode a variant of D-amino acid oxidase with the desired properties. Alternatively, D-amino acid oxidase expression is performed 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 harvested, plated replicas, cultured, induced, lysed, and screened for the desired substrate activity against D-phosphinothricin, using methods known in the art (eg, screening fluorimetric for generation of peroxide or a colorimetric test for generation of ammonia). Yeast clones or other microorganisms thus selected are cultured, DNA prepared and the entire DNA sequence of D-amino acid oxidase cloned via PCR proofreading and cloning into pCRBluntII, using the Zero Blunt TOPO kit from Invitrogen. The D-amino acid oxidase coding sequences that characterize the selected clones are determined. These D-amino acid oxidase coding sequences are subcloned again for expression in a pET vector (eg Novagen pET 24a), and transformed into E. coli BL21 DE3. Cells are grown in a fermenter in LCM50 medium containing 100 µg / ml kanamycin, induced with IPTG, harvested, disrupted, and the extract partially purified and assayed for D-amino acid oxidase activity (as detailed further). ahead). 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, encoding D-amino acid oxidase enzymes particularly targeting. In particular, genes encoding Rhodotorula gracilis D-amino acid oxidase with an arginine, serine, cysteine, lysine, asparagine or alanine replacing methionine at position 213 and / or a histidine, serine, cysteine, asparagine or alanine replacing tyrosine in position 238. The methods used are the same as those described above, except that, rather than 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 Novagen's Pet 24A, and transformed into E. coli BL21 DE3. Cells are grown in a 1.0 L fermenter, in LCM50 medium supplemented with 100 µg / ml Kanamycin, induced for expression with 1 Mm IPTG and harvested by low speed centrifugation.

LCM5 medium contains (in 1 liter)

KH2PO4 (3 g), Na2HPO4 (6 g), NaCl (0.5 g), casein hydrolyzate (Oxoid) (2 g), (NH4) 2SO4 (10 g), yeast extract (Difco (10 g), glycerol (35 g) (these ingredients are obtained in solution and autoclaved.) The following additional ingredients are filter sterilized in solutions and added to the media: MgSO4 (2.5 ml of 246.5 mg solution / ml) .Thiamine.HCl (1 ml of 8 mg / ml solution); CaCl2.2H2O (0.2 ml of 147 g / l solution); * FeSO4.7H2O / reserve citric acid (2 ml); ** trace elements in solution (5 ml), and make up to 1 liter.

* FeSO4.7H2O / citric acid stock solution per 100 ml consists of FeSO4.7H2O (0.415 mg), citric acid (0.202 mg).

** The composition of the trace element solution per 1 ml is AlCl3.6 H2O (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).

About 7 g of wet cell weight are washed in water. The cells are resuspended in an equal volume of 50mM KOH / Mops buffer at pH 7.0, containing 2mM EDTA, 2mM DTT and 0.01mM FAD. The cells are uniformly suspended using a glass homogenizer and then disaggregated using a Basic Z cell disruptor stripping head from Constant Systems (BudBrooke Rd., Warwick, U.K.), at 93,079 KPa. The crude extract is kept cold (~ 4 ºC), centrifuged at 30,000 gav for 1 h and the pellet is discarded. Some of the extracted protein is run on a Coomassie blue stained SDS-PAGE gel and, by side-by-side comparison with similarly prepared cell extracts containing only "empty" pET vector, it is estimated that 2-50% of the Total soluble protein in the extract is D-amino acid oxidase. Some of the extracted 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 standard oxygen electrode cell (calibrated at 25 ° C between zero and a saturated oxygen concentration). Optionally, Daminoacid oxidase is further purified using ion exchange, phenyl-sepharose, ammonium sulfate fractional 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. The final reaction volume in the oxygen electrode cell is 2 ml. The proportions of oxygen consumption are measured (after elimination of any drift at the baseline). The mutant form M213R (substitution of arginine for methionine) of R. gracilis D-amino acid oxidase oxidizes DLphosphinothricin at a ratio of ~ 14 nmol / min / mg protein from crude extract (the estimated purity of Daminoacid being - oxidase in the extract of 35 +/- 15% of the total protein). The mutant form M213S (substitution of arginine for methionine) of R. gracilis D-amino acid oxidase oxidizes DL-phosphinothricin at a ratio of ~ 4 nmol / min / mg protein from crude extract (the estimated purity of the D-amino acid oxidase in the extract of 35 +/- 15% of the total protein). In control experiments, the pure L-form is not oxidized at all and, depending on the concentration, the pure D-form is oxidized up to twice the proportion that the DL-form is oxidized. Under similar conditions, the native R. gracilis Daminoacid oxidase (unmutated) does not show any significant capacity (<0.4 nmol / min / mg) to oxidize DL- or D-phosphinothricin.

Example 10. Mutation and selection to generate D-amino acid oxidase genes encoding enzymes with improved specificity (kcat / Km) for D-phosphinothricin oxidation.

The sequence encoding native Rhodotorula gracilis D-amino acid oxidase is cloned into the Invitrogen carrier vector pYES6 / CT, as a HindIII / PmeI fragment, downstream of the GAL1 promoter. similarly, the sequence encoding Rhodotorula native D-amino acid oxidase is cloned into the protein expression carrier vector pAUR123 (Panvera) as an XhaI fragment downstream of the constitutive promoter ADH1. The construction of these vectors is carried out in E. coli, followed by transformation in Saccharomyces cerevisiae S288C. When appropriate, the PAT gene is used to replace blasticidin or aureobasidine antibiotic resistance genes in vectors pYES6 / CT / pAUR123, respectively, and DL-phosphinothricin, better than the antibiotic, is used to maintain selection. Furthermore, sequences encoding the M213R or M213S genes, or M213S, Y238S, or mutant forms of Rhodotorula D-amino acid oxidase, are cloned in place of the sequence encoding the wild-type.

Additional mutant D-amino acid oxidase variants are created using various methods of mutagenesis. For example, multiple variants of the D-amino acid oxidase-encoding sequence are generated by Mn2 + poisoned PCR, the mixed population is cloned against the GAL1 or ADH1 promoters of the two carrier vectors, transformed into yeast, and the selection based on the ability of the new sequence to confer on the yeast the ability to grow on D-homocysteic acid as the sole source of nitrogen. Alternatively, the mutation and selection is done directly on the transformed yeast. For example, yeasts transformed with the above plasmids are cultured in a fermenter, in the presence of a chemical mutagen such as EMS, in a culture medium with limited nitrogen, containing 10-50 mM D-homocystic acid or (if express the PAT gene), 20-100 mM DL-phosphinothricin, and are induced for D-amino acid oxidase expression (eg, cultured on galactose as a carbon source). After successive subcultures, subcultures growing in D-homocystic acid are identified

or phosphinothricin as the sole source of N, are plated, and the D-amino acid oxidase-encoding sequences are subcloned, sequenced, and expressed in E. coli for further characterization.

In a further preferred method, mutagenesis is performed on the two carrier vectors using amplification and passage through the E. coli XL-1-red strain. This strain is deficient in three major DNA repair pathways, mut S, mut D, and mut T. This produces a ~ 5000-fold increase in the rate of mutation during DNA replication. The protocol used is from Stratagene. For example, 10 ng of carrier vector is transformed into the E. coli XL1-red strain, cells are grown, and then plated onto ampicillin-containing L-agar broth for 24

h. Colony batches of> 200 transformants are pooled from each plate, scraping off the colonies from the plate into L-broth, and then grown in 1/100 and 1/1000 dilutions, and successively subcultured in L-broth. / ampicillin at 37 ° C, for 1-2 weeks, so that a large number of cell divisions have occurred. A similar procedure is performed starting from various plates. DNA mini-preparations of the carrier vector are prepared from cells grown overnight and transformed back into yeast. The transformed yeasts are grown and colonies containing improved D-amino acid oxidases are selected, as described above.

Alternatively, expression and selection of D-amino acid oxidase is performed in some microorganisms other than yeast and, for example, under the control of expression of the t7 promoter of a pET vector in an E. coli lysogen. In this case, the sequence coding for D-amino acid oxidase (optionally mutagenized by PCR) is cloned into a pET vector, transformed into E. coli XL1red, and, after several generations, transformed into a lysogen again. E. coli, such as E. coli BL212 DE3. Individual colonies can then be harvested, plated replicas plated, cultured, ITPG-induced, lysed, and screened for desired substrate activity against Dfofinotricin, using methods known in the art (eg, fluorometric screening for peroxide generation).

or a colorimetric test for ammonia generation).

Alternatively, mutagenesis and selection for sequences encoding enhanced D-amino acid oxidase is performed directly on Rhodotorula gracilis. R. gracilis is cultivated in minimal medium with D-alanine or D-glutamate as the only nitrogen source, it is subjected to successive series of mutagenesis with EMS and selection by cultivation in media under conditions of increasing rigor, in which the only source of Nitrogen varies from D-glutamate to Dhomocysteic acid. In a variant of this example, Rhodotorula gracilis is transformed with one of the yeast vectors described above, so that it expresses PAT (either when cultured in galactose or constitutively), and the final stage of rigorous selection is performed on DL- phosphinothricin or D-phosphinothricin as the sole source of nitrogen.

Optionally, the media used for yeast selection contains a low solvent concentration (eg 0.1% DMSO).

SEQUENCE LISTING

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<120> Method for selectively producing male or female sterile plants.

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Claims (18)

1. Male-or female-sterile plant production method, comprising the step of transforming plant material with a polynucleotide that encodes 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 formation and / or maturation of the male or female gamete, so that the non-phytotoxic substance causes the production of a phytotoxic that selectively prevents the formation of gametes, or renders them non-functional, in which the enzyme is expressed in male or female reproductive structures, characterized in that: (i) the non-phytotoxic substance is a D-alpha-amino acid and (ii) the enzyme is a D-amino acid oxidase.

2.

 Method according to claim 1, wherein said non-phytotoxic substance is applied mixed with another additional substance, which is selected from the group consisting of protectors, gametocides, glutathione-S-transferase inducers, cytochrome P450 inducers, herbicides, fertilizers , nematocides, synergists, fungicides, hormones, plant growth regulators, and cytochrome P450 inhibitors.

3.

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

Four.

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

5. Method according to any one of claims 3 or 4, in which the non-phytotoxic substance is a D-alpha-amino acid selected from the group consisting of D-alanine, D-valine, D-methionine, D-serine, D-leucine and Disoleucine, and said enzyme is a D-amino acid oxidase that oxidizes said amino acid to a 2-keto-acid, with concomitant reduction of oxygen to a peroxide anion. 6. Method according to any one of claims 3 or 4, wherein the non-phytotoxic substance is D-aspartate or Dglutamate and said enzyme is a D-amino acid oxidase that oxidizes said amino acid to a 2-keto-acid, with reduction concomitant from oxygen to peroxide anion. 7. Method according to the preceding claim, in which the enzyme is a mutant D-amino acid oxidase, obtainable from Rhodotorula gracilis, which comprises substitutions at positions 213 and / or 238, when compared with the wild type sequence, or it is a D-aspartate oxidase. Method according to the preceding claim, in which the oxidase obtainable from Rhodotorula has at position 213 an amino acid selected from the group consisting of Arg, Lys, ser, Cys, Asn and Ala, and / or at position 238 an amino acid selected from group consisting of His, Ser, Cys, Asn and Ala.

9.

 Method according to any one of claims 3-8, in which the enzyme is directed to another point other than the peroxisome.

10.

 Method according to claim 1 or 2, in which the enzyme is a D-amino acid oxidase and the non-phytotoxic substance is the D-enantiomer of phosphinothricin or the D-enantiomer of bialaphos.

eleven.

 Method according to either of claims 1 or 2, in which the non-phytotoxic substance is comprised in a mixture, which contains a non-phytotoxic substance, and in which the enzyme is a D-amino acid oxidase, which is capable of oxidizing a amino acid to 2-keto-acid with concomitant reduction of oxygen to peroxide anion.

12. Method according to the preceding claim, wherein the enzyme is a mutant D-amino acid oxidase, obtainable from Rhodotorula gracilis, said oxidase comprising substitutions at positions 213 and / or 238, when compared to the wild type sequence, or it is a D-aspartate oxidase. 13. Method according to the preceding claim, in which the oxidase obtainable from Rhodotorula has at position 213 an amino acid selected from the group consisting of Arg, Lys, Ser, Cys, Asn and Ala and / or, at position 238, a selected amino acid from the group consisting of His, Ser, Cys, Asn and Ala. 14. Method according to any one of claims 11-13, in which the mixture comprises D- and L-phosphinothricin and the plant material expresses a PAT gene, substantially only in green tissues and in conditionally male female floral tissue of the plant- sterile and in masculine floral fabric of the conditionally feminine-sterile plant. 15. A chimeric DNA expression cassette, comprising at least: a. A promoter element that functions in plants or plant cells, b. A DNA molecule encoding a D-amino acid oxidase, c. a transcriptional terminator element, in which the promoter element directs the expression of the male or female reproductive structures. 16. A chimeric expression cassette according to claim 15, wherein the DNA molecule encoding D Amino acid oxidase is selected from the group of DNA molecules isolated from Rhodosporidium sp., Rhodotorula sp., Trigonopsis sp., Pig, Fusarium sp., Candida sp., Schizosaccharomyces sp. and Verticilium sp. 17. An expression vector comprising a chimeric expression cassette of any one of claims 15 to 16. 10 18. A transgenic plant cell or a transgenic plant comprising an expression vector as claimed in claim 17, or a chimeric expression cassette of any one of claims 15 to 16.