Dominant gene suppression transgenes and methods of using same

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DESCRIPTION

Dominant gene deletion transgenes and methods of using the same.

Background of the invention

Field of the invention

The invention relates generally to compositions and methods for the suppression of dominant genes.

Background information

Plant breeding provides a means of combining desirable traits in a single plant variety or hybrid, including, for example, disease resistance, insect resistance, drought tolerance, improved production, and improved agronomic quality. Field crops generally reproduce by pollination, including by self-pollination (selfing; selfing), in which pollen from a flower is transferred to the same flower or to another flower of the same plant or to a genetically identical plant, and by cross-pollination. (cross; cross) in which pollen from one plant is transferred to a flower from a genetically different plant.

Plants selfed and selected for the type over many generations lead to homozygosity at almost all gene loci and produce a uniform population of true reproductive offspring. A cross between two different homozygous lines produces a uniform population of hybrid plants that can be heterozygous at many loci in the gene. A cross of two plants, each heterozygous at various loci of the gene, generates hybrid plants, which differ genetically and are not uniform.

Many crop plants, including, for example, corn, can reproduce using self-pollination or cross-pollination techniques. Corn has different male and female flowers on the same plant, located on the ear and on the ear, respectively. Natural pollination occurs in corn when the wind carries pollen from the ears to the silks that protrude above the ears. Many crop plants, including corn, are grown as hybrids, which generally show greater vigor than the parent plants from which they are derived. Thus, when generating hybrid plants it is desirable to avoid random pollination.

Hybrid plants (F1) are generated by crossing two different inbred male (P1) and female (P2) parental plants. Hybrid plants are valued because they can show better performance and vigor compared to the parent plants from which the hybrid plants are derived. Furthermore, hybrid plants (F1) generally have more desirable properties than offspring plants (F2) derived from hybrid plants. Hybrid plants are therefore commercially important, and include many agricultural crops, including, for example, wheat, corn, rice, tomatoes, and melons. Hybridization of corn has received special attention since the 1930s. The production of hybrid corn involves the development of homozygous inbred male and female lines, the crossing of these lines, and the evaluation of the crosses for improved agronomic performance. Pedigree improvement and recurrent selection are two of the breeding methods used to develop inbred lines from populations. Breeding programs combine desirable traits from two or more inbred lines, or various broad-based sources, into breeding groups from which new inbred lines are developed by selfing and selection for desired phenotypes. These new inbred lines are crossed with other inbred lines and the resulting new hybrids are evaluated to determine that they have improved production or other desirable traits, thus increasing commercial value. The first generation of the hybrid offspring, named Fj, is more vigorous than their inbred parents. This hybrid vigor, or heterosis, can manifest itself in many ways, including increased vegetative growth and increased seed production.

Hybrid seed production requires maintenance of parental seed stocks because self-crossing of hybrid plants produces offspring (F2) that, like P1 and P2, generally show less desirable characteristics than the F1 hybrid plant. Because parental plants generally have less commercial value than hybrids (F1), efforts have been made to prevent parental plants in a self-crossing ("selfing") crop, since such crossing would reduce hybrid seed production. Accordingly, methods have been developed for the selfing of a parent plant.

One method of controlling pollination is to use a parent population of plants that is male-sterile, thereby providing the female parent. Various methods have been used to control male fertility, including, for example, manual or mechanical emasculation (detasseling ), cytoplasmic male sterility, genetic male sterility, and the use of gametocides. For example, parental self-fertilization in a field can be prevented by removing anthers or removing plants from the female parent population (P2), thereby removing the P2 pollen source from the crop. P2 female plants can then be pollinated manually with P1 pollen or using mechanical means. Hybrid corn seed is generally produced by a male sterility system that incorporates manual or mechanical detasseling. Alternate stripes of two inbred lines of maize are planted in a field and the pollen-bearing spikes are removed from one of the parental lines (P2 female). Provided that the field is sufficiently isolated from foreign maize pollen sources, the ears of the cleared parental line are fertilized only with pollen from the other parental line (P1 male); resulting in hybrid seeds and plants of hybrid forms. Unfortunately, this method is time consuming and laborious. In addition, environmental variation

ES 2 339 559 T3 in plant development can result in plants that produce spikes after manual detasseling of the female parent. Therefore detasseling cannot fully ensure the male sterility of a female inbred plant. In this case, the resulting fertile female plants will successfully release pollen and some female plants will self-pollinate. This will result in the seed from the inbred female being harvested along with the desired hybrid seed. The female inbred seed is not as productive as the F seed₁. Furthermore, the presence of the female inbred seed record represents a germplasm safety hazard for the company producing the hybrid. The inbred female can also be mechanically detached. Mechanical detasseling is about as reliable as manual detasseling but is faster and less expensive. However, most detasseling machines do more damage to plants than manual detasseling, which reduces seed yields F₁. Therefore, at present no form of detasseling is fully satisfactory and there is a continuing need to seek alternative hybrid production methods that reduce production costs, increase production safety and eliminate self-pollination of the female parent during production of the female. hybrid seed.

Another method to prevent selfing of the parent plant is to use parent plants that are male-sterile or gynosterile. Genes related to male fertility have been identified in several plants and include dominant and recessive genes related to male fertility. Plants that are homozygous for a recessive male fertility-related gene do not produce viable pollen and are useful as female parent plants. However, one result of female plants that are homozygous recessive for an androfertility-related gene is that they are not capable of self-fertilization and therefore means of obtaining pollen must be provided in order to maintain the parental plant line P2. Generally, a maintainer cell line, which is heterozygous for the male fertility related gene, is generated by crossing a homozygous male fertile dominant plant with the homozygous gynosterile recessive plant. The heterozygous maintainer plants are then crossed with the recessive male-sterile homozygous plants to produce a population in which 50% of the offspring are male-sterile. The male-sterile plants are then selected for use in the generation of hybrids. As such, the method requires additional selection and breeding steps to obtain the male-sterile plants, thereby adding to the time and cost necessary to produce the hybrid plants.

To overcome the need to select male-sterile plants from male-fertile plants generated by crossing a maintainer plant line with a female plant line (male-sterile), methods have been developed to obtain male-sterile plants by expressing a cytotoxic molecule in organ cells. male reproducers of a plant. For example, a nucleic acid encoding the cytotoxic molecule can bind to a mat-specific promoter and enter plant cells, in such a way that after expression, the toxic molecule destroys the anther cells, reverting to the male-sterile plant. However, as indicated above, such female parent plants cannot self-fertilize and therefore require the preparation and use of a maintainer plant line, which, when crossed with the male-sterile female parent, restores fertility, for example , by providing a dominant male fertile gene or by providing a means to inactivate or otherwise inhibit the activity of the cytotoxic gene product (see US Patent No. 5,977,433).

Additional methods of conferring genetic male sterility have been described, including, for example, the generation of plants with multiple mutant genes at different locations in the genome that confer male sterility (see US patents and with chromosomal translocations (see US patents). United States 3,861,709 and 3,710,511). Another method of conferring genetic male-sterility includes identifying a gene necessary for male-fertility, silencing the endogenous gene, generating a transgene comprising an inducible promoter operably linked to the coding sequence of the male-fertility-related gene, and inserting the transgene back into the plant. , thus generating a plant that is male sterile in the absence of the induction agent, and which can be restored to fertile by exposing the plant to the induction agent (see US Patent No. 5,432,068)

Although the methods described above for obtaining and maintaining hybrid plant lines have been useful for breeding and agricultural purposes, they require numerous additional steps and / or lines to maintain male-sterile or gynosterile plant populations to obtain the hybrid plants. These requirements contribute to increased costs for the cultivation of hybrid plants and, consequently, increased costs for consumers. Therefore, there is a need for convenient and efficient methods for producing hybrid plants, and particularly for generating parental lines that can be crossed to obtain hybrid plants.

A reliable system of genetic male sterility would provide several advantages over other systems. In some genotypes, the laborious detasseling process can be avoided by using cytoplasmic androsterile inbred parentals (CMS). In the absence of a fertility restorer gene, plants from an inbred CMS parent are male-sterile as a result of cytoplasmic (non-nuclear) genome factors. Therefore, this CMS characteristic is inherited exclusively through the female parent in maize plants, since only the female parent provides cytoplasm to the fertilized seed. CMS plants are fertilized with pollen from another inbred plant that is not male-sterile. second inbred plant may or may not contribute genes that produce male-fertile hybrid plants. In general, de-stalked normal maize seeds and CMS-produced seeds of the same hybrid should be mixed to ensure the availability of adequate pollen loads for fertilization when cultivating the hybrids and to ensure cytoplasmic diversity.

Another type of genetic sterility is described in the and nevertheless, this form of genetic male sterility requires the maintenance of multiple mutant genes in different places in the genome and requires a marker system

ES 2 339 559 T3 complex to trace genes, making this system inconvenient. Patterson described a genetic chromosome translocation system, which can be efficient, but it is also very complex. (See Puchos for other attempts to address the drawbacks of existing sterilization systems. For example, Fabijanski, et al., Developed various methods to cause male sterility in plants (see publication no. And application P published as WO 90/08828) One method includes introducing into the plant a gene encoding a cytotoxic substance that it is expressed using a male tissue-specific promoter, Another involves an antisense system in which a critical gene for fertility and an antisense construct are identified in the gene inserted into the plant. et al. they also show various cytotoxic antisense systems. See EP 89 / 401,194 Finally, other systems use "repressor" genes that inhibit the expression of other critical genes related to androfertility. See WO 90/08829.

An even greater improvement of this system is that described in US Patent No. 5,478,369 in which a method is achieved to impart controllable male sterility to the plant by silencing a native gene that is critical for male fertility. and further introducing a functional copy of the male fertility-related gene controlled by an inducible promoter that controls expression of the gene. Therefore, the plant is constitutively sterile, becoming only fertile when the promoter is induced, which allows the expression of the gene related to male fertility.

In various circumstances, a particular plant trait is expressed in a homozygous recessive state. Difficulties arise in maintaining the homozygous state when a restoring transgenic gene must be used for maintenance. For example, it has been shown, in maize, that the MS45 gene (document to express androfertility. Plants heterozygous or hemizygous for the dominant MS45 allele are fully fertile due to the sporophytic nature of the MS45 fertility trait. A natural mutation in the MS45 gene, called ms45, confers an androsterile phenotype on plants when this mutant allele is found in homozygous state. This sterility can be reversed (ie, restore fertility), when the non-mutant form of the gene is introduced into the plant, either through normal cross-breeding or transgenic complementation methods. However, restoration of fertility by crossing eliminates the desired homozygous recessive state, and both methods completely restore male fertility and prevent the maintenance of male sterile pure maternal lines. When controlling the plant's ginofertility, the same considerations arise in which a recessive homozygous female must be maintained by crossing with a plant that contains a restoration gene. Therefore, it is of considerable value, not only to control the expression of restoration genes in a recessive genetic line, but also to control the transmission of restoration genes to offspring during the hybrid production process.

Summary of the invention

The present invention is based on the determination that the genotype of an organism (for example, a plant or a mammal) can be modified to contain dominant suppressor transgenic alleles or constructs that reduce, but not eliminate, the activity of a gene, in which the phenotype of the organism is not substantially affected. For example, plants may contain dominant suppressor transgenic alleles and / or constructs that suppress the activity of an andro-fertile gene in the plant, without rendering the plant male sterile, or may contain dominant suppressor transgenic alleles and / or constructs that suppress the activity of a gene necessary for viability, without destroying the plant. In addition, pairs of such plants having selected genotypes comprising the dominant suppressor transgenic alleles or constructs can be crossed to produce offspring showing the phenotypic change (eg, male sterility). The offspring of plants comprising suppressed male fertile genes may be useful, for example, as females in the production of hybrid plants.

Accordingly, the invention provides a method of maintaining the homozygous recessive state of a first maize plant when crossed with a second maize plant, comprising (a) providing a first male-sterile maize plant with a recessive homozygous genotype for a trait. affecting viability or fertility.

(b) introducing by transformation into a second maize plant an exogenous restoration gene construct comprising (i) a first nucleotide sequence which, if introduced into the first plant, would restore the recessive homozygous trait, said first nucleotide sequence being in a hemizygous state;

(ii) a second nucleotide sequence, operably linked to the first nucleotide sequence, that inhibits the formation, function, or dispersal of male gametes from the maintainer plant, where the second nucleotide sequence is operably linked to a promoter that preferentially directs expression in male gametes;

and providing a plant comprising said restoration gene construct as a maintainer plant;

ES 2 339 559 T3 cultivating the maintainer plant in such a way that all viable pollen produced contains the recessive allele and does not contain said restoration gene construct; and (d) pollinate the first plant with the maintainer plant to produce offspring that maintain the homozygous recessive state of the first plant.

The invention further provides

- this method in which the first nucleotide sequence restores male fertility sporophytically.

- this method in which said first nucleotide sequence is MS45.

- this method in which said first nucleotide sequence is SBMu200, BS92-7, MS1 or MS2.

- this method in which said promoter is selected from MS45, BS92-7, and SBMu200.

- this method in which the second nucleotide sequence is operably linked to an inducible promoter.

The invention also provides:

- a maintainer plant as described above which, if self-fertilizing, produces offspring in an male-sterile: male-fertile ratio of 50:50

- a maintainer plant as described above which additionally comprises a third nucleotide sequence operably linked to the first nucleotide sequence and which encodes a marker product capable of being used for the selection of the offspring comprising the restoration gene construct.

- a maintainer plant as described above in which the third nucleotide sequence encodes a gene product that provides resistance to a chemical, a gene product that provides a visual marker in vegetative or reproductive tissue, or a gene product that affects the fabric color.

The invention further provides a method of propagating the maintainer plant as described above which comprises self-pollinating said maintainer plant and selecting the offspring based on the presence of the selectable marker product.

The present invention relates to the difficulty of propagating a plant having a homozygous recessive reproductive trait without losing the homozygous recessive state in the resulting offspring. This can be achieved by introducing into a plant at least one restorative, operably linked transgenic construct (1) a first nucleotide sequence comprising a functional copy of a gene that complements the mutant phenotypic trait produced by the recessive homozygous state with (2) a second functional nucleotide sequence that interferes with formation, function, or the dispersal of male gametes from the plant and is operably linked to a preferred male gamete tissue promoter. This construct is maintained in the hemizygous state and a plant containing such a construct is referred to herein as a maintainer. When the maintainer plant containing said attached construct is used as a pollen donor to fertilize the homozygous recessive plant, the only viable male gametes provided to the homozygous recessive plant are those that contain the recessive allele, and do not contain any component of the transgenic construct. . None of the pollen grains that contain the transgenic restoration construct are viable, due to the action of the second linked gene that prevents the formation of viable pollen. Therefore, the offspring resulting from said sexual interbreeding are not transgenic with respect to this transgenic construct.

Although the non-viable pollen produced by the maintainer plant contains the transgenic restoration construct, 50% of the ovules (the female gamete) from the maintainer plant will contain the transgenic restoration construct. Thus, the maintainer plant can be propagated by self-fertilization, segregating the transgenic restoration construct in such a way that it will be included in 50% of the ear seed of a self-fertilizing maintainer plant. By linking the transgenic restoration construct with a selectable marker, 50% of the seed containing the transgene can be isolated to propagate the maintainer population, which remains homozygous for the recessive gene and hemizygous for the transgenic restoration construct.

If female gamete formation is prevented or functional, it may be desirable to link the gene capable of complementing this mutant phenotype with an inducible promoter to aid in the maintenance of the maintainer plant. When such a plant is exposed to the induction state, it will have restored female fertility, and the plant can then self-fertilize to produce offspring with the desired recessive mutant trait and restoration transgenic construct.

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Although the invention is demonstrated in plants, one of skill in the art would recognize its applicability in other non-human organisms, including mammals.

Detailed description of the invention

The present invention is demonstrated with respect to fertility in plants and more particularly with respect to androfertility in plants.

The agricultural industry produces crops that are used for food in humans and animals, and are also used in other industries to prepare products as diverse as adhesives and explosives. Corn, for example, is used for human consumption, livestock fodder (eg, fodder for cattle, dairy cattle, pigs, and birdseed for poultry), and as a raw material in industry. Food uses of corn include the consumption of corn kernels, as well as products from the dry milling and wet milling industries (eg, semolina, flour, starch, cornstarch, corn syrup, and dextrose). Corn oil is recovered from corn germ, which is a by-product of the dry milling and wet milling industries. Industrial uses of corn include the production of ethanol, cornstarch in the wet milling industry, and corn flour in the dry milling industry. Industrial applications for starch and cornmeal are based on their functional properties, including, for example, viscosity, film formation, adhesive properties, and ability to suspend particles. Corn starch and flour have application in the paper and textile industries and are also used in the manufacture of adhesives, building materials, foundry binders, laundry starch, explosives, oil well sludge, other mining applications.

Many crop plants, including rice, wheat, corn, tomatoes, and melons are grown as hybrids, which exhibit greater vigor and improved qualities compared to the parent plants. The development of hybrids in a breeding program generally requires the development of homozygous inbred lines, the crossing of these lines and the evaluation of the crosses. Pedigree breeding and recurrent selection breeding methods are used to develop inbred lines from breeding populations. For example, maize plant breeding programs combine the genetic background of two or more inbred lines (or various other sources of germplasm) into crop groups, from which new inbred lines are developed by self-pollination (self-pollination). ) and selection of the desired phenotypes. The selected inbred lines are then crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which of these has commercial potential. Thus, the development of breeding and hybrids in plants are slow and expensive processes.

Pedigree improvement begins with the crossing of two genotypes, each of which may have one or more desirable characteristics that do not exist in the other or that are complemented by the other. If the original two parents do not provide all the desired characteristics, other sources can be included in the culture population. Using this method, higher plants are selected and selfed in successive generations until homogeneous plant lines are obtained. Recurrent selection breeding such as backcrossing can be used to improve an inbred line and a hybrid can be produced using the inbred lines. Backcrossing can be used to transfer a specific desirable trait from a source or inbred line to a second inbred line lacking that trait, for example, by first crossing a higher inbred line (recurrent parent) with a donor inbred line (non-recurrent parent) carrying the appropriate gene (or genes) for the trait in question, crossing the offspring of the first backcross with the superior recurrent parent and selecting in the resulting offspring for the desired trait transferred from the non-recurring parent. After five or more generations of backcrossing with selection for the desired trait, the offspring are homozygous for the loci that control the trait being transferred and are like the superior parent for essentially all other genes. The last generation of backcross self-fertilizes to give the pure culture offspring for the gene being transferred.

A single cross hybrid (F1) results from the crossing of two inbred lines (P1 and P2), each of which has a genotype that complements the genotype of the other. In developing commercial hybrids in a maize plant breeding program, for example, only F1 hybrid plants are sought, as they are more vigorous than their inbred parents. This hybrid vigor (heterosis) can manifest itself in many polygenic traits such as higher vegetative growth and higher production. Hybrid development in a maize plant breeding program, for example, involves selecting plants from various germplasm groups for initial breeding crosses; the self-fertilization of selected plants from multi-generational breeding crosses to produce a series of inbred lines, which, despite being different from each other, are truly cultivated and highly uniform; and the crossing of the selected inbred lines with different inbred lines to produce the offspring of F1 hybrids. During the inbreeding process in corn, the vigor of the lines decreases, but is restored when two different inbreeding lines are crossed to produce the hybrid plants. An important consequence of homozygosity and homogeneity of inbred lines is that the F1 hybrid between a defined pair of inbred parental plants is always the same. Thus, once inbred parents providing a superior hybrid are identified, the hybrid seed can reproduce indefinitely as long as inbred parents are maintained.

Hybrid seed production requires the removal or inactivation of pollen produced by the female parent. Incomplete pollen removal or inactivation provides the potential for self-fertilization, increasing the risk that self-pollinated seeds will inadvertently be harvested and packed with seeds.

ES 2 339 559 T3 hybrids. Once the seed is planted, the selfed plants can be identified and selected, the selfed plants are genetically equivalent to the female inbred line used to produce the hybrid. Typically, selfed plants are identified and selected based on their decreased vigor. For example, selfing female maize plants are identified by their less vigorous appearance for vegetative and / or reproductive characteristics, including shorter plant height, small ear size, ear and kernel shape, ear color, or other. features. Selfed lines can also be identified by molecular marker analysis (see, eg, Smith and Wych, Seed Sci. Technol. 14: 1-8, 1995). Using such methods, the homozygosity of the self-pollinated line can be verified by analyzing the allelic composition at different loci in the genome.

As hybrid plants are important and valuable field crops, plant variety breeders are continually working to develop high-yielding hybrids that are agronomically based on stable inbred lines. The availability of such hybrids allows a maximum amount of the crop to be produced with the inputs used, while minimizing susceptibility to pests and environmental stresses. To achieve this goal, plant variety breeders must develop superior inbred parental lines for hybrid production by identifying and selecting genetically unique individuals that occur in a segregating population. The present invention contributes to this goal, for example by providing plants that, when crossed, generate male-sterile offspring, which can be used as female parent plants for the generation of hybrid plants.

Using traditional methods and more recent high-throughput methods, a large number of genes have been identified as giving rise to preferred spikes in their expression pattern. Correlation of the function of these genes with important biochemical or developmental processes that ultimately lead to the development of fertile pollen is difficult when strategies are limited to direct or reverse classical mutational genetic analysis. As described herein, suppression strategies in corn provide a rapid alternative means of identifying genes that are directly related to pollen development in corn. The well-characterized maize andro-fertility gene, MS45, and several anther-preferred genes of unknown function were used to assess the efficiency of male-sterility generation using post-transcriptional gene silencing (PTGS, see, for example, Kooter et al. al., (1999) Trends Plant Sci. 4: 340-346) or transcriptional gene silencing (TGS; see, for example, Mette et al. (2000) EMBO J 19: 5194-5201).

To examine PTGS, RNAi constructs containing hairpins having stem structures composed of inverted repeats of the cDNA sequences expressed in the anther and a loop containing a non-homologous coding sequence or a maize cleavable intron were introduced into maize. .

To examine TGS as a strategy to inactivate gene function in the anther, a second series of constructs was generated in which promoters from anther-specific gene sequences formed the stem and a non-homologous sequence formed the loop. Constructs were expressed using constitutive promoters and anther preferred promoters.

Contrasting fertility phenotypes were observed, depending on the type of hairpin construction expressed. The plants expressing the PTGS constructs were male-fertile. In contrast, the plants expressing the TGS constructs were male-sterile and lacked MS45 mRNA and protein. Furthermore, the sterility phenotype of plants containing the hsRNA specific for the MS45 promoter (ie, TGS constructs) was reversed when MS45 was expressed from heterologous promoters in these plants. These results demonstrate that TGS provides a tool to rapidly correlate gene expression with the function of unknown genes such as anther-expressed monocot genes.

As used herein, the term "endogenous", when used in reference to a gene, means a gene that is normally present in the genome of cells of a specified organism and is present in its normal state in cells ( that is, it occurs in the genome in the state in which it normally occurs in nature). The term "exogenous" is used herein to refer to any material that is introduced into a cell. The term "exogenous nucleic acid molecule" or "transgene" refers to any nucleic acid molecule that is not normally present in a cell genome or is introduced into a cell. Said exogenous nucleic acid molecules are generally recombinant nucleic acid molecules, which are generated using recombinant DNA methods as described herein or otherwise known in the art. As described herein, a non-human transgenic organism may contain, for example, a first transgene and a second transgene. Said first and second transgene can be introduced into a cell, for example, a progenitor cell of a transgenic organism, as individual nucleic acid molecules or as a single unit (for example, contained in different vectors or contained in a single vector, respectively). . In either case, it can be confirmed that a cell from which the transgenic organism originated contains both transgenes using known and routine methods such as marker gene expression or nucleic acid hybridization or PCR analysis. Alternatively, or additionally, confirmation of the presence of the transgenes may occur later, for example, after regeneration of a plant from a putatively transformed cell.

An endogenous gene related to the fertility of a plant of a breeding pair described herein may be inactivated due, for example, to (1) a mutation of the endogenous gene such that the function of a product encoded by the gene is suppressed. (for example, the gene product is not expressed or is expressed at a level

ES 2 339 559 T3 which is insufficient to mediate its full effect on the plant or the plant cell); or (2) to the expression of an exogenous nucleic acid molecule that reduces or inhibits the expression of the gene product encoded by the endogenous gene. As such, the term "inactivated" is used broadly herein to refer to any manipulation of an endogenous gene, or a cell containing the gene, such that function mediated by a product encoded by the gene is suppressed. Additionally it should be recognized that regardless of whether the inactivated endogenous gene has reduced activity or is totally inactive, the desired relevant phenotype is maintained.

Mutation of an endogenous gene that results in suppression of the function of the gene can be effected, for example, by deleting or inserting one or more nucleotides in the nucleotide sequence of the gene (for example, in the promoter, in the coding sequence or in the intron), substituting one or some nucleotides in the gene for other different nucleotides or deactivating the gene (for example, by homologous recombination using an appropriate targeting vector). Plants with such mutations in both alleles can be obtained, for example, using crossing methods as described herein, or otherwise known in the art. Inactivation of an endogenous gene that results in suppression of gene function can also be accomplished by introducing into plant cells a transgene that suppresses the expression of the endogenous gene or a product expressed from the endogenous gene (e.g., a encoded polypeptide), or a transgene that encodes a product (e.g., an RNA) that suppresses the expression of the endogenous gene or a product encoded by the endogenous gene in the cells of the plant in which the gene is normally expressed.

By way of example, the inactivation of endogenous genes related to fertility can be carried out by expressing hairpin RNA molecules (hRNA) in the cells of the reproductive organs of a plant (for example: cells of the stamen where endogenous genes of the fertility are male fertile genes). The stamen, which comprises the male reproductive organ of plants, includes various types of cells, including, for example, the filament, anther, mat, and pollen. The hsRNAs useful for the purposes of the present invention are designed to include inverted repeats of a promoter of the endogenous gene to be inactivated; shRNAs that can suppress the expression of a gene have been described (see, for example, Matzke et al. (2001) Curr. Opin. Genet. Devel 11: 221227; Scheid et al (2002) Proc. National Acad. Sci., USA 99: 13659-13662; Waterhouse and Helliwell (2003) Nature Reviews Genetics 4: 29-38; Aufsaftz et al (2002) Proc. Nat'l Acad. Sci. 99 (4): 16499-16506; Sijen et al., Curr. Biol. (2001) 11: 436-440). As described herein, the use of stamen-specific or stamen-preferred promoters, including anther-specific promoters, pollen-specific promoters, mat-specific promoters, and the like, allows the expression of hsRNA in plants (particularly in cells. reproductive reproductive organs of the plant), where hsRNA suppresses the expression of an endogenous gene related to fertility, thereby inactivating the expression of the endogenous fertility gene. Thus, the use of a specific shRNA to suppress a promoter that drives the expression of a fertility gene provides a means of inactivating an endogenous fertility gene.

The terms "first", "second", "third" and "fourth" are used herein solely to clarify the relationships of various cells and molecules or to distinguish different types of a molecule, and unless expressly stated otherwise , it is not intended to indicate any particular order, importance or quantitative characteristic. For example, and unless specifically stated otherwise, reference to a "first" plant that contains a "first endogenous gene" is intended to indicate only that the specified gene is present in the specified plant. By means of a second example and unless specifically stated otherwise, reference to a "first plant containing a first transgene and a second transgene" is intended solely to indicate that said plant contains two exogenous nucleic acid molecules that are different from each other. .

As used herein, the term "nucleic acid molecule" or "polynucleotide" or "nucleotide sequence" broadly refers to a sequence of two or more deoxyribonucleotides or ribonucleotides that are linked by a phosphodiester bond. Thus, the terms include RNA and DNA, which can be a gene or a part thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence or the like and can be single- or double-stranded, as well as a DNA / RNA hybrid. Furthermore, the terms are used herein to include naturally occurring nucleic acid molecules, which can be isolated from a cell, as well as synthetic molecules, which can be prepared, for example, by chemical synthesis methods or by enzymatic methods such as by polymerase chain reaction (PCR). The term "recombinant" is used herein to refer to a nucleic acid molecule that is manipulated outside of a cell, including two or more linked heterologous nucleotide sequences. The term "heterologous" is used herein to refer to a nucleotide sequence that is not normally linked in nature or, if linked, is linked in a manner other than described. For example, reference to a transgene comprising a coding sequence operably linked to a heterologous promoter means that the promoter is one that does not normally drive expression of the nucleotide sequence in a specified cell in nature.

In general, the nucleotides comprising an exogenous nucleic acid molecule (transgene) are naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2'-deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose. However, a nucleotide sequence or nucleic acid molecule can also contain nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides. Such nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs (Lin et al., Nucl. Acids Res 22: 5220-5234, 1994; Jellinek et al., 34: 11363 Biochemistry 34: 11363-11372, 1995; Pagratis et al., Nature Biotechnol 15: 68-73, 1997). Likewise, the covalent bond that joins the nucleotides of a

ES 2 339 559 T3 nucleotide sequence is generally a phosphodiester bond, but it can also be, for example, a thiodiesel bond, a phosphorothioate bond, a peptide-like bond, or any other bond known to those skilled in the art to be useful. to join nucleotides to produce synthetic polynucleotides (see, eg, Tam et al., Nucl. Acids. Res. 22: 977-986,1994; Ecker and Crooke, BioTechnology 13: 351360,1995). Incorporation of non-naturally occurring nucleotide analogs or nucleotide or analog bonding can be especially useful when the nucleic acid molecule is to be exposed to an environment that may contain nucleolytic activity, including, for example, a plant tissue culture medium or in a plant cell, as modified molecules may be less susceptible to degradation.

A nucleotide sequence containing naturally occurring nucleotides and phosphodiester linkages can be chemically synthesized or can be produced using recombinant DNA methods, using, as a template, an appropriate polynucleotide. By comparison, a nucleotide sequence containing nucleotide analogs or covalent bonds other than phosphodiester bonds is generally chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and therefore it can be used to produce said polynucleotide recombinantly from a suitable template (Jellinek et al., above, 1995).

An exogenous nucleic acid molecule can comprise operably linked nucleotide sequences such as a promoter operably linked to a nucleotide sequence encoding an hpRNA, or a promoter linked to a nucleotide sequence encoding an androfertility gene product. The term "operably linked" is used herein to refer to two or more molecules that, when linked together, generate a molecule that shares characteristic features of each of the individual molecules. For example, when used in reference to a promoter (or other regulatory element) and a second nucleotide sequence encoding a gene product, the term "operably linked" means that the regulatory element is positioned relative to the second nucleotide sequence. , so that the transcription or translation of the isolated nucleotide sequence is under the influence of the regulatory element. When used in reference to a fusion protein comprising a first polypeptide and one or more additional polypeptides, the term "operably linked" means that each polypeptide component of the fusion protein (chimeric) exhibits some or all of the characteristic functions of the polypeptide. component (eg, a cell compartment localization domain and an enzyme activity). In another example, two operably linked nucleotide sequences, each of which encodes a polypeptide, may be such that the coding sequences are in reading phase and therefore, after transcription and translation, result in production of two polypeptides, which can be two different polypeptides or a fusion protein.

When an exogenous nucleic acid molecule includes a promoter operably linked to a nucleotide sequence encoding an RNA or polypeptide of interest, the exogenous nucleic acid molecule can be referred to as an exogenous expressible nucleic acid molecule (or transgene). The term "expressible" is used herein because, although such a nucleotide sequence can be expressed from the promoter, it does not really need to be expressed at a particular time. For example, when a promoter for an expressible transgene is an inducible promoter that lacks basal activity, an operably linked nucleotide sequence encoding an RNA or polypeptide of interest is expressed only after exposure to an appropriate induction agent.

Transcriptional promoters generally act in a position and orientation dependent manner and are usually positioned at or within about five nucleotides to about fifty nucleotides 5 '(upstream) of the transcriptional start site of a gene in nature. By comparison, enhancers can act relatively independent of position and orientation and several hundred or thousands of nucleotides can be placed upstream or downstream from a transcription initiation site, or in an intron within the coding region of a gene, while still being operably linked to the coding region in order to enhance transcription. The relative positions and orientations of various regulatory elements, in addition to those of a promoter, include the positioning of a transcribed regulatory sequence such as an internal ribosome entry site, or a translated regulatory element such as a cellular compartmentalization domain in a framework. suitable reading are well known, and methods for operably linking such elements are routine in the art (see, for example, Sambrook et al., "Molecular Cloning: A laboratory manual ”(Cold Spring Harbor Laboratory Press 1989); Ausubel et al., "Current Protocols in Molecular Biology" (John Wiley and Sons, Baltimore MD 1987 and supplements during 1995)).

Promoters useful for expressing a nucleic acid molecule of interest can be any of a range of naturally occurring promoters, known to be operative in plants or animals, as desired. Promoters that drive expression in cells of the male or female reproductive organs of a plant are useful for generating a transgenic plant or pair of breeding plants of the invention. Promoters useful in the present invention can include constitutive promoters, which are generally active in most or all tissues of a plant; inducible promoters, which are generally inactive or have a low basal expression level, and can be induced to relatively high activity after contact of the cells with an appropriate induction agent; tissue-specific (or tissue-preferred) promoters, which are generally expressed in a single or a few particular cell types (eg, plant anther cells); and specific developmental or phase promoters, which are only active for a defined period during the growth or development of a plant. Promoters can often be modified, if necessary, to vary the level of expression. Some embodiments comprise promoters exogenous to the species being manipulated. For example, the Ms45 gene introduced

ES 2 339 559 T3 in maize germplasm ms45ms45 can be driven by a promoter isolated from another plant species; A hairpin construct can then be designed to drive the exogenous plant promoter, reducing the possibility of the hairpin interacting with endogenous maize non-target promoters.

Exemplary constitutive promoters include the cauliflower mosaic virus (CaMV) 35S promoter (Odell et al. (1985) Nature 313: 810-812), the corn ubiquitin promoter (Christensen et al. (1989) Plant Mol Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675-689); the core promoter of the Rsyn7 promoter and other constitutive promoters described in WO 99/43838 and US Patent No. 6,072,050: rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); pEMU (Last et al: Theor (1991). Appl. Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J 3: 2723-2730); ALS promoter (US Patent No. 5,659,026); rice actin promoter (US Patent No. 5,641,876; WO 00/70067), maize histone promoter (Brignon et al., Plant Mol Biol 22 (6): 1007-1015 (1993 ), Rasco-Gante et al., Plant Cell Rep. 21 (6): 569-576 (2003)) and the like. Other constitutive promoters include, for example, those described in US Patent Nos. 5,608,144 and 6,177,611 and in PCT Publication Wo 03/102198.

Tissue-specific, tissue-preferred or phase-specific regulatory elements further include, for example, the AGL8 / FRUITFULL regulatory element, which is activated after flower induction (Hempel et al., Development 124: 3845-3853, 1997) ; Root-specific regulatory elements such as the regulatory elements of the RCP1 gene and the LRP1 gene (Tsugeki and Fedoroff, Proc. Natl. Acad., USA 96: 12941-12946, 1999; Smith and Fedoroff, Plant Cell 7: 735-745, 1995); flower-specific regulatory elements such as the regulatory elements of the LEAFY gene and the APETALA1 gene (Blazquez et al., Development 124: 3835-3844, 1997; Hempel et al., above, 1997); Seed-specific regulatory elements such as the oleosin gene regulatory element (Plant et al., Plant Mol. Biol. Biol. 25: 193-205, 1994) and specific regulatory element of the dehiscence zone. Other tissue-specific or phase-specific regulatory elements include the Zn13 promoter, which is a pollen-specific promoter (Hamilton et al., Plant. Mol. Biol. 18: 211-218, 1992); the promoter of UNUSUAL FLORAL ORGANS (UFO), which is active in the apical meristem of the shoot; the promoter active in the stem meristem (Atanassova et al., Plant J. 2: 291, 1992), the cdc2 promoter and the cyc07 promoter (see, for example, Ito et al., Plant. Mol. Biol. 24: 863-878, 1994; Martínez et al., Proc. Natl. Acad. Sci., USA 89: 7360, 1992); the preferred meri-5 and H3 meristem promoters (Medford et al., Plant Cell 3: 359, 1991; Terada et al., Plant J. 3: 241, 1993); preferred meristem and phloem promoters of related Myb genes in barley (Wissenbach et al., Plant J. 4: 411, 1993); cyc3aAt and cyc1At from Arabidopsis (Shaul et al. (1996) Proc. Natl. Acad. Sci. 93: 4868-4872); CYS and CYM from C roseus cyclins (Ito et al. (1997) Plant J. 11: 983-992) and CyclinBI from Nicotiana ((Trehin et al. (1997) Plant Mol. Biol. 35: 667-672); the promoter of the APEPALA3 gene, which is active in the floral meristem (Jack et al., Cell 76: 703, 1994; Hempel et al., previously 1997); a promoter a member of the agamous type family (AGL), eg, AGL8, which is active in the shoot meristem after the flowering transition (Hempel et al., previously 1997); promoters of the zone of floral abscission; L1 specific promoters; enhanced ripening tomato polygalacturonase promoter (Nicholass et al., Plant Mol. Biol. 28: 423-435 (1995)), the E8 promoter (Deikman et al., Plant Physiol. 100: 2013-2017 (1992)) and the fruit-specific 2A1 promoter, maize U2 and U5 sRNA promoters, the Z4 promoter of a gene encoding the 22 kD zein Z4 protein, the Z10 promoter of a gene that encodes a 10 kD zein protein, a Z27 promoter of a gene encoding a 27 kD zein protein, the A20 promoter of the gene encoding a 19 kD zein protein, and the like. Using well known methods (see, for example, US Patent No. 5,589,379) additional tissue specific promoters can be isolated. Stem specific promoters include preferred stem meristem promoters such as the promoters described in Weigel et al. (1992) Cell 69: 843-859 (Accession No. M91208); Accession No. AJ131822; Accession No. Z71981; Accession No. AF049870 and preferred stem promoters described in McAvoy et al. (2003) Acta Hort. (ISHS) 625: 379-385. Preferred inflorescence promoters include the chalcone synthase promoter (Van der Meer et al. (1992) Plant J. 2 (4): 525-535), anther-specific LAT52 (Twell et al. (1989) Mol. Gen. Genet. 217: 240-245), pollen-specific Bp4 (Albani et al (1990) Plant Mol Biol. 15: 605), corn pollen-specific Zm13 gene (Hamilton et al. (1992) Plant Mol. Biol. 18 : 211-218; Guerrero et al. (1993) Mol. Gen. Genet. 224: 161-168), microspore-specific promoters such as the apg gene promoter (Twell et al., Sex. Plant Reprod. 6: 217224 (1993)), and mat-specific promoters such as the TA29 gene promoter (Mariani et al., Nature 347: 737,1990; US Patent No. 6,372,967) and other stamen-specific promoters such such as the promoter of the MS45 gene, the promoter of the 5126 gene, the promoter of the BS7 gene, the promoter of the PG47 gene (US 5,412,085; US 5,545,546; Plant J 3 (2): 261-271 (1993)) , the promoter of the SGB6 gene (US 5,470,359), the promoter of the G9 gene (5,8937,850; 5,589,610), the SB200 gene promoter (WO 02/26789), or the like (see Example 1). Tissue-preferred promoters of interest additionally include a gene expressed in sunflower pollen SF3 (Baltz et al. (1992) The Plant Journal 2: 713-721), pollen-specific genes from B. napus (Amoldo et al. ( 1992) J. Cell. Biochem, Abstract No. Y101204). Additional tissue preferred promoters include those indicated by Yamamoto et al. (1997) Plant J. 12 (2): 255-265 (psaDb); Kawamata et al. (1997) Plant Cell Physiol. 38 (7) 792-803 (PsPAU); Hansen et al. (1997) Mol. Gen Genet. 254 (3): 337-343 (ORF13); Russell et al. (1997) Transgenic Res. 6 (2): 157-168 (waxy or ZrnGBS; 27kDa zein, ZmZ27; osAGP; osGT1); Rinehart et al. (1996) Plant Physiol. 112 (3): 1331-1341 (Fbl2A from cotton); Van Camp et al. (1996) Plant Physiol. 112 (2): 525-535 (Nicotiana SodA1 and SodA2); Canevascini et al. (1996) Plant Physiol. 112 (2): 513-524 (Nicotiana Itp1); Yamamoto et al. (1994) Plant Cell Physiol. 35 (5): 773-778 (Pinus cab-6 promoter); Lam (1994) Results Probl. Cell Differ. 20: 181196; Orozco et al. (1993) Plant Mol Biol. 23 (6): 1129-1138 (spinach rubisco activase (Rca)); Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90 (20): 9586-9590 (PPDK promoter); and Guevara-Garcia et al. (1993) Plant J. 4 (3): 495505 (Agrobacterium pmas promoter). A tissue specific promoter that is active in cells of the male and female reproductive organs may be useful in the present invention.

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"Seed preferred" promoters include "seed specific" promoters (those promoters active during seed development such as seed reserve protein promoters) as well as "seed germination" promoters (those promoters active during seed development). seed germination). See Thompson et al. (1989) BioEssays 10: 108. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced messenger), cZ19B1 (19 kDa corn zein), mi1ps (myo-inositol-1-phosphate synthase); see WO 00/11177 and US Patent No. 6,225,529. Gamma-zein is an endosperm specific promoter. Globulin-1 (Glob-1) is a representative embryonic specific promoter. For dicots, seed specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lecithin, cruciferin, and the like. For monocots, seed specific promoters include, but are not limited to, 15 kDa corn zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunk 2, globulin 1, etc. See also WO 00/12733 and US Patent 6,528,704, which describe seed-preferred promoters of the endl and end2 genes. Other embryo-specific promoters are described in Sato et al. (1996) Proc. Natl. Acad. Sci. 93: 8117-8122 (rice homeobox, OSH1); and PostmaHaarsma et al. (1999) Plant Mol. Biol. 39: 257-71 (rice KNOX genes). Other endosperm specific promoters are described in Albani et al. (1984) EMBO 3: 1405-15; Albani et al. (1999) Theor. Appl. Gen. 98: 1253-62; Albani et al. (1993) Plant J. 4: 343-55; Mena et al. (1998) The Plant Journal 116: 53-62 (barley DOF); Opsahl-Ferstad et al. (1997) Plant J12: 235-46 (corn Esr); and Wu et al. (1998) Plant Cell Physiology 39: 885-889 (rice GluA-3, GluB-1, NRP33, RAG-1).

An inducible regulatory element is one that is capable of directly or indirectly activating the transcription of one or more DNA sequences or genes in response to an inducer. The inducer can be a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenolic compound or a physiological stress, such as imposed directly by heat, cold, salinity or toxic elements or indirectly by the action of a pathogenic agent or disease such as a virus; or other biological or physical agent or environmental state. A plant cell containing an inducible regulatory element can be exposed to an inducer by external application of the inducer to the cell or plant such as by spraying, watering, heating, or the like. An induction agent useful for inducing the expression of an inducible promoter is selected based on the particular inducible regulatory element. In response to exposure to an induction agent, the transcription of the inducible regulatory element is generally initiated de novo or increased above the basal or constitutive expression level. Typically the protein factor that specifically binds to an inducible regulatory element to activate transcription is present in an inactive form which is then directly or indirectly converted by the inducer to the active form. Any inducible promoter can be used in the present invention (See Ward et al., Plant Mol. Biol. 22: 361-366, 1993).

Examples of inducible regulatory elements include a metallothionein regulatory element, an inducible copper regulatory element, or an inducible tetracycline regulatory element, the transcription of which can be effected in response to divalent metal ions, copper, or tetracycline, respectively (Furst et al., Cell 55: 705-717, 1988; Mett et al., Proc. Natl. Acad. Sci., USA 90: 4567-4571, 1993; Gatz et al., Plant J. 2: 397-404, 1992; Roder et al. ., Mol. Gen. Genet. 243: 32-38, 1994). Inducible regulatory elements also include an ecdysone regulatory element or a glucocorticoid regulatory element, the transcription of which can be effected in response to ecdysone or other steroids (Christopherson et al., Proc. Natl. Acad. Sci, USA 89: 6314-6318, 1992; Schena et al, Proc. Natl. Acad. Sci, USA 88: 10421-10425, 1991; US Patent No. 6,504,082); a cold-sensitive regulatory element or a thermal shock regulatory element, the transcription of which can be effected in response to exposure to cold or heat, respectively (Takahashi et al. Plant Physiol. 99: 383-390, 1992); the promoter of the alcohol dehydrogenase gene (Gerlach et al, pNaS USA 79: 2981-2985 (1982); Walker et al, PnAs 84 (19): 6624-6628 (1987)), inducible by anaerobic conditions and the inducible light promoter from from the pea rbcS gene or the pea psaDb gene (Yamamoto et al. (1997) Plant J. 12 (2): 255-265); an inducible light regulating element (Feinbaum et al., Mol. Gen. Genet. 226: 449, 1991; Lam and Chua, Science 248: 471, 1990; Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90 (20): 9586-9590; Orozco et al. (1993) PlantMol. Bio. 23 (6): 1129-1138), a plant hormone inducible regulatory element (Yamaguchi-Shinozaki et al. Plant Mol. Biol. 15: 905,1990; Kares et al, Plant Mol. Biol. 15: 225, 1990) , and the like. An inducible regulatory element can also be the promoter of the maize In2-1 or ln2-2 gene, which responds to herbicidal benzene sulfonamide protectants (Hershey et al, Mol. Gen. Gene. 227: 229-237, 1991; Gatz et al. al, Mol. Gen. Genet. 243: 32-38, 1994), and the Tn 10 transposon Tet repressor (Gatz et al, Mol. Gen. Genet. 227: 229-237, 1991). Stress inducible promoters include salt / water stress inducible promoters such as P5CS (Zang et al. (1997) Plant Sciences 129: 81-89); cold-inducible promoters, such as, cor15a (Hajela et al. (1990) Plant Physiol. 93: 1246-1252), cor15b (Wlihelm et al. (1993) Plant Mol Biol 23: 1073-1077), wsc120 (Ouellet et al. (1998) FEBS Lett. 423-324-328), ci7 (Kirch et al. (1997) Plant Mol Biol. 33: 897909), ci21A (Schneider et al. (1997) Plant Physiol. 113: 335-45); drought-inducible promoters, such as, Trg-31 (Chaudhary et al (1996) Plant Mol. Biol. 30: 1247-57), rd29 (Kasuga et al. (1999) Nature Biotechnology 18: 287-291); osmosis-inducible promoters, such as Rab17 (Vilardell et al. (1991) Plant Mol. Biol. 17: 985-93) and osmotin (Raghothama et al, (1993) Plant Mol Biol 23: 1117-28); and heat-inducible promoters, such as heat shock proteins (Barros et al. (1992) Plant Mol. 19: 665-75; Marrs et al. (1993) Dev. Genet. 14: 27-41), smHSP (Waters et al. (1996) J. Experimental Botany 47: 325-338), and the heat shock inducible element of the parsley ubiquitin promoter (WO 03/102198). Other stress inducible promoters include rip2 (US Patent No. 5,332,808 and US Publication No. 2003/0217393) and rd29a (Yamaguchi-Shinozaki et al. (1993) Mol. Gen. Genetics 236: 331-340 ). Certain promoters are inducible by injury, including the Agrobacterium pmas promoter (Guevara-Garcia et al. (1993) Plant J. 4 (3): 495-505) and the Agrobacterium ORF13 promoter (Hansen et al, (1997) Mol Gen. Genet. 254 (3): 337-343).

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Other regulatory elements active in plant cells and useful in the methods or compositions of the invention include, for example, the regulatory element of the nitrite reductase gene from spinach (Back et al. Plant Mol. Biol. 17: 9, 1991); a gamma zein promoter, an oleosin ole16 promoter, a globulin I promoter, an actin I promoter, an actin cl promoter, a sucrose synthetase promoter, an INOPS promoter, an EXM5 promoter, a globulin2 promoter , ab-32, an ADPG-pyrophosphorylase promoter, a Ltpl promoter, a Ltp2 promoter, an oleosin ole17 promoter, an oleosin ole18 promoter, an actin 2 promoter, a pollen-specific protein promoter, a pollen-specific pectate lyase gene promoter or a PG47 gene promoter, and an anther-specific RTS2 gene promoter, a SGB6 gene promoter or G9 gene promoter, a mat-specific RAB24 gene promoter, a alpha anthranilate synthase subunit, an alpha zein promoter, a beta anthranilate synthase subunit promoter, a dihydrodipicolinate synthase promoter, a Thi I promoter, an alcohol dehydrogenase promoter, a cab binding protein promoter, an H3C4 promoter, a RUBISCO SS starch branching enzyme promoter, an actin 3 promoter, an actin7 promoter, a regulatory protein GF14-12 promoter, a ribosomal protein L9 promoter , a cellulose biosynthetic enzyme promoter, an S-adenosyl-L-homocysteine ​​hydrolase promoter, a superoxide dismutase promoter, a Kkinase receptor promoter, a phosphoglycerate mutase promoter, a root-specific RCc3 mRNA promoter, a glucose-6 phosphate isomerase promoter, a pyrophosphate-fructose 6-phosphate-1-phosphotransferase promoter, a beta-ketoacyl-ACP synthase promoter, a 33 kDa photo system 11 promoter , an oxygen-developing protein promoter, a 69 kDa vacuolar ATPase subunit promoter, a glyceraldehyde-3-phosphate dehydrogenase promoter, a maturation inducible-type protein promoter and ABA, a phenylalanine ammonium lyase promoter, an adenosine triphosphatase S-adenosyl-L-homocysteine ​​hydrolase promoter, a chalcone synthase promoter, a zein promoter, a globulin-1 promoter, an auxin-binding protein promoter, a UDP glucose flavonoid glycosyl transferase gene promoter, an NTI promoter, an actin promoter, and an opaque 2 promoter.

An exogenous nucleic acid molecule can be introduced into a cell as a naked DNA molecule, it can be incorporated into a matrix such as a liposome, or a particle such as a viral particle, or it can be incorporated into a vector. Incorporation of the polynucleotide into a vector can facilitate manipulation of the polynucleotide or introduction of the polynucleotide into a plant cell. Accordingly, the vector can be derived from a plasmid or it can be a viral vector such as a T-DNA vector (Horsch et al. Science 227: 1229-1231 (1985)). If desired, the vector can include components of a plant transposable element, for example, a Ds transposon (Bancroft and Dean, Genetics 134: 1221-1229, 1993) or an Spm transposon (Aarts et al, Mol. Gen. Genet. 247: 555-564, 1995). In addition to containing the transgene of interest, the vector may also contain various nucleotide sequences that facilitate, for example, rescue of the vector from a transformed plant cell; passage of the vector into a host cell, which may be a plant, animal, bacterial, or insect host cell; or the expression of a coding nucleotide sequence in the vector, including all or part of a rescued coding region. Thus, a vector may contain any of a variety of other transcriptional and translational elements, including constitutive and inducible promoters, enhancers, and the like (see, for example, Bitter et al, Meth. Enzymol. 153: 516-544, 1987). For example, a vector can contain elements useful for passage, growth, or expression in a bacterial system, including a bacterial origin of replication; a promoter, which can be an inducible promoter; and the like. A vector may also contain one or more restriction endonuclease cleavage and recognition sites, including, for example, a poly linker sequence, to facilitate insertion or removal of a transgene.

In addition, or alternatively, a nucleotide sequence relevant to a fertility-related gene (for example, an hpRNA comprising an inverted repeat of a fertility-related gene promoter, or a coding sequence for a fertility-related gene, alone or operably linked to a heterologous promoter), an exogenous nucleic acid molecule or a vector containing said transgene, it may contain one or more other expressible nucleotide sequences encoding an RNA or polypeptide of interest. For example, the additional nucleotide sequence can encode an antisense nucleic acid molecule; an enzyme such as β-galactosidase, β-glucuronidase, luciferase, alkaline phosphatase, glutathione α-transferase, chloramphenicol acetyltransferase, guanine xanthine phosphoribosyltransferase and neomycin phosphotransferase; a viral polypeptide or a peptide part thereof or a plant growth factor or hormone.

In certain embodiments, the expression vector contains a gene encoding a selection marker that is functionally linked to a promoter that controls the initiation of transcription. For a general description of expression vectors and reporter genes in plants, see Gruber et al., "Vectors for Plant Transformation" in Methods of Plant Molecular Biology and Biotechnology 89-119 (CRC Press, 1993). In the use of the term, this means including all types of selection markers, assessable or selective. Expression of such a nucleotide sequence can provide a means of selecting a cell that contains the construct, for example, by conferring a desirable phenotype on a plant cell that contains the nucleotide sequence. For example, the additional nucleotide sequence may be, or encode, a selectable marker which, when present or expressed in a plant cell, provides a means of identifying the plant cell that contains the marker.

A selectable marker provides a means to screen a population of organisms or cells of an organism (eg, plants or plant cells) to identify those that have the marker and therefore the transgene of interest. A selectable marker generally confers a selective advantage on the cell or an organism (eg, a plant) containing the cell, eg, the ability to grow in the presence of a negative selective agent such as an antibiotic or, in the case of a plant, a herbicide. A selective advantage

ES 2 339 559 T3 may also be due, for example, to an enhanced or novel ability to use an added compound as a nutrient, growth factor or energy source. A selective advantage can be conferred by a single polynucleotide, or its expression product, or by a combination of polynucleotides whose expression in a plant cell provides the cell with a selective positive advantage, a selective negative advantage, or both. It should be recognized that expression of the transgene of interest (eg, encoding an hpRNA) also provides a means of selecting for cells that contain the encoding nucleotide sequence. However, the use of an additional selectable marker, which, for example, allows a plant cell to survive otherwise toxic conditions, provides a means to enrich transformed plant cells containing the desired transgene. Examples of selectable or titratable genes known in the art can be found, for example, in Jefferson et al. (1991) in PlantMolecular Biology Manual, ed. Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet et al. Mol. Cell. Biol. 7: 725-737, 1987; Goff et al., EMBO J. 9: 2517-2522, 1990; Kain et al., BioTechniques 19: 650-655, 1995; and Chiu et al., Curr. Biol. 6: 325-330, 1996.

Examples of selectable markers include those that confer resistance to antimetabolites such as herbicides or antibiotics, eg, dihydrofolate reductase, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13: 143-149, 1994; see also Herrera Estrella et al., Nature 303: 209-213, 1983; Meijer et al., Plant Mol. Biol. 16: 807-820, 1991); neomycin phosphotransferase, which confers resistance to neomycin aminoglycosides, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2: 987-995, 1983) and hygro, which confers hygromycin resistance (Marsh, Gene 32: 481-485, 1984; see also Waldron et al., Plant Mol. Biol. 5: 103-108, 1985; Zhijian et al., Plant Science 108: 219-227, 1995); trpB, which allows cells to use indole instead of tryptophan; hisD, which allows cells to use histinol instead of histidine (Hartman, Proc. Natl. Acad. Sci., USA 85: 8047, 1988); mannose-6-phosphate isomerase allowing cells to use mannose (WO 94/20627); ornithine decarboxylase, which confers resistance to the ornithine decarboxylase inhibitor, 2- (difluoromethyl) -DL-ornithine (DFMO; McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.); and Aspergillus terreus deaminase, which confers Blasticidin S resistance (Tamura, Biosci. Biotechnol. Biochem. 59: 2336-2338, 1995). Additional selectable markers include, for example, a mutant EPSPV synthase, which confers resistance to glyphosate (Hinchee et al., BioTechnology 91: 915-922, 1998), a mutant acetolactate synthase, which confers resistance to imidazolinone or sulfonylurea (Lee et al. al., EMBO J. 7: 1241-1248, 1988), a mutant psbA, which confers atrazine resistance (Smeda et al., Plant Physiol. 103: 911-917, 1993), or a mutant protoporphyrinogenic oxidase (see US Patent No. 5,767,373), or other markers that confer resistance to a herbicide such as glufosinate. Examples of suitable selectable marker genes include, but are not limited to, genes encoding chloramphenicol resistance (Herrera Estrella et al., EMBO J. 2: 987-992, 1983); streptomycin (Jones et al., Mol. Gen. Genet. 210: 86-91, 1987); spectinomycin (Bretagne-Sagnard et al., Transgenic Res. 5: 131-137, 1996); bleomycin (Hille et al., Plant Mol. Biol. 7: 171-176, 1990); sulfonamide (Guerineau et al., Plant Mol. Biol. 15: 127-136, 1990); bromoxynil (Stalker et al., Science 242: 419-423, 1988); glyphosate (Shaw et al., Science 233: 478-4.81, 1986); phosphinothricin (DeBlock et al., EMBO J. 6: 2513-2518, 1987), and the like. One option for using a selective gene is a DNA encoding glufosinate resistance and in one embodiment may be the phosphinothricin acetyltransferase ("PAT") gene, the maize-optimized pAt gene, or the bar gene controlled by CaMV promoters. 35S or ubiquitin. Genes confer resistance to bialaphos. See Gordon-Kammetal., Plant Cell 2: 603; 1990; Uchimiya et al., BioTechnology 11: 835, 1993; White et al., Nucl. Acids Res. 18: 1062, 1990; Spencer et al., Theor. Appl. Genet. 79: 625-631, 1990; and Anzai et al., Mol. Gen. Gen. 219: 492, 1989). An aversion to the PAT gene is the corn optimized PAT gene, described in US Patent No. 6,096,947.

Additionally, markers that facilitate the identification of a plant cell containing the polynucleotide encoding the marker include, for example, luciferase (Giacomin, Plant Sci. 116: 59-72, 1996; Scikantha, J. Bacteriol. 178: 121, 1996), green fluorescent protein (Gerdes, FEBS Lett. 389: 44-47, 1996; Chalfie et al., Science 263: 802, 1994), and other fluorescent protein oβ-glucuronidase variants (Jefferson, Plant Mol. Biol. Rep. 5: 387, 1987; Jefferson et al., EMBO J. 6: 3901-3907, 1987; Jefferson, Nature 342 (6251): 837-838, 1989); genes in maize that regulate pigment production (Ludwig et al., Science 247: 449, 1990; Grotewold et al., PNAS 88: 4587-4591, 1991; Cocciolone et al., Plant J 27 (5): 467 -478, 2001; Grotewold et al., Plant Cell 10: 721-740, 1998); β-galactosidase (Teeri et al., EMBO J. 8: 343-350, 1989); luciferase (Ow et al., Science 234: 856-859, 1986); chloramphenicol acetyltransferase (CAT) (Lindsey and Jones, Plant Mol. Biol. 10: 43-52, 1987); and many others described in this document or others known in the art. Such markers can also be used as reporter molecules. Those skilled in the art can make many variations on promoters, selectable markers, and other components of the construct.

The term "plant" is used broadly herein to include any plant or a part of a plant, at any stage of development, including a plant cutting, a plant cell, a plant cell culture, a plant organ, a plant seed and a seedling. A plant cell is the structural and physiological unit of the plant, comprising a protoplast and a cell wall. A plant cell may be in the form of a single isolated cell or aggregate of cells such as a friable callus or a cultured cell or it may be part of a higher organized unit, for example, a plant tissue, a plant organ or a plant. Thus, a plant cell can be a protoplast, a gamete-producing cell, or a cell or collection of cells that can regenerate into a whole plant. Thus, a seed, which comprises multiple plant cells and is capable of regeneration into a whole plant, is considered a plant cell for the purposes of this disclosure. A plant tissue or organ can be a seed, a protoplast, a callus, or any other group of plant cells that is organized into a structural or functional unit. Particularly useful parts of a plant include harvestable parts and parts useful for the propagation of the offspring of the plants.

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A harvestable part of a plant can be any useful part of a plant, for example flowers, pollen, seedlings, tubers, leaves, stems, fruit, seeds, roots and the like. A part of a plant useful for propagation includes, for example, seeds, fruits, cuttings, seedlings, tubers, rhizomes, and the like.

A transgenic plant can regenerate from a genetically modified plant cell, that is, a whole plant can regenerate from a plant cell; a group of plant cells; a protoplast; a seed; or a part of a plant such as a leaf, a cotyledon, or a cutting. Protoplast regeneration varies between plant species. For example, in the maturation and germination phase, a protoplast suspension can be prepared and, in certain species, embryo formation can be induced from the protoplast suspension. Culture media generally contain various components necessary for development and regeneration, including, for example, hormones such as auxins and cytokinins; and amino acids such as glutamic acid and proline, depending on the particular plant species. Effective regeneration will depend, in part, on the medium, genotype, and culture history and is reproducible if these variables are controlled for.

Regeneration can occur from callus, explants, organs, or plant parts. The transformation can be carried out in the context of regeneration of part of the plant or organ. (see Meth. Enzymol. Vol. 118; Klee et al. Ann. Rev. Plant Physiol. 38: 467 (1987)). Using the regeneration-transformation method from leaf discs, for example, the discs are grown on selective media, followed by shoot formation in about two to four weeks (see Horsch et al., Previously 1985). The growing shoots are excised from the callus and transplanted into an appropriate selective root induction medium. Rooted seedlings are transplanted into the soil as soon as possible after the appearance of the roots. The seedlings can be replanted if required, until they reach maturity.

In seed-propagated crops, mature transgenic plants can self-pollinate to produce a homozygous inbred plant. The resulting inbred plant produces seeds that contain the introduced transgene and can be grown to produce plants that express the polypeptide. Methods for the improvement and selection of cross-breeding plants that have desirable characteristics or other characteristics of interest include those described herein and others well known to plant variety breeders.

In various aspects of the present invention, one or more transgenes are introduced into cells. When used in reference to a transgene, the term "introduction" means transferring the exogenous nucleic acid molecule to a cell. A nucleic acid molecule can be introduced into a plant cell by a variety of methods. For example, the transgene can be contained in a vector, it can be introduced into a plant cell using a direct gene transfer method such as electroporation or micro-projectile-mediated transformation or using Agrobacterium-mediated transformation. As used herein, the term "transformed" refers to a plant cell that contains an exogenously introduced nucleic acid molecule.

One or more exogenous nucleic acid molecules can be introduced into plant cells using any of the known and routine methods for transformation into plants, including biological and physical protocols for transformation into plants (see, for example, Miki et al., " Procedures for Introducing Foreign DNA into Plants "; In Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, Eds. (CRC Press, Inc., Boca Raton, 1993) pages 67-88). Furthermore, expression vectors and in vitro culture methods for transformation of plant tissues or cells and plant regeneration are well known and routine (see, eg, Gruber et al., "Vectors for Plant Transformation"; Id. pages 89-119).

Suitable methods of plant cell transformation include microinjection, Crossway et al. (1986) Biotechniques 4: 320-334; electroporation, Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83: 5602-5606; Agrobacterium-mediated transformation, see for example, Townsend et al. in US Patent 5,563,055; direct gene transfer, Paszkowski et al. (1984) EMBO J. 3: 2717-2722; and ballistic acceleration of particles, see for example, Sanford et al. US Patent 4,945,050; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6: 923-926. See also Weissinger et al. (1988) Annual Rev. Genet. 22: 421-477; Sanford et al. (1987) Particulate Science and Technology 5: 27-37 (onion); Christou et al. (1988) Plant Physiol. 87: 671-674 (soybean); McCabe et al. (1988) Bio / Technology 6: 923-926 (I am bean); Datta et al. (1990) Biotechnology 8: 736740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85: 4305-4309 (corn); Klein et al. (1988) Biotechnology 6: 559-563 (maize); Klein et al. (1988) Plant Physiol. 91: 440-444 (corn); Fromm et al. (1990) Biotechnology 8: 833-839; Hooydaas-Van Slogteren et al. (1984) Nature (London) 311: 763-764; Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. GP Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9: 415-418; and Kaeppler et al. (1992) Theor. Appl. Genet. 84: 560-566 (whisker-mediated transformation); D.Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12: 250-255 and Christou et al. (1995) Annals of Botany 75: 407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (maize via Agrobacterium tumefaciens); all incorporated herein by reference.

Agrobacterium-mediated transformation provides a useful method for introducing a transgene into plants (Horsch et al., Science 227: 1229 1985). A. tumefaciens and A. rhizogenes are pathogenic bacteria present in the soil that genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for the genetic transformation of the plant (see, for example,

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Kado, Crit. Rev. Plant Sci.10: 1, 1991; see, also, Moloney et al., Plant Cell Reports 8: 238, 1989; US Patent No. 5,591,616; WO 99/47552; Weissbach and Weissbach, "Methods for Plant Molecular Biology" (Academic Press, NY 1988), section VIII, pages 421-463; Grierson and Corey, "Plant Molecular Biology" 2^to Ed. (Blackie, London 1988), Chapters 7-9; see, also, Horsch et al., previously 1985).

With respect to A. tumefaciens, the wild-type form contains a Ti plasmid that directs the production of the tumorigenic crown gall that develops in host plants. Transfer of the tumor-inducing region of T-DNA from the Ti plasmid to a plant genome requires the virulence genes encoded by the Ti plasmid as well as T-DNA borders, which are a series of direct DNA repeats that delineate the region to be transferred. An Agrobacterium-based vector is a modified form of a Ti plasmid, in which tumor induction functions are replaced by a nucleotide sequence of interest that is introduced into the host plant. Methods of using Agrobacterium-mediated transformation include cocultivation of Agrobacterium with cultured isolated protoplasts; the transformation of plant cells or tissues with Agrobacteriu; and the transformation of seeds, tips or meristems with Agrobacterium. Furthermore transformation in the plant by Agrobacterium can be carried out using vacuum infiltration of a suspension of Agrobacterium cells (Bechtold et al., CR Acad. Sci. Paris 316: 1194, 1993).

Agrobacterium-mediated transformation can employ cointegrated vector or binary vector systems, in which the components of the Ti plasmid are divided between a helper vector, which permanently resides in the Agrobacterium host and carries the virulence genes, and a shuttle vector, containing the gene of interest linked to T-DNA sequences. Binary vectors are well known in the art (see, for example, De Framond, BioTechnology 1: 262, 1983; Hoekema et al., Nature 303: 179, 1983) and are commercially available (Clontech; Palo Alto CA) . For transformation, Agrobacterium can be co-cultured, for example, with plant cells or injured tissue such as leaf tissue, root explants, hypocotyls, cotyledons, parts of the stem or tubers (see, for example, Glick and Thompson, "Methods in Plant Molecular Biology and Biotechnology ”(Boca Ratón FL, CRC Press 1993)). Injured cells within plant tissue that have been infected by Agrobacterium can develop organs de novo when grown under appropriate conditions, the resulting transgenic shoots eventually giving rise to transgenic plants containing the introduced polynucleotide.

Agrobacterium-mediated transformation has been used to produce a variety of transgenic plants, including, for example, transgenic cruciferous plants such as Arabidopsis, mustard, rapeseed, and flax; transgenic legume plants such as alfafa, peas, soybeans, clover and white clover; transgenic nightshade plants such as eggplant, petunia, potato, tobacco and tomato (see, for example, Wang et al., "Transformation of Plants and Soil Microorganisms" (Cambridge, University Press 1995)). In addition, Agrobacterium-mediated transformation can be used to introduce an exogenous nucleic acid molecule into apple, poplar, belladonna, black currant, carrot, celery, cotton, cucumber, grape, horseradish, lettuce, bluebell, striped melon, neem, poplar , strawberry, sugar beet, sunflower, walnut, asparagus, rice, wheat, sorghum, barley, corn and other plants (see, for example, Glick and Thompson, previously 1993; Hiei et al., Plant J. 6: 271-282, 1994; Shimamoto, Science 270: 1772-1773, 1995).

Suitable A. tumefaciens strains and vectors as well as Agrobacteria transformation and suitable growth and selection media are well known in the art (GV3101, pMK90RK), Koncz, Mol. Gen. Genet. 204: 383-396, 1986; (C58C1, pGV3850kan), Deblaere, Nucl. Acid Res. 13: 4777, 1985; Bevan, Nucleic Acid Res. 12: 8711, 1984; Koncz, Proc. Natl. Acad. Sci. USA 86: 8467-8471, 1986; Koncz, Plant Mol. Biol. 20: 963-976, 1992; Koncz, Specialized vectors for gene tagging and expression studies. In: Plant Molecular Biology Manual Vol. 2, Gelvin and Schilperoort (Eds.), Dordrecht, The Netherlands: Kluwer Academic Publ. (1994), 1-22; European Patent A-1 20 516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters BV, Alblasserdam (1985), Chapter V; Fraley, Crit. Rev. Plant. Sci., 4: 1-46; An, EMBO J. 4: 277-287, 1985).

As noted herein, the present invention provides vectors capable of expressing genes of interest controlled by regulatory elements. In general, vectors must be functions in plant cells. Sometimes it may be preferred to have vectors that are functional in E. coli (eg, protein production for antibody generation, DNA sequence analysis, insert construction, obtaining nucleic acid quantities). Vectors and procedures for cloning and expression in E. coli are described in Sambrook et al., (Above).

The transformation vector, comprising the promoter of the present invention operably linked to an isolated nucleic acid sequence in an expression cassette, may also contain at least one additional nucleotide sequence for a gene that is co-transformed in the organism. Alternatively, the additional sequence (or sequences) can be provided in another transformation vector.

When the exogenous nucleic acid molecule is contained in a vector, the vector may contain functional elements, for example "left border" and "right border" sequences of Agrobacterium T-DNA, which allow stable integration into a genome of plant. Furthermore, methods and vectors are known that allow the generation of marker-free transgenic plants, for example, where a selectable marker gene is lost at a certain stage of plant development or plant reproduction and include, for example, methods co-transformation (Lyznik, Plant Mol. Biol. 13: 151-161, 1989; Peng, Plant Mol. Biol. 27: 91-104,1995), or methods using enzymes capable of promoting homologous recombination in plants (see, for example, document

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WO97 / 08331; Bayley, Plant Mol. Biol. 18: 353-361, 1992; Lloyd, Mol. Gen. Genet. 242: 653-657, 1994; Maeser, Mol. Gen. Genet. 230: 170-176, 1991; Onouchi, Nucl. Acids Res. 19: 6373-6378, 1991; see, also Sambrook et al., previously 1989).

Direct gene transfer methods can also be used to introduce the desired transgene (or transgenes) into cells, including plant cells that are refractory to Agrobacterium-mediated transformation (see, eg, Hietal., Plant J. 6: 271 -282, 1994; US Patent No. 5,591,616). Such methods include direct gene transfer (see European Patent A 164 575), injection, electroporation, biolistics methods such as particle bombardment, pollen-mediated transformation, virus-mediated transformation in plant RNA, transformation mediated by liposomes, transformation using enzyme-damaged or degraded immature embryos or enzyme-damaged or degraded embryogenic calli, and the like. Direct gene transfer methods include micro-projectile-mediated transformation (biolistics) methods, where micro-projectiles measuring 1 to 4 mm carry the transgene on the surface. A vector, particularly an expression vector containing the transgene (or transgenes) of interest, is introduced into the plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m / s, sufficient to penetrate the plant cell walls and membranes (see, eg, Sanford et al., Part. Sci. Technol. 5: 27,1987; Sanford, Trends Biotech. 6: 299, 1988, Klein et al., BioTechnology 6: 559-563, 1988; Klein et al., BioTechnology 10: 268, 1992). In maize, for example, numerous target tissues can be bombarded with DNA-coated microprojectiles to produce transgenic plants, including, for example, callus (Type I or Type II), immature embryos, and meristematic tissue.

Other methods for the physical delivery of a transgene in plants use sonication of the target cells (Zhang et al., BioTechnology 9: 996, 1991); liposomes or spheroplast fusion (Deshayes et al., EMBO J. 4: 2731, 1985; Christou et al., Proc Natl. Acad. Sci., USA 84: 3962, 1987); precipitation or incubation of CaCl₂ with polyvinyl alcohol or poly-L-omitin (Hain et al., Mol. Gen. Genet. 199: 61, 1985; Draper et al., Plant Cell Physiol. 23: 451, 1982); and electroporation of protoplasts and whole cells and tissues (Donn et al., In "Abstracts of Vlllth International Congress on Plant Cell and Tissue Culture" IAPTC, A2-38, p. 53, 1990; D'Halluin et al., Plant Cell 4: 1495-1505, 1992; Spencer et al., Plant Mol. Biol. 24: 51-61, 1994).

A direct gene transfer method such as electroporation can be particularly useful for introducing exogenous nucleic acid molecules into a cell such as a plant cell. For example, plant protoplasts can be electroporated in the presence of a recombinant nucleic acid molecule, which can be in a vector (Fromm et al., Proc. Natl. Acad. Sci., USA 82: 5824, 1985). High field strength electrical impulses reversibly permeabilize the membranes allowing the introduction of nucleic acid. Electroporated plant protoplasts reform the cell wall, divide and form a plant callus. Microinjection can be performed as described in Potrykus and Spangenberg (eds.), Gene Transfer To Plants. Springer Verlag, Berlin, NY (1995). An introduced transformed plant cell containing the recombinant nucleic acid molecule that can be identified due to the presence of a selectable marker included in the construct.

As mentioned above, micro-projectile-mediated transformation also provides a useful method for introducing exogenous nucleic acid molecules into a plant cell (Klein et al., Nature 327: 70-73, 1987). This method uses micro projectiles such as gold or tungsten, which are coated with the desired nucleic acid molecule by precipitation with calcium chloride, spermidine or polyethylene glycol. Micro-projectile particles are accelerated at high speed in plant tissue using a device such as the BIOLISTIC PD-1000 particle gun (BioRad; Hercules CA). Micro-projectile-mediated delivery ("particle bombardment") is especially useful for transforming plant cells that are difficult to transform or regenerate using other methods. Methods for transformation using biolistic methods are well known (Wan, Plant Physiol. 104: 37-48, 1984; Vasil, BioTechnology 11: 1553-1558, 1993; Christou, Trends in Plant Science 1: 423-431, 1996). Micro-projectile-mediated transformation has been used, for example, to generate a variety of transgenic plant species, including cotton, tobacco, corn, wheat, oats, barley, sorghum, rice, hybrid poplar, and papaya (see Glick and Thompson, above. 1993; Duan et al., Nature Biotech. 14: 494-498, 1996; Shimamoto, Curr. Opin. Biotech. 5: 158-162, 1994).

A rapidly transforming regeneration system for the production of transgenic plants such as a system that produces transgenic wheat in two or three months (see European Patent No. EP 0709462A2) may also be useful to produce a transgenic plant according to a method of the invention, thus allowing a faster identification of the functions of the genes. Transformation of most dicot plants is possible with the methods described above. The transformation of monocotyledonous plants can also be carried out using, for example, biolistic methods as described above, transformation of chloroplasts, electroporation or partially permeabilized cells, introduction of DNA using glass fibers, Agrobacterium-mediated transformation and the like.

Plasti transformation can also be used to introduce a nucleic acid molecule into a plant cell (US Patent Nos. 5,451,513, 5,545,817 and 5,545,818; WO 95/16783; McBride et al., Proc. Natl. Acad. Sci., USA 91: 7301-7305, 1994). Chloroplast transformation involves the introduction of regions of cloned plastid DNA flanking a desired nucleotide sequence, for example, a selectable marker together with the polynucleotide of interest, into a suitable target tissue, using, for example, a biolistic transformation method or by protoplasts (eg, calcium chloride or PEG-mediated transformation).

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Flanking regions of one to 1.5 kb ("targeting sequences") facilitate homologous recombination with the plastome genome, and allow the replacement or modification of specific regions of the plastome. Using this method, point mutations in the chloroplast 16S rRNA and rps 12 genes can be used that confer resistance to spectinomycin and streptomycin and can be used as selectable markers for transformation (Svab et al., Proc. Natl. Acad. Sci., USA 87: 8526-8530, 1990; Staub and Maliga, Plant Cell 4: 39-45, 1992), producing stable homoplasmic transformants, at a frequency of approximately one per 100 target leaf bombardments. The presence of cloning sites between these markers allows the creation of a plastid targeting vector for the introduction of foreign genes (Staub and Maliga, EMBO J. 12: 601-606, 1993). Substantial increases in transformation frequency are obtained by replacing the recessive rRNA or antibiotic resistance genes of r proteins with a dominant selectable marker, the bacterial aadA gene that encodes the detoxifying enzyme aminoglycoside-3'-adenyltransferase for spectinomycin (Svab and Maliga, Proc. Natl. Acad. Sci., USA 90: 913917, 1993). Generally, after transformation, approximately 15 to 20 cycles of cell division are required to reach a homoplastid state. Plastid expression, in which genes are inserted by homologous recombination into all of the several hundred copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over genes expressed in the nucleus to allow high levels of expression that can easily exceed 10% of the total soluble protein in the plant.

Cells that have been transformed can be grown in plants in conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5: 81-84. These plants can then be grown and pollinated with the same strain or different transformed strains and result in plants having the expression of the desired phenotypic characteristics which can then be identified. To ensure that the expression of the desired phenotypic characteristics is maintained and stably inherited, two or more generations can be cultured.

Plants suitable for the purposes of the present invention may be monocots or dicots and include, but are not limited to, corn, wheat, barley, rice, sweet potato, soybean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, bell pepper, celery, pumpkin, zucchini, hemp, pumpkin, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soy, tomato, sorghum, sugar cane, beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arab / dops / s thal / ana, and woody plants such as conifers and deciduous trees . Thus, a transgenic plant or a genetically modified plant cell of the invention can be an angiosperm or gymnosperm.

Angiosperms are divided into two broad classes based on the number of cotyledons, which are the seed leaves that generally store or absorb food; a monocotyledonous angiosperm has only one cotyledon and dicotyledonous angiosperms have two cotyledons. Angiosperms produce a variety of useful products including materials such as wood, rubber, and paper; fibers such as cotton and linen; herbs and medicines such as quinine and vinblastine; ornamental flowers such as roses and orchids which are included within the scope of the present invention; and food products such as grains, oils, fruits, and vegetables. Angiosperms include a variety of flowering plants, including, for example, cereal plants, legume plants, oilseed plants, hardwood trees, ornamental fruit and flower plants, the general classes of which are not necessarily exclusive. Cereal plants, which produce an edible grain, include, for example, corn, rice, wheat, barley, oats, rice, dactyl, guinea grass, and sorghum. Legume plants include members of the pea family (Fabaceae) and produce a characteristic fruit known as a legume. Examples of legumes include, for example, soybeans, peas, chickpeas, moths, broad beans, kidney beans, lima beans, lentils, pinto peas, dry beans, and peanuts as well as alfalfa, foot bird clover, white clover, and sainfoin. . Oil plants, which have seeds that are useful as an oil source, include soybeans, sunflower, rapeseed (canola) oil, and cottonseed. Angiosperms also include hardwood trees, which are woody perennial plants that generally have only one stem (trunk). Examples of tree bugs include alder, ash, poplar, linden, beech, birch, cherry, cotton, elm, eucalyptus, hickory, carob, maple, oak, persimmon, poplar, sycamore, hickory, redwood, and willow. Trees are useful, for example, as a source of pulp, paper, structural material, and fuel.

Angiosperms produce seeds enclosed within a mature, adult ovary. An angiosperm fruit may be suitable for human or animal consumption or for collecting seeds to propagate the species. For example, hops is a member of the blackberry family that is prized for its flavor in malt liquor. Fruiting angiosperms also include grape, orange, lemon, grapefruit, avocado, date, peach, cherry, olive, plum, coconut, apple and pear and blackberry, blueberry, raspberry, strawberry, pineapple, and tomato, cucumber, and eggplant plants. . An ornamental flower is an angiosperm grown for its decorative flower. Examples of commercially important ornamental flowers include rose, lily, tulip and chrysanthemum plants, snapdragon, camellia, carnation, and petunia and may include orchids. It will be recognized that the present invention can also be practiced using gymnosperms, which do not produce seeds in a fruit.

Some embodiments of this invention overcome the problem of maintaining recessive homozygous reproductive traits when using a transgenic restoration strategy, reducing the number of plants, plantations and stages necessary for the maintenance of plants with said traits.

Homozygosity is a genetic state that exists when identical alleles reside at corresponding loci on homologous chromosomes. Heterygosity is a genetic state that exists when different alleles reside at loci

ES 2 339 559 T3 corresponding on homologous chromosomes. Hemizygosity is a genetic state that exists when there is only one copy of a gene (or set of genes) with no allelic homologue on the sister chromosome.

Maintaining the recessive homozygous state for male sterility is achieved by introducing into a plant a transgenic restoration construct that is attached to a sequence that interferes with the formation, function, or dispersal of the male gametes of the plant, to create a plant " maintainer ”or“ donor ”. The restoration transgene, after introduction into a plant that is homozygous recessive for the male-sterile genetic trait, restores the genetic function of that trait. Due to the linked gene driven by a male gamete-specific promoter, all pollen containing the restoration transgene is rendered unviable. All viable pollen produced contains a copy of the recessive allele but does not contain the restoration transgene. The transgene is maintained in the hemizygous state in the maintainer plant.

The pollen of the maintainer plant can be used to fertilize plants that are homozygous for the recessive trait, and the offspring will therefore maintain their homozygous recessive state. The maintainer plant containing the restoration transgene construct is propagated by self-fertilization, with half of the resulting seed being used to produce more plants that are recessive homozygous for the gene of interest and hemizygous for the restoration transgenic construct.

The maintainer plant serves as a pollen donor for the plant that has the homozygous recessive trait. The maintainer plant is optimally produced from a plant that has the homozygous recessive trait and that also has nucleotide sequences introduced into it that would restore the trait created by the homozygous recessive alleles. In addition, the restoration sequence is linked to nucleotide sequences that interfere with the function, formation, or dispersal of male gametes. The gene can function to impede the formation of male gametes or impede the function of male gametes by any of a variety of well-known modalities and is not limited to a particular methodology. By way of example, but not limitation, this may include the use of one or more genes that express a cytotoxic product for male gametes (See, eg, US patents. Nos. 5,792,853 and 5,689,049; and document PCT / EP89 / 00495); they inhibit the formation of the product of another gene important for the formation, function or dispersal of male gametes (see, US patents 5,859,341 and 6,297,426); combine with another gene product to produce a substance that prevents the formation, function, or dispersal of male gametes (see U.S. Patents: 6162,964; 6,013,859; 6,281,348; 6,399,856; 6,248,935; 6,750 .868; and 5,792,853); are antisense to or cause co-suppression of a gene critical for the formation, function, or dispersal of male gametes (see U.S. Patents; Nos .: 6,184,439; 5,728,926; 6,191,343; 5,728,558; and 5,741.84), or the like.

Normally, to produce more plants that have a recessive state, one could cross the recessive plant with another recessive plant, or self-pollinate a recessive plant. This may not be desirable for some recessive traits and may be impossible for recessive traits that affect reproductive development. Alternatively, the homozygous plant could be crossed with a second plant that has the restoration gene, but this requires additional crossing to secrete the restorer gene to reach the recessive phenotypic state again. On the other hand, in one embodiment the invention provides a process in which the homozygous recessive state can be maintained, by crossing it with the maintainer plant. This method can be used in any situation where it is desired to continue the recessive state. This results in a relatively simple and cost-effective system for maintaining a homozygous recessive plant population.

When the homozygous recessive state is one that produces male sterility, the maintainer plant must necessarily contain a functional restoration transgenic construct capable of complementing the mutation and rendering the homozygous recessive plant capable of producing viable pollen. Binding of this male fertile restoration gene with a second functional nucleotide sequence that interferes with the formation, function, or dispersal of male gametes results in a pollen-producing maintainer plant that contains only the recessive allele of the restored gene in its native locus due to the pollen-specific cytotoxic action of the second nucleotide sequence. This viable pollen fraction is non-transgenic with respect to the restoration transgene construction.

For example, to produce male sterile female plants for use in the process of producing hybrids that are sterile as a result of being homozygous for a mutation in the MS45 gene, a gene that is essential for male fertility is desirable. Said mutant MS45 allele is designated as ms45. A plant that is homozygous for ms45 (represented by the annotation ms45 / ms45) exhibits the homozygous recessive male-sterile phenotype and produces non-functional pollen. See US Patent Nos: 5,478,369; 5,850,014; 6,265,640; and 5,824,524. In both inbred and hybrid plant production processes, it is highly desirable to maintain this homozygous recessive state. When sequences encoding the MS45 gene are introduced into a plant in a homozygous state, sporophytic restoration of androfertility occurs. (Cigan et al. (2001) Sex Plant. Repro 14: 135-142) By the method of the invention, a plant that is homozygous recessive ms45 / ms45 can have a functional MS45 gene introduced inside it, and therefore, androfertility is restored. This gene can be linked to a second gene that functions to process non-functional pollen or that prevents its formation, or that produces a lethal product in pollen, and that is linked to a promoter that directs its expression in male gametes. This results in a viable pollen-producing plant containing ms45 without the restoration transgenic construct.

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An example is a construct that includes the MS45 gene operably linked to the 5126 promoter, a preferred male tissue promoter (See US Patent No. 5,837,851) and further linked to the cytotoxic DAM methylase gene controlled by the PG47 promoter (See US Patent Nos. 5,792,853; 5,689,049). The resulting plant produces pollen, but viable pollen only contains the gene for ms45. Therefore, it can be used as a pollinator to fertilize the recessive homozygous plant (ms45 / ms45), and 100% of the offspring produced will remain male-sterile as a result of the maintenance of homozygosity for ms45. The progeny will not contain the introduced transgenic restoration construct.

Obviously, many variations in male fertility are available to this method. Any other critical gene related to male fertility can be used in this system. For example and without limitation, such genes can include the SBMu200 gene (also known as SB200 or MS26) described in WO 02/26789; the BS92-7 gene (also known as BS7) described in WO 02/063021; the MS2 gene described in Albertsen and Phillips, "Developmental Cytology of 13 Genetic Male Sterile Loci in Maize" Canadian Journal of Genetics & Cytology 23: 195-208 (Jan. 1981); or the Arabidopsis MS2 gene described in Aarts et al., "Transposon Tagging of a Male Sterility Gene in Arabidopsis", Nature, 363: 715-717 (Jun 24, 1993); and the Arabidopsis M1 gene described in Wilson et al., "The Arabidopsis MALE STERILITY (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PDH-finger family of transcription factors", Plant J., 1:27 -39 (Oct 28, 2001).

A desirable result of the process of the invention is that the plant having the restorative nucleotide sequence can self-fertilize; that is, the pollen of the plant is transferred to the flower of the same plant to achieve the propagation of the restorer plants. (It should be noted that "self-fertilization" includes both the situation in which the pollen-producing plant is fertilized with that same pollen and the situation in which pollen from a genetically identical plant or group of plants pollinates a plant that is a genetically identical individual, or a group of such genetically identical plants). The transgenic restoration construct will not be present in pollen, but will be included in 50% of the ovules (the female gamete). The seed resulting from self-fertilization can be planted, and the seed having the restoration transgenic construct selected. The selection process can be performed by one or more of any of the known processes, the most common being that the restoration nucleotide sequence is linked to a marker gene. The marker can be titratable or selectable and allows the identification of the seed that comprises the restoration sequence, and / or of those plants produced from the seed that has the restoration sequence.

In one embodiment of the invention, it is possible to provide that the promoter driving the restoration gene is inducible. This allows additional control in the process, when desired, by providing that the plant having the restorative nucleotide sequences is constitutively male sterile. This type of male sterility is indicated in US Patent No. 5,859,341. In order for the plant to be fertile, the induction substance must be provided, and the plant will be fertile. Again, when combined with the process of the invention, as described above, the single pollen produced will not contain the restorative nucleotide sequences.

The gamete that controls the transmission of the restoration nucleotide sequences may be the female gamete, rather than the male gamete. The process is the same as described above, except in cases where it is also desired to maintain the plant having the self-fertilizing restorative nucleotide sequences, in which case it will be useful to provide that the promoter driving the restoration gene is inducible, in this way female fertility can be triggered by exposure to the induction substance, and seeds can be formed. Controlling female fertility in this manner is described in US Patent No. 6,297,426. Examples of genes that impact female fertility include the branched teosinte1 (Tb1) gene, which increases apical dominance, resulting in multiple spikes and suppression of female tissue. Hubbard et al. (2002) Geneticas 162: 1927-1935; Doebley et al (1997) Nature 386: 485-488 (1997). Another example is called "barren 3" or "ba3". This mutant was isolated from a mutant maize plant infected with the striated wheat mosaic virus and is described in Pan and Peterson, J Genet. And Breed. 46: 291-294 (1992). The plants develop normal spikes, but do not have any ear buds along the stems. In Coe and Beckett, Maize Genet. Coop Newslett 61: 46-47 (1987) describes sterile stems in the fastigiata variety. Other examples include the barren stalk1 gene (Gallavotti et al., Nature 432: 630-635 (2004)); lethal ovule mutant (Vollbrecht, Maize Genetics Cooperation Newsletter 68:23 (1994)); and defective pistil mutant (Miku and Mustyatsa, Genetika 14 (2): 365-368 (1978)).

To control the expression of the regions of the restoration transgenic construct encoding specific proteins and functions, any promoter element compatible with the plant can be employed. These can be promoters from plant genes, such as, for example, the ubiquitin promoter, the ribulose-1, 5-bis-phosphate carboxylase small subunit promoter, or tumor-inducing plasmid promoters from Agrobacterium tumefaciens, such as nopaline synthase and octopine synthase promoters, or viral promoters such as the 19S and 35S promoters from cauliflower mosaic virus (CaMV) or the 35S promoter from scrofularia mosaic virus. See, Kay et al., (1987) Science 236: 1299 and European Patent Application No. 0 342 926. See International Application WO 91/19806 for a review of illustrative plant promoters suitably employed in the present invention. The range of available plant-compatible promoters includes inducible promoters and tissue-specific ones.

The invention contemplates the use of promoters that provide tissue-preferred expression, including promoters that preferentially express male or female gamete tissue of the plant. The invention does not require that any particular gamete tissue preferred promoter be used in the process and any of the

ES 2 339 559 T3 are many such promoters known to those of skill in the art. By way of example, but not limitation, such a promoter is the 5126 promoter that preferentially drives expression of the gene to which it is linked for male plant tissue, as described in US Patent No. 5,837. 851 and 5,689,051. Other examples include the MS45 promoter described in US Patent No. 6,037,523; the SF3 promoter described in US Patent No. 6,452,069; the BS92-7 or BS7 promoter described in WO 02/063021; the SBMu200 promoter described in WO 02/26789; and the regulatory element SGB6 described in US Patent No. 5,470,359 and TA39 (Koltunow et al. (1990) "Different temporal and spatial gene expression patterns occur during anther development". Plant Cell 2: 1201-1224; Goldberg et al., (1993) Anther development: basic principles and practical applications. Plant Cell 5: 1217-1229; and US Patent No. 6,399,856. See also Nadeau et al., Plant Cell 8 (2): 213-39 (1996); and Lu et al., Plant Cell 8 (12): 2155-68 (1996).

The P67 promoter indicated in SEQ ID NO: 1 is 1112 nucleotides long. This promoter was isolated from a genomic clone corresponding to a maize EST sequence. The sequence showed limited homology to the putative pectin methyl esterase.

The pollen specificity of P67 expression has been confirmed by RT-PCR and Northern blot analysis of RNA samples from different tissues including leaf, root, mature pollen grains / anther, spike in vacuole phase, spikelets, ear. , sheath, silk and embryo. The results indicate a high level of specificity for expression in developmental pollen, particularly in the uni-nucleated middle stage.

Southern blot analysis has shown that the clone represents single or low copy genes in the maize genome. Using the oat chromosome substitution line, chromosome mapping revealed that the sequence localizes to maize chromosome 1.

The clone was used to screen a bacterial artificial chromosome (BAC) library in corn. BAC positive clones have been found and subcloned in the phagemid pBluescript KS s. Subclones corresponding to the cDNA sequences have been identified and sequenced. The transcriptional start site has been determined using a rapid amplification strategy mediated by RNA ligase at the 5 'end. The promoter region was designated P67.

The P95 promoter indicated in SEQ ID NO: 2 is 1092 nucleotides in length. This promoter was isolated from a genomic clone corresponding to a maize EST sequence. The sequence showed limited homology to the putative L-ascorbate oxidase.

The specificity of P95 expression with respect to pollen has been confirmed by RT-PCR and Northern blot analysis of RNA samples from different tissues including leaf, root, mature pollen grains / anther, spike in vacuole phase, spikelet. , cob, pod, silk and embryo. The results indicate a high level of specificity for expression in developmental pollen, particularly in the uni-nucleated medium state.

Southern blot analysis has shown that the clone represents single or low copy genes in the maize genome. Using the oat chromosome substitution line, chromosome mapping revealed that the sequence is located on Chromosomes 6 and 8 of maize.

The clone was used to screen a BAC library in corn. Positive BAC clones have been found and subcloned in the phagemid pBluescript KS s. Subclones corresponding to the cDNA sequences have been identified and sequenced. The transcriptional start site has been determined using a rapid amplification strategy mediated by RNA ligase at the 5 'end. The promoter region was designated P95.

Using well known techniques, additional promoter sequences can be isolated based on their sequence homology with respect to SEQ ID NO: 1 or SEQ ID NO: 2. In these techniques, all or part of a known promoter sequence is used as a probe that selectively hybridizes to other sequences present in a population of cloned genomic DNA fragments (ie, libraries) from the selected organism. Methods that are readily available in the art for hybridization of nucleic acid sequences can be used to obtain sequences that correspond to these promoter sequences in species including, but not limited to, maize (Zea mays), rapeseed (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticumaestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solarium tuberosum), peanut (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus (Citrus spp.), Cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), Avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olives (Olea europaea), oats, barley, vegetables, ornamental and coniferous plants. Preferably the plants include corn, soybeans, sunflower, safflower, rapeseed, wheat, barley, rye, alfalfa, and sorghum.

The entire promoter sequence or parts thereof can be used as a probe capable of specifically hybridizing to the corresponding promoter sequences. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique and preferably are at least about 10 nucleotides in length and more preferably at least about 20 nucleotides in length. These probes can be used to amplify corresponding promoter sequences from a selected organism.

ES 2 339 559 T3 by the well-known polymerase chain reaction (PCR) process. This technique can be used to isolate additional promoter sequences from a desired organism or as a diagnostic test to determine the presence of the promoter sequence in an organism. Examples include hybridization screening of plaque DNA libraries (both plaques and colonies; see, for example, Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, eds., Academic Press).

In general, sequences that correspond to a promoter sequence of the present invention and that hybridize to a promoter sequence described herein will have at least 50%, 55%, 60%, 65%, 70%, 75% homology. 80%, 85%, 90%, 95% even 98% or more with the described sequence.

Fragments of a particular promoter sequence described herein may function to promote pollen-preferred expression of an isolated operably linked nucleotide sequence. These fragments will comprise at least about 20 contiguous nucleotides, preferably at least about 50 contiguous nucleotides, more preferably at least about 75 contiguous nucleotides even more preferably at least about 100 contiguous nucleotides of the particular promoter nucleotide sequences described herein. The nucleotides of such fragments will normally comprise the TATA recognition sequence of the particular promoter sequence. Such fragments can be obtained by using restriction enzymes to cleave the naturally occurring promoter sequences described herein; synthesizing a nucleotide sequence from the naturally occurring DNA sequence; or by using PCR technology. See particularly, Mullis et al. (1987) Methods Enzymol. 155: 335-350, and Erlich, ed. (1989) PCR Technology (Stockton Press, New York). Again, variants of these fragments, such as those resulting from site-directed mutagenesis, are included in the compositions of the present invention.

Thus, nucleotide sequences comprising at least about 20 contiguous nucleotides from the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 are included. These sequences can be isolated by hybridization, PCR, and the like. Such sequences include fragments capable of directing the preferred expression of pollen, fragments useful as probes to identify similar sequences, as well as elements responsible for tissue or temporal specificity.

Biologically active variants of the promoter sequence are also included in the compositions of the present invention. A regulatory "variant" is a modified form of a promoter in which one or more bases have been modified, removed, or added. For example, a routine way to remove part of a DNA sequence is to use an exonuclease in conjunction with DNA amplification to produce nested unidirectional deletions of double-stranded DNA clones. A commercial kit for this purpose is marketed under the trade name Exo-Size^TM (New England Biolabs, Beverly, Mass.). Briefly, this procedure involves incubating exonuclease III with DNA to progressively remove nucleotides in the 3 'to 5' direction at 5 'overhangs, blunt ends, or enzymatic cuts in the DNA template. However, exonuclease III cannot remove nucleotides at the 3 'end, on blunt ends or 4 base cuts in the DNA template. The timed digestion of a clone with this enzyme produces nested unidirectional deletions.

An example of a regulatory sequence variant is a promoter formed by causing one or more deletions in a larger promoter. Deletion of the 5 'part of a promoter to the TATA box near the transcription start site can be performed without canceling the promoter activity, as described in Zhu et al., The Plant Cell 7: 1681-89 (1995 ). Such variants must retain promoter activity, particularly the ability to direct expression in specific tissues. Biologically active variants include, for example, the native regulatory sequences of the invention that have one or more nucleotide substitutions, deletions, or insertions. Activity can be measured by Northern blot analysis, reporter activity measurements when using transcriptional fusions, and the like. See, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2^to ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), incorporated herein by reference.

The nucleotide sequences for the pollen-preferred promoters described in the present invention, as well as the variants and fragments thereof, are useful in the genetic engineering of any plant when they are operably linked to an isolated nucleotide sequence whose expression is controlled to achieve a desired phenotypic response.

The nucleotide sequence operably linked to the regulatory elements described herein may be an anti-sense sequence for a target gene. By "antisense DNA nucleotide sequence" is meant a sequence that is in reverse orientation to the normal 5 'to 3' orientation of that nucleotide sequence. When delivered within a plant cell, expression of an antisense DNA sequence prevents normal expression of the DNA nucleotide sequence for the target gene. The antisense nucleotide sequence encodes a transcribed RNA that is complementary and capable of hybridizing to endogenous messenger RNA (mRNA) produced by transcription of the DNA nucleotide sequence for the target gene. In this case, the production of the native protein encoded by the target gene is inhibited to achieve a desired phenotypic response. In this manner, the regulatory sequences claimed herein can be operably linked to antisense DNA sequences to reduce or inhibit the expression of a native or exogenous protein in the plant.

Many nucleotide sequences are known to inhibit pollen formation or function or dispersal, and any sequence that achieves this inhibition will suffice. United States Patent No. 6,399,856 includes a description of

ES 2 339 559 T3 encryption relating to genes that can correctly impact development or function and includes dominant negative genes such as cytotoxin genes, methylase genes, and growth inhibition genes. Dominant negative genes include the A-chain diphtheria toxin gene (Czako and An (1991) Plant Physiol. 95 687692); cell cycle division mutants such as CDC in maize (Colasanti et al., (1991) Proc. Natl. Acad. Sci. USA 88, 3377-3381); the WT gene (Farmer et al., Hum. Mol. Genet. 3, 723-728, 1994); and P68 (Chen et al., Proc. Natl. Acad. Sci. USA 88, 315-319, 1991). A suitable gene may also encode a protein involved in inhibiting pistil development, pollen-receiving stigma interactions, pollen tube growth or fertilization, or a combination thereof. In addition, genes that interfere with the normal accumulation of starch in pollen or affect the osmotic balance within the pollen may also be suitable. These include, for example, the corn alpha-amylase gene, the corn beta-amylase gene, debranching enzymes such as Sugary1 and pullulanase, glucanase and SacB.

In an illustrative embodiment, the DAM-methylase gene is used, the expression product that catalyzes the methylation of adenine residues in plant DNA. Methylated adenines will not affect cell viability and will only be found in tissues where the DAM-methylase gene is expressed, since these methylated residues are not found endogenously in plant DNA. Examples of so-called "cytotoxic" genes have been described above and may include, but are not limited to, the pectate lyase pelE gene, from Erwinia chrysanthermi (Kenn et al. (1986) J. Bacteriol 168: 595); the A chain diphtheria toxin gene (Greenfield et al (1983) Proc. Natl. Acad. Sci. USA 80: 6853, Palmiter et al. (1987) Cell 50: 435); the T-urf13 gene from cmsT maize mitochondrial genomes (Braun et al. (1990) Plant Cell 2: 153; Dewey et al. (1987) Proc. Natl. Acad. Sci. USA 84: 5374); the CytA toxin gene from Bacillus thuringiensis Israeliensis causing cell membrane disruption (McLean et al. (1987) J. Bacteriol 169: 1017, US Patent No. 4,918,006); DNases, RNases, (US Patent No. 5,633,441); proteases, or genes that express anti-sense RNA.

Furthermore, the methods of the invention are useful for maintaining the recessive homozygous state of traits other than those that impact plant fertility. The state-restoring gene of interest would be introduced into a plant linked to a nucleotide sequence that inhibits pollen formation, function, or dispersal and that may additionally bind to a preferred male gamete tissue promoter or to a gene encoding a marker. , for example, a seed specific marker. The viable pollen produced by the plant into which the construct is introduced contains only the recessive allele of the gene of interest and none of the restoration transgenic sequences. Half of the female gametes of the hemizygous transgenic plant contain the transgene and can be self-pollinated or pollinated by a plant comprising the recessive alleles. Half of the seeds produced will carry the transgene and can be identified by the attached marker. The hemizygous state can be maintained by selfing the hemizygous plant; half of the progeny will contain the transgene and therefore the trait of interest.

The genes of interest reflect markets and commercial interests and those involved in crop development. The crops and markets of interest change, and as developing nations open up to new markets, new crops and technologies also emerge. Furthermore, as understanding of agricultural traits and characteristics such as yield and heterosis increases, the choice of genes for transformation will change accordingly.

Regulation of male fertility is necessarily measured in terms of its effectiveness in individual cells. For example, removal of 99.99% of pollen grains is necessary to achieve reliable sterility for commercial use. However, the success of suppressing or restoring the expression of other traits can be achieved less rigorously. Within a particular tissue, for example, cell expression of 98%, 95%, 90%, 80% or less results in the desired phenotype.

This invention has utility for a variety of recessive genes, not limited to those in which the expression of the homozygous recessive trait compromises the ability of plants to maintain their full reproductive capacity. General categories of genes of interest include, for example, genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in internal management such as heat shock proteins. More specific categories of transgenes, for example, include genes that encode traits important for agricultural traits, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Genes of interest generally include those involved in oil, starch, carbohydrate, or nutrient metabolism as well as those that affect grain size, sucrose loading, and the like. Agriculturally important traits such as oil, starch and protein content can be further genetically altered using traditional farming methods. Modifications include increasing the content of oleic acid, saturated or unsaturated oils, increasing the levels of lysine and sulfur, disposing of essential amino acids, and also modifying starch. In United States Patent Application Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389. modifications of the hordothionin protein are described. Another example is the 2S soy albumin-encoded sulfur and / or lysine-rich seed protein described in US Patent No. 5,850,016 and the barley chymotrypsin inhibitor, described in Williamson et al., Eur. J Biochem. 165: 99-106. (1987). Other important genes encode growth factors and transcriptional factors.

Agronomic traits can be improved by altering the expression of genes that: affect growth and development, especially during environmental stress. These include, for example, genes encoding cytokinin biosynthesis enzymes, such as isopentenyl transferase; genes encoding cytokinin catabolic enzymes, such

ES 2 339 559 T3 as cytokinin oxidase; genes encoding polypeptides involved in cell cycle regulation, such as CyclinD or cdc25; genes encoding cytokinin receptors or sensors, such as CRE1, CKI1 and CKI2, histidine phosphotransmitters, or cytokinin response regulators.

Insect resistance genes can encode resistance to pests that leave a large yield trail, such as rootworm, cutworm, European corn borer, and the like. Such genes include, for example: Bacillus thuringiensis endotoxin genes, US Patent No. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al. (1986) Gene 48: 109; lectins, Van Damme et al. (1994) Plant Mol. Biol. 24: 825; and the like.

Genes encoding disease resistance traits include: detoxification genes, such as against fumonosin (WO 9606175 filed June 7, 1995); genes for avirulence (avr) and disease resistance (R), Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; Mindrinos et al. (1994) Cell 78: 1089; and the like.

Commercial traits can also be encoded in a gene (or genes) that could alter or increase for example starch for the production of paper, textiles and ethanol or provide the expression of proteins with other commercial uses. Another important commercial use for transformed plants is the production of polymers and bioplastics as described in US Patent No. 5,602,321 issued February 11, 1997. Genes such as B-ketothiolase, PHBase (polyhydroxybutyrate synthase) and acetoacetyl-CoA reductase (see Schubert et al. (1988) J. Bacteriol 170 (12): 5837-5847) facilitate the expression of polyhydroxyalkanoates (PHA).

Exogenous products include enzymes and plant products as well as those from other sources including prokaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, and the like. The protein level of the seeds can be increased, in particular modified seed proteins having improved amino acid distribution to improve the nutritional value of the seed. This is achieved by the expression of said proteins that have an enhanced amino acid content.

The expression cassettes of the invention, comprising a promoter and the isolated nucleic acid sequence of interest, may also include, at the 3 'end of the isolated nucleotide sequence of interest, a plant functional transcriptional and translational termination region. . The termination region may be native to the promoter nucleotide sequence of the cassette, it may be native to the DNA sequence of interest, or it may come from another source.

Other convenient termination regions are available from the A. tumefaciens Ti plasmid, such as the octopine synthase and nopaline synthase termination regions. See also: Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. 1989) Nucleic Acids Res. 17: 7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15: 9627-9639.

Expression cassettes may additionally contain 5 'leader sequences. Such leader sequences can act to enhance translation. Translation leader sequences are known in the art and include: picornavirus leader sequences, for example: EMCV leader (Encephalomyocarditis 5 'noncoding region), Elroy-Stein et al. (1989) Proc. Nat. Acad. Sci. USA 86: 6126-6130; potyvirus leaders, eg TEV (Tobacco Etch Virus) leader, Allison et al. (1986); MDMV (Corn Dwarf Mosaic Virus) leader, Virology 154: 9-20; human immunoglobulin heavy chain binding protein (BiP), Macejak et al. (1991) Nature 353: 90-94; Untranslated leader of alfalfa mosaic virus coat protein mRNA (AMV RNA 4), Jobling et al. (1987) Nature 325: 622-625); tobacco mosaic virus (TMV) leader, Gallie et al. (1989) Molecular Biology of RNA, pages 237-256; and leader of the corn chlorotic spot virus (MCMV) Lommel et al. (1991) Virology 81: 382-385. See also Della-Cioppa et al. (1987) Plant Physiology 84: 965-968. The cassette can also contain sequences that enhance translation and / or mRNA stability such as introns.

In cases where it is desirable that the expressed product of the isolated nucleotide sequence is directed to a particular organelle, particularly the plasmid, amyloplast or endoplasmic reticulum, or is secreted on the cell surface or extracellularly, in the expression cassette it may further comprise a coding sequence for a transit peptide. Such transit peptides are well known in the art and include, but are not limited to: the transit peptide for acyl transporter protein, RUBISCO small subunit, plant EPSP synthase, and the like.

In preparing the expression cassette, the various DNA fragments can be manipulated to provide the DNA sequences in the proper orientation and, where appropriate, in the proper reading frame. For this purpose, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. To this end, techniques such as in vitro mutagenesis, primer repair, restriction digestion, hybridization, and re-substitutions, such as transitions and transversions, may be involved.

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The following expressions are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window", (c) "percent sequence identity" and ( d) "substantial identity".

(a) As used herein, "reference sequence" is a defined sequence used as the basis for a sequence comparison. A reference sequence can be a substring or the entirety of a specific sequence; for example, a segment of a full-length promoter sequence, or the promoter sequence (b). As used herein, "comparison window" refers to a contiguous and specific segment of a polynucleotide sequence, in which the sequence polynucleotide can be compared to a reference sequence and wherein the part of the polynucleotide sequence in the comparison window can comprise additions or deletions (i.e. gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In general, the comparison window is at least 20 contiguous nucleotides in length, and optionally may be 30, 40, 50, 100 or more nucleotides in length. Those skilled in the art will understand that to avoid high similarity to a reference sequence due to the inclusion of gaps in the polynucleotide sequence, a gap penalty is typically introduced and subtracted from the number of couplings.

(c) As used herein, "percent sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e. gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where there is an identical nucleic acid base in the two sequences to produce the number of coupled positions, dividing the number of coupled positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percent sequence identity (d) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90% and even more preferably at least 95% compared to a reference sequence, using one of the alignment programs described using conventional parameters.

Sequence alignment methods for comparison are well known in the art. Genetic comparisons can be determined by BLAST searches (Basic Local Alignment Search Tool; Altschul, SF, et al., (1993) J. Mol. Biol. 215: 403-410; see also www.ncbi.nlm.nih. gov / BLAST /) with default parameters to identify with respect to sequences contained in the BLAST database "GENEMBL". A sequence can be analyzed to determine its identity with respect to all publicly available DNA sequences contained in the GENEMBL database using the BLASTN algorithm with the default parameters.

In order to define the present invention, GAP (Global Alignment Program) is used. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to look for the alignment of two complete sequences that maximizes the number of couplings and minimizes the number of gaps. The default gap creation penalty values ​​and gap extension penalty values ​​in Version 10 of the Wisconsin Package software package^® (Accelrys, Inc., San Diego, CA) for the protein sequences are 8 and 2, respectively. For nucleotide sequences, the default gap creation penalty is 50 while the default gap extension penalty is 3. Percent similarity is the percentage of symbols that are similar. The symbols across the gaps are ignored. A similarity is scored when the matrix score value for a pair of symbols is greater than or equal to 0.50, the threshold of similarity. The scoring matrix used in Version 10 of the Wisconsin Package® software package (Accelrys, Inc., San Diego, CA) is BLOSUM62 (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915).

Large amounts of nucleic acids of the present invention can be produced by replication in a suitable host cell. Natural or synthetic nucleic acid fragments encoding a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of being introduced and replicated in a prokaryotic or eukaryotic cell. Normally, nucleic acid constructs will be suitable for replication in a unicellular host, such as a yeast or bacterium, but the introduction (with or without genome integration) of cultured eukaryotic cell lines of mammals or plants or other is also intended. . The purification of nucleic acids produced by the methods of the present invention is described, for example, in Sambrook et al., Molecular Cloning. A Laboratory Manual, 2^to Ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) or Ausubel et al., Current Protocols in Molecular Biology, J. Wiley and Sons, NY (1992).

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Nucleic acid constructs prepared for introduction into a prokaryotic or eukaryotic host may comprise a host-recognized replication system, including the nucleic acid fragment that is intended to encode the desired protein and will also preferably include transcription and regulatory sequences. initiation of transcription operably linked to the coding protein segment. Expression vectors may include, for example, an origin of replication or autonomously replicating sequences (ARS) and expression control sequences, a promoter, an enhancer, and necessary processing information sites, such as binding sites. ribosome, RNA splicing and binding sites, polyadenylation sites, transcription terminator sequences, and mRNA stabilizing sequences. Signs of secretion can also be included if appropriate. Such vectors can be prepared by means of standard recombinant techniques well known in the art and described, for example, in Sambrook et al., Molecular Cloning. A Laboratory Manual, 2^to Ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) or Ausubel et al., Current Protocols in Molecular Biology, J. Wiley and Sons, NY (1992).

Vectors for introduction of genes by both recombination and extrachromosomal maintenance are known in the art and any suitable vector can be used. Methods for introducing DNA into cells such as electroporation, calcium phosphate co-precipitation, and viral transduction are known in the art and the choice of method is within the competence of one of ordinary skill in the art (Robbins, Ed., Gene Therapy Protocols, Human Press, NJ (1997)).

Gene transfer systems known in the art may be useful in practicing the present invention. These include viral and non-viral transfer methods. Various viruses have been used as gene transfer vectors, including polyoma, i.e., SV40 (Madzak et al., J. Gen. Virol., 73: 1533-1536 (1992)), adenovirus (Berkner, Curr. Top Microbiol Immunol, 158: 39-61 (1992), Berkner et al., Bio Techniques, 6: 616-629 (1988), Gorziglia et al., J. Virol., 66: 4407-4412 (1992); Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584 (1992); Rosenfeld et al., Cell, 68: 143-155 (1992); Wilkinson et al., Nucl. Acids Res., 20: 2233-2239 (1992); Stratford Perricaudet et al., Hum. Gene Ther., 1: 241-256 (1990)), vaccinia viruses (Mackett et al., Biotechnology, 24: 495499 (1992)), associated adenoviruses (Muzyczka, Curr. Top. Microbiol. Immunol., 158: 91-123 (1992); Ohi et al., Gene, 89: 279-282 (1990)), herpes viruses including HSV and EBV (Margolskee, Curr. Top. Microbiol. Immunol., 158: 67-90 (1992); Johnson et al., J. Virol., 66: 2952-2965 (1992); Fink et al., Hum. Gene Ther., 3: 11-19 (1992); Breakfield et al., Mol. Neurobiol., 1: 337-371 ( 1987;) Fresse et al., Biochem. Pharmacol., 40: 2189-2199 (1990)), and bird retrovirus (Brandyopadhyay et al., Mol. Cell Biol., 4: 749-754 (1984); Petropouplos et al., J. Virol., 66: 3391-3397 (1992)), murine (Miller, Curr. Top. Microbiol. Immunol., 158: 1-24 (1992); Miller et al., Mol. Cell Biol., 5: 431- 437 (1985); Sorge et al., Mol. Cell Biol., 4: 1730-1737 (1984); Mann et al., J. Virol., 54: 401-407 (1985)), and of human origin ( Page et al., J. Virol., 64: 5370-5276 (1990); Buchschalcher et al., J. Virol., 66: 2731-2739 (1992)).

Non-viral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Graham et al., Virology, 52: 456-467 (1973); Pellicer et al., Science, 209: 14141422 (1980)), mechanical techniques, eg microinjection (Anderson et al., Proc. Natl. Acad. Sci. USA, 77: 53995403 (1980); Gordon et al., Proc. Natl. Acad. Sci. USA, 77: 7380-7384 (1980); Brinsteretal., Cell, 27: 223-231 (1981); Constantini et al., Nature, 294: 92-94 (1981)), Membrane fusion mediated transfer methods by liposomes (Feigner et al., Proc. Natl. Acad. Sci. USA, 84: 7413-7417 (1987 ); Wang et al., Biochemistry, 28: 95089514 (1989); Kaneda et al., J. Biol. Chem., 264: 12126-12129 (1989); Stewart et al., Hum. Gene Ther., 3: 267-275 (1992); Nabel et al., Science, 249: 1285-1288 (1990); Lim et al., Circulation, 83: 2007-2011 (1992)), and direct DNA uptake and receptor-mediated DNA transfer (Wolff et al., Science, 247: 1465-1468 (1990); Wu et al. , BioTechniques, 11: 474-485 (1991); Zenke et al., Proc. Natl. Acad. Sci. USA, 87: 3655-3659 (1990); Wu et al., J. Biol. Chem., 264: 16985-16987 (1989); Wolff et al., BioTechniques, 11: 474485 (1991); Wagner et al., 1990; Wagner et al., Proc. Natl. Acad. Sci. USA, 88: 42554259 (1991); Cotten et al., Proc. Natl. Acad. Sci. USA, 87: 4033-4037 (1990); Curiel et al., Proc. Natl. Acad. Sci. USA, 88: 8850-8854 (1991); Curiel et al., Hum. Gene Ther., 3: 147-154 (1991)).

Example 1

Fork RNA promoter expression affects plant fertility

This example demonstrates that the fertility or fertility potential of plants can be modified by the expression of hairpin RNA molecules (hsRNA) specific to promoters of genes that encode proteins involved in androfertility-related pathways.

ShpRNA promoter constructs were generated by linking a ubiquitin promoter to an inverted repeat of the desired promoter, including a segment of the NOS promoter between the inverted repeat sequences. The expression of each construct generated a specific shRNA for one of the following promoters: MS45, 5126, BS7, SB200, and PG47. Nucleic acid molecules and methods for preparing maize constructs and transformation have been previously described (Cigan et al. (2001) Sex Plant Reprod. 14: 135-142). The offspring (T1 generation) of the transformed plants (T0) were analyzed.

Of 32 transformation events comprising the hsRNA specific for the promoter of the MS45 gene, 29 produced T1 plants that were male-sterile.

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Of 32 transformation events comprising the hsRNA specific for the 5126 gene promoter, 29 produced T1 plants that were male-sterile.

Of 32 transformation events that comprised hsRNA specific for the BS7 gene promoter, 23 produced T1 plants that produced a small amount of non-viable pollen ("killer" phenotype) or were male-fertile but produced only a small amount of viable pollen (phenotype "Spill").

Of 31 transformation events that comprised the hsRNA specific for the SB200 gene promoter, 13 produced T1 plants of the killer or shedder phenotype.

Of 24 transformation events comprising hsRNA specific for the PG47 gene promoter linked to a construct to provide herbicide resistance, 15 revealed no transmission of herbicide resistance to the T1 seedling using pollen from primary transformants. This is consistent with the expected post-meiotic expression of PG47.

RNA from plant anthers expressing the various hRNAs was analyzed by northern blot. For each target, six independent events in the T1 generation were analyzed to determine whether shRNA expression reduced the continuous levels of steady-state RNA of the target genes. The anthers were brought to the tetrad release state for the early uninucleated stage of microspore development. Poly A + RNA was isolated, separated by electrophoresis, transferred to membranes, and sequentially hybridized with specific probes for MS45, 5126, bS7, SB200, NOS, and actin (RNA loading control). No MS45, 5126 or BS7 transcripts were detected in plants expressing hsRNA specific for these endogenous promoters. Only a slight reduction of SB200 RNA was observed in plants expressing SB200 shRNA.

Protein immunoblot analysis of anther proteins was also performed essentially as previously described (Cigan et al., Sex Plant Reprod. 14: 135-142, 2001). For each target, six independent events in the T1 generation were analyzed to determine whether expression of the promoter shRNA reduced the steady-state protein levels of the target genes. The anthers were brought to the previous state, cultured in Laemelli buffer, separated by electrophoresis and reacted sequentially with antibodies specific for the MS45, BS7, SB200 or 5126 proteins. The result was similar to that of the northern blot, no MS45, 5126 or BS7 proteins were detected in plants expressing hRNA specific for these endogenous promoters and only a slight reduction of SB200 protein was observed for events comprising SB200hp.

These results demonstrate that promoter shRNA expression can selectively suppress endogenous gene expression in plant cells. Furthermore, the results show that the deletion of different genes involved in the male sterility of plants can affect the plant phenotype in various ways, including the degree of male fertility.

Example 2

Expression of the exogenous MS45 gene product restores fertility

This example demonstrates that plants rendered male sterile by expression of a hairpin construct of the MS45 promoter can restore fertility by expression of an exogenous MS45 gene construct.

Constructs were prepared containing the MS45 coding sequence operably linked to a ubiquitin heterologous promoter (UBI), 5126, SB200, or BS7; these constructs were introduced into ms45ms45 plant cells. Regenerated plants and their offspring were fertile, demonstrating that the native MS45 promoter can be substituted for a constitutive or anther-preferred promoter to confer an male-fertile phenotype on ms45 maize mutants. (See also Cigan et al., Sex Plant Reprod. 14: 135-142, 2001).

Additionally, plants containing the UBI: MS45 or 5126: MS45 construct were crossed with plants that were male sterile due to the expression of a promoter shRNA of the MS45 gene. The offspring were tested by PCR for the presence of the hp construct and UBI: MS45 or 5126: MS45. RNA hybridization analysis was performed and fertile phenotypes were scored.

Northern blot analysis of RNA obtained from the leaves of the offspring plants revealed that MS45 was expressed from the ubiquitin promoter in the offspring containing 7 of 12 hp obtained from the UBI: MS45 cross. Furthermore, the expression of MS45 from the UBI promoter correlates with the fertility observed in the descendant plants. These results demonstrate that MS45 is expressed from the constitutive ubiquitin promoter and that constitutive expression of the MS45 gene product confers male fertility in offspring plants.

Additionally, RNA from the anthers of these MS45hp maize plants containing 5126: MS45, BS7: MS45 or UBI: MS45 was analyzed. Anthers were brought to the tetrad release stage to the early uninucleated stage of microspore development and poly A + RNA was collected, electrophoresed, and sequentially hybridized.

ES 2 339 559 T3 with probes for MS45, SB200 and BS7. MS45 was expressed in the anthers of male fertile offspring both driven by the constitutive UBI promoter and anther-specific 5126 or BS7 promoters, with anther harvest timing likely affecting signal strength. MS45 RNA was not observed in male sterile plants containing only hairpin. These results demonstrate that the suppression of MS45 expression due to MS45 shRNA can be overcome by MS45 expression from a heterologous promoter that drives expression at least in anther cells.

The promoter expressing the MS45 gene can come from a source other than maize and can be, for example, any plant promoter capable of transcribing MS45 such that expression of the transcription unit produces male fertile plants. For example, rice genes and maize Arabidopsis homologs MS45, 5126, BS7 and MS26 have been isolated and identified. Overall there is a significant similarity between the coding regions, with conservation of the intronic regions. Importantly, the corresponding promoters from rice and corn have approximately 50 to 60% identity, suggesting that these promoters may function sufficiently in the maize mat to transcribe the MS45 gene. To test this, each of the promoters from rice MS45, rice BS7, rice MS26, and Arabidopsis 5126 were fused to the MS45 coding region of maize and tested for the ability of the construct to confer fertility when transformed into ms45ms45 mutants. . Using this test system, a high frequency of male fertile plants was observed for all four constructs.

In certain aspects, it is advantageous to use promoters other than corn to express the MS45 gene. For example, when promoter hRNAs from some species reduce the function of the target gene such that the plant is non-viable or non-reproductive, a promoter from a different species can be used to transcriptionally express the function of the complementation gene (eg, MS45). , thus overcoming this potential problem. Additionally, shRNA constructs can be generated to direct the non-maize promoters to suppress the expression of the MS45 gene and a means to reduce or eliminate function and render the plant male sterile by directing the non-maize promoter used in the MS45 expression cassette. For example, a homozygous recessive ms45 plant can be transformed with a MS45 rice promoter homolog by driving expression of the MS45 gene (MS45r :: MS45), rendering the plant male fertile. To suppress the expression of this MS45r :: MS45 cassette, a second maize plant can be generated which is heterologous to the maize MS45 mutation and expresses an MS45r promoter shRNA. As there are no equivalent target sequences of the endogenous MS45 rice promoter in this maize plant, this plant would be male-fertile. The second plant can be crossed with the homozygous ms45 plant containing the MS45r :: MS45 construct and progeny screened for the MS45r :: MS45 and MS45r hpRNA constructs. In this situation, the function of the MS45r :: MS45 gene is suppressed by the presence and expression of the MS45rHP, resulting in an androsterile plant.

The use of such constructs is based on the discovery that the hp 5126 promoter from rice in maize does not result in male-sterile plants. This is in contrast to results obtained using a maize 5126 hp promoter (see Example 1) and suggests that expression of the rice 5126 promoter hairpin is not capable of suppressing the endogenous maize 5126 gene.

Taken together, the present Examples demonstrate that endogenous genes related to plant fertility can be inactivated using shRNA-mediated suppression and that a fertile phenotype can be restored in phenotypically sterile plants.

Example 3

Promoter-specific hairpin RNA suppresses transgene-mediated transmission of herbicide resistance

This example demonstrates that pollen from hemizygous plants for a UBI: PG47 hairpin construct is unfeasible as determined by non-transmission of herbicide resistance for T1 crosses when a herbicide resistance gene binds to the PG47 hairpin construct.

In plant cells, a specific shRNA for the promoter of the PG47 gene was introduced, comprising an inverted repeat of the promoter of the PG47 gene driven by a ubiquitin promoter (UBI: PG47hp), linked to a 35S: PAT construct. Pollen from plants expressing the transgene, representing 24 transformation events of a single copy or less, was transported to the ears of wild-type corn. The seed set in the pods was very good and comparable to that observed when wild-type pollen was used. For each event, 32 seeds were planted in the soil and the seedlings were sprayed 5 days after germination with LIBERTY 2X herbicide to detect transmission of UBI: PG47hp linked to 35S: PAT.

It was expected that if the PG47-specific hpRNA functioned in the post-meiotic division of microspores, then viability would be normal, and 50% of the pollen would carry the transgene, providing resistance to the herbicide in 50% of the offspring. However, if the function of PG47 is necessary for pollen viability and the hairpin construction can suppress the expression of the PG47 gene product, then 50% of the pollen grains would be unviable; all viable pollen would lack the transgene and would be unable to transmit resistance to the herbicide. Non-functioning UBI: PG47hp constructs would be detectable by the presence of herbicide resistant plants.

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Fifteen of the 24 events tested were sensitive to the herbicide. These results demonstrate that the UBI: PG47hp constructs suppress the expression of the PG47 gene in pollen, rendering 50% of the pollen unviable and preventing the transmission of herbicide resistance operably linked to the suppression construct.

Example 4

Plants containing multiple promoter-specific hairpin RNAs suppress multiple target promoters

Plants containing 5126HP (ie a transgene encoding a 5126 promoter shRNA) are used as pollen receptors for pollen from plants expressing BS7HP. In plants containing both 5126HP and BS7HP, the endogenous expression of 5126 and BS7 is suppressed, leading to a stronger sterility phenotype than that observed with either construct alone. Plants are selected to contain either 5126HP or BS7HP or both, are grown to maturity, and the fertility phenotypes of these resulting plants are determined.

Alternatively or in addition to crossover as a means of combining hairpin constructs, one such construct, for example 5126HP, can be placed under the transcriptional control of an inducible promoter. In the absence of induction, these BS7HP-containing plants are capable of producing enough pollen on their own. However, after 5126HP induction, these plants are male-sterile and can be used as females during hybrid production. This process depends on the combined expression of the hairpin constructs (HP) to give a sterile plant, whereas the expression of a single HP construct does not confer sterility.

In certain embodiments, the expression of both shRNAs can be brought under the transcriptional control of a single promoter. In this case, the shRNAs can be designed to contain multiple target promoters within the same encoded RNA. For example, the 5126 promoter region can be juxtaposed to the BS7 promoter region and placed under the transcriptional control of a single ubiquitin promoter or other constitutive, developmental or tissue preferred promoter, resulting in the expression of an RNA containing a hybrid hairpin 5126 and BS7 that directs the suppression of both endogenous genes 5126 and BS7. Any combination and number of various promoters that multiply the target and different promoters can be used in this scheme. For example, a promoter that regulates plant height genes and a promoter vital to a reproductive process can be combined, resulting in sterile plants of short stature.

Example 5

Maintenance of inbred plants and production of hybrid plants containing complementation constructs and target promoters that suppress promoter-specific hairpin RNAs

This example demonstrates how an inbred plant containing two constructs, a promoter-specific dominant hairpin RNA (shRNA) construct and an MS45 gene expressed from a tissue-specific promoter, can be maintained and used in male-sterile female production. for the production of hybrids.

Both A1 and A2 inbred plants are homozygous recessive ms45ms45. Fertility is restored in A1 inbred plants by introduction of a transgene expressing the MS45 coding region using the 5126 promoter. A1 inbred plants also contain a construct that expresses BS7HP. These plants can self-fertilize and maintain independently of the A2 inbred plants. In A2 inbred plants, fertility is restored by expression of the MS45 coding region using the BS7 promoter. Inbred A2 plants also contain a construct that expresses 5126HP. These plants can self-fertilize and maintain independently of inbred A1 plants.

To generate seeds to obtain inbred females for hybrid production, the A1 inbred plant is removed and fertilized using pollen from the A2 inbred plant. The seed resulting from this cross is planted and all the descendant plants are male-sterile due to the presence of the homozygous alleles ms45 and 5126HP and BS7HP that suppress the genes for restoration of fertility, 5126-MS45 and BS7-MS45, respectively. These plants are used as females in the production of hybrids and are pollinated with plants bearing the wild-type MS45 gene resulting in an F1 hybrid seed. All the plants from these seeds are heterozygous for the MS45 gene and therefore male-fertile.

This example demonstrates that plants containing both the dominant suppression and restoration constructs can be maintained and used in a hybrid seed production strategy to generate fertile and gynosterile inbred hybrid plants.

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Example 6

Utility of plants containing pollen-specific target promoters that suppress promoter-specific hairpin RNAs and MS45 complementation constructs for hybrid plant production and inbred plant maintenance

This example demonstrates how a method that comprises the use of two constructs, a dominant hairpin RNA (hRNA) construct specific for a pollen-specific promoter and a restoration transgene, allows the propagation of a plant that has a recessive homozygous reproductive trait. without loss of the recessive homozygous state in the resulting offspring, for use in the production of sterile plants for the production of hybrids. This is achieved by introducing into a plant at least one restorative transgenic construct, operably linked to a first nucleotide sequence comprising a functional copy of a gene that complements the mutant phenotypic trait produced by the recessive homozygous state with a second functional nucleotide sequence. that interferes with the formation, function or dispersal of the male gametes of the plant. This construct is kept in the hemizygous state and a plant containing such a construct is called a maintainer. Interference with male gamete formation, function, or dispersal can be achieved by linking sequences that interfere with male gamete formation, function, or dispersal with a preferred promoter from gamete tissue. As the transgene is in a hemizygous state, only half of the pollen grains produced contain the restorative transgenic construct and none of these are viable due to the action of a second gene that prevents the formation of viable pollen. Therefore, when the maintainer plant containing said attached construct is used as a pollen donor to fertilize the homozygous recessive plant, only the viable male gametes provided to the homozygous recessive plant are those that contain the recessive allele, but do not contain any components. of the transgenic construct. The offspring resulting from said sexual interbreeding are not transgenic with respect to this transgenic construct.

Although the non-viable pollen produced by the maintainer plant contains the transgenic restoration construct, 50% of the ovules (the female gamete) will contain the transgenic restoration construct. Therefore, the maintainer plant can be propagated by self-fertilization, segregating the transgenic restoration construct so that it will be contained in 50% of the seeds of a self-fertilized maintainer plant. By linking the transgenic restoration construct with a selectable marker, 50% of the seed containing the transgene can be isolated to propagate the maintainer population that remains homozygous for the recessive gene and hemizygous for the transgenic restoration construct. In this case, a single inbred plant can be kept.

The A1 inbred plant is homozygous recessive for the fertility gene ms45. A1 inbred plants contain a construct in which male fertility is restored by expressing the MS45 coding region using a tissue specific promoter, for example the native MS45 promoter. Inbred A1 plants also contain a hairpin construct directed to suppress a pollen-expressing promoter, in this example, a PG47HP-expressing construct operably linked to the MS45 restoration construct and a selectable or identifiable marker, eg, a marker that confers herbicide resistance and / or a construct that serves as a visual or detectable marker to scan the plant and / or seed. These plants are fertile and can self-fertilize and maintain themselves. The seed of these plants will secrete 50:50 for the transgene because only non-transgenic pollen is viable and capable of fertilizing an ovule, of which 50% contains the construct.

To generate seeds to obtain female inbred plants for the production of hybrids, only non-transgenic plants of A1 inbred plants were kept in a row; these plants are homozygous recessive ms45 and male-sterile. In an adjacent row, transgenic and non-transgenic plants were grown from the A1 inbred plant. In this row, fertility is secreted one by one (fertile to sterile); fertile plants are used to pollinate sterile plants in the adjacent row. The seed of this cross is not transgenic for the operably linked restorer, hsRNA and explorable marker constructs and all offspring are male-sterile due to the presence of the homozygous ms45 allele. These plants are used as females in the production of hybrids and are pollinated with plants bearing the wild-type MS45 gene resulting in hybrid F1 seeds. All plants from these seeds are heterozygous and for the MS45 gene and therefore male-fertile.

This example demonstrates that plants containing a dominant pollen suppression hairpin construct and a fertility restoration construct can be maintained as inbred plants and used in a hybrid seed production strategy to generate inbred gynosterile plants and fertile hybrid plants. .

Example 7

Combinations

Two or more components of the constructs described herein can be combined in various ways to create systems for controlling gene expression. Said combinations can be made by joining said components within a single vector, using multiple vectors in simultaneous or sequential transformations and / or cultivating plants that comprise one or more components. The following Table 1 describes possible components

ES 2 339 559 T3 tes. Table 2 provides illustrative, but not exhaustive, combination representations useful in controlling male fertility.

For example, the components may include promoters or coding regions other than those indicated and the order of the components within the constructs may be different from those shown. Furthermore, a construct could comprise combinations of individual promoter / coding sequences and a promoter that directs the transcription of multiple components of the coding sequence. As an example of the latter, a construct could comprise a constitutive promoter that directs the transcription of a sequence that encodes MS45 as well as a polynucleotide that encodes a gene product involved in the production or regulation of an identifiable marker (eg, pigment). to create a broadcast product. This would allow screening of transformants using any plant tissue, even if expression of MS45 results in male fertility.

Within any of the constructs, one or more hairpin components of the promoter could be included, for example within an intron of any of the encoded genes or within a 5 'or 3' noncoding region or as an initial or terminal extension. A hairpin can drive a single promoter, or two or more promoters, within a single transcribed RNA. The hairpin configurations of pollen promoters and / or polynucleotides encoding pollen disrupting polypeptides may serve to prevent transmission of the transgene through male gametes.

Pollen-specific or pollen-preferred promoters ("Poll-P") include, for example, PG47, P95 (beginning between the middle and last uninucleated stages; see SEQ ID NO: 2), and P67 (similar profile to P95, most strongly expressed mean uninucleated state, see SEQ ID NO: 1).

Mat-specific ("Tisp-P") or mat-preferred ("Tap-P") promoters include, for example, MS45 (US Patent 6,037,523); 5126 (US Patent 5,837,851); Bs7 (WO 02/063021); and SB200 (WO 02/26789).

Other tissue-preferred or tissue-specific promoters useful in the invention include, for example, Br2 (Science 302 (5642): 71-2, 2003), CesA8, and LTP2 (Plant J 6: 849-860, 1994).

Constitutive promoters ("ConstP") include, for example, the CaMV 35S promoter (WO 91/04036 and WO 84/02913); and the corn ubiquitin promoter.

Male fertile ("MF") genes useful in the invention include, for example, MS45 (Cigan et al., Sex. Plant Repro. 14: 135-142 (2001); US Patent 5,478,369) and MS26 (publication US Patent 20030182689).

Pollen suppression genes ("Cytotox") useful in the invention include DAM (GenBank J01600, Nucleic Acids Res. 11: 837-851 (1983); alpha-amylase (GenBank L25805, Plant Physiol. 105 (2): 759 -760 (1994)); D8 (Physiol. Plant. 100 (3): 550-560 (1997)); SacB (Plant Physiol. 110 (2): 355-363 (1996)), lipases and ribonucleases. Herein In this aspect, a single polypeptide or a fusion of two or more polypeptides is contemplated to generate a fusion product. Selectable marker systems useful in practicing the invention include, for example, herbicide resistance conferred by PAT or MoPAT.

Identifiable marker systems useful in the practice of the invention, for example in the identification of transgenic seeds among the offspring of a selfed maintainer line, include GFP (Gerdes (1996) FEBS Lett. 389: 44-47; Chalfie et al. (1994) Science 263: 802), RFP.DSred (Dietrichetal. (2002) Biotechniques 2 (2): 286-293), KN1 (Smith et al. (1995) Dev. Genetics 16 (4): 344-348) , CRC, P, (Bruce et al. (2000) Plant Cell 12 (1): 65-79, and Sugary1 (Rahman et al. (1998) Plant Physiol. 117: 425-435; James et al. (1995) Plant Cell 7: 417-429; U18908).

Forked configurations can comprise, for example, PG47hp, P95hp or P67 hp. A hairpin can drive a single promoter or it can drive two or more promoters via a single transcribed RNA. The hairpin can be located at any appropriate position within the construct, such as within an intron or any of the encoded genes or within 5 'or 3' non-coding regions.

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TABLE 1

<td>Symbol</td><td>Description</td><td>Example</td>

<td></td><td></td><td></td>

<td>Poll-P</td><td>Pollen promoter</td><td>PG47, P95, P67</td>

<td>Tisp-P</td><td>Tissue specific promoter</td><td>Br2, CesA8, LTP2</td>

<td>Tap-P</td><td>Mat promoter</td><td>Ms45, 5126, Bs7, Sb200</td>

<td>ConstP</td><td>Constitutive promoter</td><td>35S, Ubi</td>

<td>MF</td><td>Fertility gene</td><td>Ms45, Ms26</td>

<td>Cytotox</td><td>Cytotoxic gene</td><td>DAM, Alpha-Amylase, D8, SacB</td>

<td>HerbR</td><td>Herbicide resistance</td><td>PAT, MoPAT</td>

<td>Exploration</td><td>Identifiable marker</td><td>RFP, GFP, KN1.CRC, Su1</td>

<td>HP</td><td>Fork</td><td>PG47hp, P95hp, P67hp</td>

TABLE 2

<td>Description</td><td>Components (edit)</td>

<td></td><td></td>

<td>Cytotox only. + Selection</td><td>Poll-P: Citotox / Tap-P: MF / ConstP: Herb R</td>

<td>Cytotox only. + Selection + Exploration</td><td>Poll-P: Citotox / Tap-P: MF / ConstP: Herb R / Tisp-P: Exploration</td>

<td>Cytotox. Double + Selection</td><td>Poll-P: Citotox / Poll-P: C¡totox / TapP: MF / ConstP: Herb R</td>

<td>Cytotox only. + Exploration</td><td>Poll-P: C¡totox / Tap-P: MF / Tisp- Q: Exploration</td>

<td>Cytotox. double + scan</td><td>Poll-P: Citotox / Poll-P: C¡totox / Tap- P: MF / Tisp-P: Exploration</td>

<td>Fork + Cytotox only. + Selection</td><td>ConstP: HP / Poll-P: Citotox / TapP: MF / ConstP: HerbR</td>

<td>Fork + Cytotox only. +</td><td>ConstP: HP / Poll-P: C¡totox / Tap-P: MF / T¡sp-</td>

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<td>Exploration</td><td>Q: Exploration</td>

<td>Fork + Selection</td><td>ConstP: HP / Tap-P: MF / ConstP: Herb R</td>

<td>Fork + Scan</td><td>ConstP: HP / Tap-P: MF / Tisp-P: Scan</td>

<td>Hairpin / Male fertile fusion + Exploration</td><td>ConstP: HP + MF / Tisp-P: Scan</td>

<td>Fork / Male fertile fusion + Selection</td><td>ConstP: HP + MF / ConstP: Herb R</td>

<td>Hairpin included / Androfért¡l + Selection</td><td>ConstP: MF included HP / ConstP: Herb R</td>

<td>Hairpin Included / Androdertile + Exploration</td><td>ConstP: MF included HP / Tisp-P: Scan</td>

<td>Fork included / Scan</td><td>Tap-P: MF / ConstP: Scan included HP</td>

<td>Fork included only cytotox. / Scan</td><td>Poll-P: Citotox / Tap- Q: MF / ConstP: Scan included HP</td>

<td>Constitutive fertility / Fork scan included</td><td>ConstP: (MF + Scan) included HPTap-P: Citotox / ConstP: (MF + Scan) included HP</td>

Example 8

Selection based on visual marker

The experiments described below were designed to investigate whether the maize p1 gene could be used as a visual marker to detect the seed bearing a bound transgene, when expressed from various non-p1 promoters. As part of the experimental design, the coloration of the transformed plant seed was tested as well as the coloration of the seed generated by crossing the pollen of the transformed plant, to examine the maternal and paternal inheritance of the p1 gene.

The maize p1 gene has been shown to be a Myb-related transcriptional activator that regulates the a1 and c2 genes to produce 3-deoxy flavonoids such as C-glycosyl flavones, 3-deoxyantocyanins, flavan-4-ols, and flobaphenes (Grotewold et al. al., PNAS 88: 4587-4591 (1991)). The synthesis of these and related compounds results in the coloration of the floral organs including pericarp, ear, silks, husks and glumes of the spikes (Cocciolone et al., Plant J 27 (5): 467-478 (2001)) . Typically the expression of this gene is maternal; that is, the crossing of the p1 gene does not confer coloration to the reproductive parts until the cultivation of the seed in the next generation. As it has been shown that the p1 gene imparts color to non-reproductive tissues of maize by constitutive expression in BMS (Black Mexican Sweet) cells (Grotewold et al., PI Cell 1998), the expression of the p1 gene was investigated by placing the gene p1 under the transcriptional control of the preferred END2 and LTP2 maize seed promoters. Constitutive promoters from Actin rice and Ubiquitin corn were also used to transcriptionally regulate the p1 gene. These vectors would test whether expression of the p1 gene would confer sufficient color differences to use as a visual marker.

The following vectors were introduced into corn by Agrobacterium transformation and tested for seed color of both the transformed plant and pollen-pollinated ears of the transformed plants.

23030 End2: P1-UbimoPAT

23066 Actin: P1-UBImoPat

23069 LTP2: P1-UBImoPat

ES 2 339 559 T3

23528

End2: P1-35SPAT

23535

LTP2: P1-35S: PAT

23537

UBI: P1-35S: PAT

Transformation with PHP23030 and PHP23069 has produced plants which are shown to secrete colored seed both in the ears of the transformed primary plants and in the ears pollinated by pollen from these transformed plants. For PHP23030, 12 of the 14 independent events used for far crossing demonstrated segregation of brown grains between yellow grains at a segregation ratio of approximately 1: 1. The ears of the primary transformants were pollinated with pollen from non-transformed plants and the grains of these ears were also segregated brown: yellow in an approximate 1: 1 ratio. Identical results were observed with three of the four events generated with PHP23069.

Single copy PHP23030 5 event brown and yellow seeds were sorted and planted to test brown seed germination and co-segregation of the bound herbicide resistance marker, 35SPAT, with the colored kernels. In this small trial, the majority (&gt; 95%) of the brown seeds produced herbicide resistant plants, while 39 of the 40 germinated yellow seedlings were sensitive to herbicides.

Close examination of the brown seeds of PHP23030 revealed that the aleurone layer was fluorescent green, while the endosperm of the brown seed of PHP23069 showed intense green fluorescence when compared to the yellow segregated seed from the same ear. This is consistent with the observation of green fluorescence observed in BMS cells bombarded with 35S: P1 (Grotewold et al., Plant Cell 10 (5): 721-740 (1998)). Furthermore, examination of the PHP23528 (End2: P1-35SPAT) and PHP23535 (LTP2: P1-35S: PAT) transformed calli revealed, unlike the non-transformed GS3 calli, that both calli containing PHP23528- and PHP23535 were fluorescent green. . The observation of green fluorescence in these transformed calli and the co-segregation of brown kernels with the herbicide selectable marker in transformed plants indicate that p1 expression of at least seed-preferred promoters can be used as a visual marker to identify maize tissues. transformed.

Example 9

Alternatives for pollen cytotoxicity

As shown in Tables 1 and 2, alteration of pollen function can be achieved by any of a number of methods, including the targeted degradation of starch in the pollen grain or interference with the accumulation of starch in pollen development. . For example, a construct comprising the alpha-amylase coding region is operably linked to a pollen-specific promoter. The native secretory signal peptide region may be present, may be removed, or may be replaced by an amyloplastid-targeted signal peptide. In other embodiments, a construct may comprise a pollen-specific promoter operably linked to a coding region for beta-amylase or for a debranching enzyme such as Sugary1 (Rahman et al. (1998) Plant Physiol. 117: 425-435; James et al. (1995) Plant Cell 7: 417-429; U18908) or pullulanase (Dinges et al. (2003) Plant Cell 15 (3): 666-680; Wu et al. (2002) Archives Biochem. Biophys. 406 (1): 21-32).

For example, hairpin constructs are created that drive the promoter from the maize Sugary1 gene. Due to the loss of activity of the starch debranching enzyme, sugary1 mutants have shrunken kernels. Constitutive expression of the inverted promoter repeat can cause loss of Su1 promoter activity and result in inherited modified grain morphology.