RO120270B1 - Inducing male sterility at plants, by avidin high level expression - Google Patents

Abstract

The invention relates to inducing male sterility, at plants, by avidin high level expression. Male sterile plants may be produced by increasing avidin endogene concentration in the plant tissues. This effect may be reached by producing transgenic plants containing an expression vector, where a promotor is operationally bound to a DNA sequence encoding the avidin. There are also revealed methods for restoring male fertility.

Description

The invention relates to a method for inducing male fertility in plants, using DNA molecules that encode avidin. More specifically, this invention is directed to methods for producing transgenic, sterile male plants expressing avidin. Moreover, this invention is directed to methods for restoring male fertility to the offspring of sterile male plants.

Pollen fertility control is essential in the production of hybrid crop plants. See, for example, J. M. Poehlman, Breeding Field Crops, 3rd ed. (Van Nostrand and Reinhold, New York, NY, 1987), which is incorporated herein by reference. In hybrid maize production, for example, pollen fertility control is usually performed by removing male inflorescence or mat, before pollen spread. Manual removal of the inflorescence is extremely laborious. Although the mechanical removal of the inflorescences is less laborious than the manual removal of the inflorescences, it is less achievable and requires further examination of the crop, and possibly, the manual remediation of the removal of the inflorescences. Both methods of removing the inflorescences produce a loss of yield of female parents.

Most plants of interest, however, have both functional organs, male and female, in the same flower; therefore, neutering is not a simple procedure. Although it is possible to manually remove pollen-forming organs before the pollen spreads, this form of hybrid production is extremely laborious and costly.

Also, pollen fertility can be controlled by applying a foliar spray, which has male gametocidal properties, Cross et al., Sex Plant Reprod. 4: 235 (1991). For example, the application of etrel to anthers results in additional mitosis in pollen, wheat, degeneration of tapestry cells in barley and pollen malformed or aborted in Eragrostis, Bennetet al., Plant Cell Environ. 6:21 (1983); Berthe et al., Crop Sci. 18:35 (1978). However, the chemical approach is laborious and presents potential problems with the toxicity of the chemicals introduced into the environment.

In addition, commercial production of hybrid seed, through the use of gametocides, is limited by the cost and accessibility of the chemicals, as well as the operational safety and durability of the applications. A serious limitation of gametocides is that they have phytotoxic effects, the severity of which is genotype dependent. Other limitations include the fact that these chemicals may not be effective for crops with a long flowering period, because the newly produced flowers may not be affected. As a result, repeated application of chemicals is required.

Many current commercial hybrid seed production systems for field crops are based on a genetic means of controlling pollination. Plants that are used as females either fail to spread pollen, either produce pollen that is biochemically incapable of self-fertilization, or fail to produce pollen. Plants that are incapable of self-fertilization are called self-incompatible. The difficulties associated with using a self-incompatibility system include the accessibility and propagation of the female self-incompatible line and the stability of self-compatibility. In some situations, the auto-incompatibility can be overcome chemically or the immature flower buds can be manually pollinated before the biochemical mechanism, which blocks the pollen, is activated. Self-incompatible systems, which can be inactivated, are often very vulnerable to the stress of climatic conditions, which interrupt or reduce the efficiency of biochemical blocking for self-pollination.

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Of greater interest, for commercial seed production, are genetic systems based on pollen control that cause male sterility. These systems are of two general types: (1) male nuclear sterility, which is pollen failure, 3 due to mutations in one or more nuclear genes, or (2) genetic male sterility, commonly referred to as cytoplasmic male sterility (CMS), in that pollen formation 5 is blocked or fails due to an alteration in a cytoplasmic organelle, which is generally mitochondria. 7

Nuclear sterility can be either dominant or recessive. Dominant sterility can be used for hybrid seed formation only if vegetative or clonal propagation of the female line is possible. Recessive sterility can be used if the sterile and fertile plants are easily separated. The commercial utility of dominant and recessive sterility systems 11 is limited, however, by the cost of clonal propagation and, respectively, the female rows of self-fertilizing plants. 13

Although there are hybridization schemes that involve the use of CMS, there are limitations to its commercial value. An example of a CMS system is a specific mutation 15 in cytoplasmic localized mitochondria, which may, when in its own nuclear background, lead to failure of mature pollen formation, in some situations, the nuclear fund may compensate for 17 cytoplasmic mutation and normal formation occurs. of pollen. Restorative genes, nuclear specific, allow pollen formation in plants with CMS mitochondria. In general, the use of CMS 19 for commercial seed production involves the use of three breeding lines: a sterile male line (parental female), a holding line, which is isogenic to the sterile male line 21, but contains fully functional mitochondria, and a male parent line.

The male parent line may or may not contain specific restoring genes in the cytoplasm.

For crops such as vegetables, for which hybrid seed recovery is 25 without significance, a CMS system can be used without restoration. For crop plants for which the fruit or seed of the hybrid is the commercial product, the fertility of the hybrid seed must be restored by specific restorative genes in the male parent, or the sterile male hybrid must be pollinated. The pollination of unrestored hybrids can be achieved by including a small percentage of male fertile plants for pollination. In most species, the CMS character is inherited from the maternal line, because all 31 cytoplasmic organs are inherited only from the egg cell.

CMS systems have limitations, which may make it impossible to use them as a unique solution 33 to produce sterile male plants. For example, a particular type of CMS in maize (T-cytoplasm) confers sensitivity to the toxin produced by infection with a particular fungus. Although 35 still used for many cultivated plants, CMS systems can be interrupted under certain environmental conditions. 37

The technical problem, which the invention solves, is to provide a means of control for pollen, other than manual, mechanical, chemical, or by traditional genetic methods, but by a method of producing sterile male plants through -a heterologous expression of avidin and by providing a chimeric gene comprising a DNA sequence encoding 41 avidin, which is operatively linked to a plant promoter sequence.

Induction of male sterility in plants by the expression of high levels of avidin, 43 according to the invention, solves this problem and removes the disadvantages above, in that it comprises an isolated DNA molecule, comprising a nucleotide sequence, which co-difficult avidin. operably linked to an anther-specific promoter sequence, wherein said promoter sequence directs the avidin gene transcription into the tissue of the anthers of the plant, an expression vector 47, comprising the isolated DNA molecule, vectordeexpression, wherein said sequence

EN 120270 Β1 anther specific promoter comprises an anterior box selected from the group consisting of:

(a) a DNA molecule having the nucleotide sequence of SEQ ID NO 1;

(b) a DNA molecule having the nucleotide sequence of SEQ ID NO: 2;

(c) a DNA molecule having the nucleotide sequence of SEQ ID NO: 3; and (d) a functional fragment of (a), (b) or (c), wherein said anther-specific promoter further comprises a core promoter selected from the group consisting of;

(a) 35S CaMV core promoter;

(b) a DNA molecule having the nucleotide sequence of SEQ ID NO: 4;

(c) a DNA molecule having the nucleotide sequence of SEQ ID NO: 5;

(d) a DNA molecule having the nucleotide sequence of SEQ ID NO: 6 and (e) a fragment of (b), (c) or (d), a vector further comprising a Lex A gene operator sequence, a transgenic plant comprising said DNA molecule, transgenic plant in which said plant is a maize plant, transgenic plant, wherein said anther-specific promoter sequence comprises an anterior box selected from the group consisting of:

(a) a DNA molecule having the nucleotide sequence of SEQ ID NO: 1;

(b) a DNA molecule having the nucleotide sequence of SEQ ID NO: 2;

(c) a DNA molecule having the nucleotide sequence of SEQ ID NO: 3 and (d) a fragment of (a), (b) or (c), a transgenic plant wherein said anther-specific promoter further comprises a core promoter selected from the group consisting of:

(a) 35S CaMV core promoter;

(b) a DNA molecule having the nucleotide sequence of SEQ ID NO: 4;

(c) a DNA molecule having the nucleotide sequence of SEQ ID NO: 5;

(d) a DNA molecule having the nucleotide sequence of SEQ ID NO: 6; and (e) a fragment of (b), (c) or (d), a transgenic plant comprising a nucleotide sequence encoding avidin operably linked to a promoter and a Lex A operator, a method of producing an androsteric transgenic plant, consisting of introducing into the plant cells an expression vector comprising a nucleotide sequence encoding operably linked avidin to a tissue-specific promoter, wherein said promoter controls the expression of said sequence encoding avidin; and in regenerating a transgenic plant from said plant cells, wherein the expression of said sequence encoding avidin in the plant causes androsterility, a method in which said tissue-specific promoter sequence is an anther-specific promoter, a method in which said anther-specific promoter comprises an earlier box selected from the group consisting of:

(a) a DNA molecule having the nucleotide sequence of SEQ ID NO: 1;

(b) a DNA molecule having the nucleotide sequence of SEQ ID NO: 2;

(c) a DNA molecule having the nucleotide sequence of SEQ ID NO: 3; and (d) a fragment of (a), (b) or (c), wherein said anther-specific promoter further comprises a core promoter selected from a group consisting of:

(a) 35S CaMV core promoter;

(b) a DNA molecule having the nucleotide sequence of SEQ ID NO: 4;

(c) a DNA molecule having the nucleotide sequence of SEQ ID NO: 5;

(d) a DNA molecule having the nucleotide sequence of SEQ ID NO: 6; and (e) a fragment of (b), (c) or (d), a method of using an avidin gene for the production of an androfert hybrid plant, comprising the steps:

(a) the production of a first androsterin parent plant, comprising said DNA molecule, acting as a first foreign gene, in which avidin expression causes androsterility;

(B) the production of a second transgenic parental plant expressing a second foreign gene 1; and (c) cross-fertilization of said first parent with said second parent, for the production of a hybrid plant, wherein said hybrid plant expresses said second foreign gene and wherein the product of said second foreign gene reduces expression 5 of avidin in said hybrid plant, thereby producing an androfertile hybrid plant, method in which said second foreign gene is selected from the group consisting of a 7 antisense gene, a ribozyme gene and an external guide sequence gene, method in which molecule of

Said DNA of said first parent plant further comprises a LexA operator 9, which is operatively linked to the DNA molecule promoter and wherein said second foreign gene encodes the LexA repressor, a method for restoring androfertility in a plant 11 comprising applying on the leaves of said plant a biotin solution, an isolated DNA molecule, wherein said nucleotide sequence encoding avidin comprises 13 the DNA fragment of pPHI5168 extending from the restriction site Mscl, immediately above the 5 'end of said nucleotide sequence. encoding avidin up to the Hindll restriction site at the 3 'end of said nucleotide sequence encoding avidin, expression vector comprising the isolated DNA molecule, transgenic plant in which said non-cleotidic sequence encoding avidin, comprises the DNA fragment of pPHI5168, which extends from and the restriction tul Mscl, immediately above the 5 'end of said nucleotide sequence 19 encoding avidin, to the Hindll restriction site at the 3' end of said nucleotide sequence encoding avidin, a method in which said nucleotide sequence 21 encodes avidin, , comprises the DNA fragment of pPHI5168, which extends from the restriction site Mscl, immediately above the 5 'end of said nucleotide sequence encoding avidin, to the Hindll restriction site at the 3' end of said nucleotide sequence encoding avidin, method wherein said DNA molecule, which encodes 25 avidin, comprises the DNA fragment of pPHI5168, which extends from the Mscl restriction site, immediately above the 5 'end of said nucleotide sequence encoding avidin, 27 to the Hindll restriction site at the end 3 'of said nucleotide sequence encoding avidin, ve expression vector, wherein said expression vector is pPHI5168. 29 below, the figures accompanying the invention are briefly presented.

FIG. 1 shows plasmid PHI5168, which encodes avidin; 31

FIG. 2 shows plasmid PHI610, which encodes the bar gene.

1. Definitions.33 In the following description, a number of terms have been used extensively. The following definitions are given to facilitate understanding of the invention.35

A structural gene is a DNA sequence that is transcribed into messenger RNA (mRNA), which is then translated into an amino acid sequence characteristic of a specific polypeptide 37.

A foreign gene refers, in the present description, to a DNA sequence that is functionally linked to at least one heterologous regulatory element. For example, any gene, other than the structural gene 5126 from maize, is considered to be a foreign gene, if expression 41 of that gene is controlled by a regulatory element specific to the anthers, of gene 5126.

A promoter is a DNA sequence that directs the transcription of a gene, such as a structural gene, an antisense gene, a ribozyme gene, or an external guide sequence gene. Usually, a promoter is located in the 5 'region of a gene, proximal to the transcriptional start site 45. If a promoter is a promoter that can be induced, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent, if the promoter is a constitutive promoter. A plant promoter is a promoter sequence that will direct the transcription of a gene into a plant tissue. 49

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A core promoter contains nucleotide sequences essential for promoter functioning, including the TATA box and transcription start. By this definition, a core promoter may or may not have detectable activity in the absence of the specific sequences that may intensify the activity or confer tissue-specific activity. For example, the SGB6 core promoter consists of approximately 38 nucleotides toward the 5'-µl transcriptional start site of the SGB6 gene, while the 35S core promoter of the Cauliflower Mosaic Virus (CaMV) consists of approximately 33 nucleotides to the 5'-µl start site. 35S genome transcriptome.

A tissue-specific promoter is a DNA sequence that, when functionally linked to a gene, directs a higher level of transcription of that gene into a specific tissue than some or all other tissues in the body. For example, an anthers-specific promoter is a DNA sequence, which directs a higher level of transcription of an associated gene into the tissue of the anthers of the plant. The promoter specific for SGB6 anthers, for example, may direct the expression of a foreign gene in the tissue of the anthers, but not in the tissue of the root or beetle.

As used herein, an anthers-specific promoter comprises 2 functional elements, the anthers box and a core promoter. A particular anthers box may have the functional characteristics of a classic intensifier. The combination of an anther box and a core promoter can stimulate gene expression to a larger size than a single core promoter. This is true even for a chimera-specific chimeric promoter containing an anterior box and a core promoter derived from different genes. Such chimeric regulatory elements, specific to the anthers, are described below.

An operator is a DNA molecule that is located at 5'i of a gene and contains a nucleotide sequence that is recognized and linked to a repressor protein. Binding of a repressor protein to its related operator results in inhibition of gene transcription. For example, the LexA gene encodes a repressor protein that binds to the LexA operator.

An isolated DNA molecule is a fragment of DNA that has been separated from the DNA of an organism. For example, a cloned DNA molecule encoding an avidin gene is an isolated DNA molecule. Another example of an isolated DNA molecule is a chemically synthesized DNA molecule or enzyme-produced cDNA that is not integrated into the genomic DNA of an organism.

An enhancer is a DNA regulatory element, which can increase transcription efficiency.

Complementary DNA (cDNA) is a single-stranded DNA molecule, which is formed from a mRNA template by the reverse transcriptase enzyme. Usually, a complementary primer is used on portions of the mRNA to initiate reverse transcription. For those skilled in the art, the use of the term cDNA also refers to a double-stranded DNA molecule, consisting of such a single-stranded DNA molecule and its complementary DNA strand.

The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a structural gene, the expression involves the transcription of a structural gene into the mRNA and the translation of the mRNA into one or more polypeptides.

A cloning vector is a DNA molecule, such as a plasmid, cosmid, or bacteriophage, which has the ability of autonomous replication in a host cell. Typically, cloning vectors contain one or a small number of restriction endonuclease recognition sites, at which foreign DNA sequences can be inserted in a determinable manner, without losing essential vector biological function, as well as a marker gene. which is suitable for use in identifying and selecting cells transformed with donor vectors. Typically, marker genes include genes that provide tetracycline resistance or ampicillin resistance.

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An expression vector is a DNA molecule, comprising a gene that is expressed 1 in a host cell. Typically, the expression gene is placed under the control of certain regulatory elements, including constitutive or induced promoters, tissue-specific regulatory elements and enhancers. Such a gene is called to be operatively linked to regulatory elements. 5

A recombinant host can be any prokaryotic or eukaryotic cell, which contains either a cloning vector or an expression vector. Also, these terms include 7 those prokaryotic or eukaryotic cells, which have been processed by genetic engineering to contain the gene (s) cloned (s) in the chromosome or genome of the host cell. 9

A transgenic plant is a plant that has one or more plant cells that contain a foreign gene. In eukaryotes, RNA polymerase II catalyzes the transcription of a structural gene to produce mRNA. A DNA molecule may be designated to contain an RNA polymerase 13 II template, wherein the RNA transcript has a sequence that is complementary to that of a specific mRNA. The RNA transcript is called an antisense RNA and a DNA sequence encoding 15 antisense RNA is called an antisense gene. Antisense RNA molecules are capable of binding to mRNA molecules, resulting in inhibition of mRNA translation. 17

A ribozyme is an RNA molecule that contains a catalytic center. The term includes RNA enzymes, self-assembly RNAs and self-cleaving RNAs. A DNA sequence that encodes a ribozyme is called a ribozyme gene.

An external guide sequence is an RNA molecule that directs endogenous ribozyme, 21 RNase P, to a particular mRNA species, which results in mRNA cleavage by RNase P. A DNA sequence that encodes an external guide sequence, is called a guide gene sequence. external.

2. Overview. 25

The present invention provides methods for generating male sterile plants by preparing transgenic plants expressing avidin. Avidin may be constitutively expressed, 27 in a non-tissue-specific manner, or may be expressed specifically in the anthers. Also, methods for restoring male fertility are ensured.29

An avidin gene is isolated and inserted into an expression vector suitable for introduction into a plant tissue. The expression vector further comprises a promoter that may be a constitutive promoter, a non-tissue-specific promoter, such as the ubiquitin promoter, a tissue-specific promoter, such as an anthers-specific promoter, 33, or a promoter that can be induced. The expression vector is introduced into plant tissue by standard methods and the transgenic plants are selected and propagated. Transgenic plants, which express avidin, are male sterile. Male fertility can be restored by coexpression, in transgenic plants, of a second gene that inhibits avidin 37 gene transcription or inhibits avidin mRNA translation. Alternatively, male fertility can be restored by spraying developing plants with biotin solutions. 39

3. Isolation of DNA molecules encoding avidin.

The nucleotide sequences, which encode avidin, can be isolated by known methods. For example, a cDNA encoding avidin of egg white from hens may be isolated from a cDNA bank in the hen's oviduct, by the methods described by Gope et al., Nucleic Acids Res., 43

15: 3595 (1987), which is incorporated herein by reference. Alternatively, genomic clones of the avidin gene can be obtained from genomic DNA of organisms known to produce 45 avidin. For example, a chicken avidin gene can be cloned from chicken genomic DNA by the methods described in Keinanen et al., Eur. J. Biochem. 220: 615 (1994). 47

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Also, avidin genes can be isolated using polymerase chain reaction (PCR), using standard methods, see, Erlich, PCR Technology: Principles and Applications for DNA Amplification (Stockton Press, NY, 1989) and Innis et al., PCR Protocols : a Guide to Methods and Applications (Academic Press, San Diego, 1990). Methods for PCR amplification of long nucleotide sequences such as the ELONGASE ™ system (LifeTechnologies, Inc., Gaithersburg, MD) can also be used to obtain longer nucleotide sequences, such as genomic clones encoding avidin. PCR primers, complementary to the 5 'and 3' terminals of known avidin gene sequences, can be synthesized, using commercial synthesizers for oligonucleotides, such as those provided by Applied Biosystems (Foster City, CA). In a preferred embodiment, the primers include additional nucleotide sequences, which contain cleavage sites of the restriction endonuclease. The presence of such sites allows direct cloning of PCR products into appropriate cloning vectors, after treatment with an appropriate restriction enzyme. See, Finney, Molecular Cloning of PCR Products in Current Protocols in MolecularBiology, Ausubel et al., Editors (John Wiley & Sons, New York, 1987) p. 15.7.1.

The DNA template, for PCR, can be either cDNA or genomic DNA and can be prepared from a suitable organism, using methods well known in the art, Sambrook et al., Molecular Cloning: a Laboratory Manual, second edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NI 1989).

Genomic DNA can be prepared directly from avian, reptilian or amphibian tissue using commercial reagents such as Triazol ™ (Life Technologies, Inc., Gaithersburs, MD). Alternatively, avidin-encoding cDNA can be prepared using mRNA extracted from chicken oviduct tissue, using a commercially available kit (Pharmacia, Piscataway, NJ). The preparation of mRNA is used as a template for cDNA synthesis, using poly (dT) or hexameric primers by standard techniques, see, Sambrook et al., Supra. Then, cDNA synthesis is performed using a commercially available kit (Pharmacia) and the cDNA is used directly for PCR, using the method of Saiki et al., Science, 239: 487 (1988).

Genomic DNA or cDNA fragments isolated by the methods described above may be inserted into a vector, such as a bacteriophage or cosmid vector, according to conventional techniques, such as the use of restriction enzyme digestion, to provide appropriate terminals, use of alkaline phosphatase treatment to avoid unwanted DNA molecule binding and ligation with appropriate ligands. Techniques for manipulating such vectors are described by Ausubel et al., (Editors), Current Protocols in Molecular Biology, p.3.0.5.-3.17.5 (1990) [Ausubel].

Alternatively, nucleotide sequences encoding avidin may be obtained by synthesizing avidin-encoding DNA molecules using long, mutually initiated oligonucleotides, see, for example, Ausubel on p. 8.2.8. at 8.2.13, see also Wosnick et al., Gene, 60: 115 (1987). Current techniques, which use the polymerase chain reaction, provide the ability to synthesize 1.8-kilobase genes, Adang et al., Plant Molec. Biol. 21: 1131 (1993); Bambotetal., PCR Methods and Applications 2: 266 (1993). The nucleotide sequence of a synthesized avidin gene can be based on any known DNA molecule, which encodes avidin, see, for example, Gope et al., Supra.

These clones can be analyzed, using a variety of standard techniques, such as restriction analysis, Southern analysis, primer extension analysis, and DNA sequence analysis. The primer extension assay or nuclear protection assay Sl, for example, can be used to locate the likely start site of the donated gene transcript, Ausubel on pages 4.8.1 -4.8.1; Walmsley et al., Quantitative and Qualitative Analysis of Exogenous Gene Expression by the Sl Nuclease Protection Assay, in Methods in MolecularBiology, vol. 7: Gene Transferring Expression Protocols, Murray (editors) pp. 271-281 (Humana Press Inc. 1991).

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4. Avidin gene donation in an expression vector and preparation of 1 transgenic plants.

Once an avidin gene has been isolated, it is placed in an expression vector by 3 standard methods, see, Sambrook et al., Supra. The selection of a suitable expression vector will depend on the method of introducing the expression vector into the host cells. Typically, an expression vector contains: (1) prokaryotic DNA elements, which code for a bacterial replication origin and an antibiotic resistance gene, to ensure growth and selection of the expression vector in the bacterial host; (2) a cloning site for insertion of an exogenous DNA sequence, for example, an avidin gene; (3) eukaryotic DNA elements that control the initiation of exogenous gene transcription, such as a promoter; (4) DNA elements that control the processing of transcripts, such as a transcription termination / polyadenylation sequence; and (5) a gene encoding a marker protein (for example, a reporter gene), wherein the gene is operatively linked to DNA elements that control transcription initiation. 13 General descriptions of plant expression vectors and reporter genes can be found in Gruber et al., Vectors for Plant Transformation in Methods in Plant Molecular Biology and 15 Biotechology, Glick et al., (Pp. 89-119 (CRC Press, 1993).

In a preferred embodiment of the present invention, a plant location promoter system is used. Plant ubiquitin promoters are well known in the art, see, for example, EPAO 342 926, which is incorporated herein by reference. The ubiquitin promoter is a constitutive motor.

In another preferred embodiment of the invention, a inducible promoter is used, 21 to allow induced regulation of avidin expression. An example of such inducible promoter is the glutathione S-transferase system in maize, see, Viegand et al., Plant 23

Mol. Biol. 7: 235 (1986). Also, this method allows reversible induction of male sterility. Thus, avidin expression and, at the same time, male sterility can be controlled as desired by activating the promoter. When the promoter is not activated, avidin is not expressed and the plants are male sterile. 27

Expression vectors, which contain an avidin gene, can be introduced into protoplasts or intact tissues, such as immature embryos and meristems, or into callus cultures or isolated cells. Preferably, expression vectors are introduced into intact tissues. General methods of growing plant tissues are provided, for example, by Miki et al., Procedures for 31 Introducing Foreign DNA into Plants in Methods in Plant Molecular Biology and Biotechnology, Glick et al., (Editors), p. 67- 88 (CRC Press, 1993) and by Phillips et al., 33

Cell / Tissue Culture and in Vitro Manipulation in Corn and Corn Improvement, 3rd edition, Sprague et al., (Editors) pp. 345-387 (American Society of Agronomy, Inc. et al., 1988). 35

Methods of introducing expression vectors into plant tissue include direct infection or co-cultivation of plant tissue with Agrobacterium tumefaciens, Horsch et al., 37 Science 227 1229 (1985). Preferably, a disassembled Ti plasmid is used as a vector for foreign DNA sequences. The transformation can be accomplished according to the procedures described, for example, in EPA Patents116 718 (1984) and 270822 (1988). Preferred Ti plasmid vectors contain the foreign DNA sequence between the border sequences or at least located upstream 41 of the right border sequence.

Other types of vectors can be used to transform plant cells, using 43 procedures, such as direct gene transfer (see, for example, PCT application WO 85/01856 and European application 275 069), protoplast transformation in vitro (de for example, patent 45 US 4684 611), virus-mediated plant transformation (e.g., European application 067553 and US patent 4407 956) and liposome-mediated transformation (e.g. 47

RO 120270 Β1 US Patent 4536475). Appropriate methods for corn processing are provided by Fromm et al., Bio / Techology 8: 833 (1990) and by Gordon-Kamm et al., The Plant Cell 2: 603 (1990). Standard methods for rice processing are described by Christou et al., Trends in Biotechnology 10: 239 (1992) and by Lee et al., Proc. Nat'l Acad. Sci. USA 88: 6389 (1991). Wheat can be transformed, using methods that are similar to corn or rice processing. Moreover, Casas et al., Proc. Nat'l Acad. Sci. US 90: 11212 (1993), describes a method for sorghum transformation, while Wan et al., Plant Physiol. 104: 37 (1994) describe a method for processing barley.

In general, direct transfer methods are preferred for processing a monocotyledonous plant, in particular a cereal, such as rice, corn, sorghum, barley or wheat. Suitable direct transfer methods include microproject mediated release, DNA injection, electroporation, and the like, see, for example, Gruber et al., Supra, Miki et al., Supra, and Klein et al., Bio / Technology 10: 268 ( 1992). More preferably, expression vectors are introduced into the tissues of a monocotyledonous plant, using micro-projectile-mediated release with a bio-ballistic device.

5. Adjusting male fertility.

A. Production of sterile male plants.

In order to induce male sterility, according to the present invention, an expression vector is constructed, in which a DNA sequence, encoding avidin, is functionally linked to DNA sequences that regulate gene transcription in a plant tissue. The general requirements of an expression vector are described above. In order to achieve male sterility through the expression of avidin, it is preferable that avidin is not expressed only transiently, but that the expression vector is introduced into the embryonic tissue of the plant, in such a way that the avidin is expressed at last. stage of development in the adult plant. For example, mitotic stability can be achieved using plant viral vectors that ensure epichromosomal replication.

A preferred method of achieving mitotic stability is provided by integrating expression vector sequences into the host chromosome. Such mitotic stability can be ensured by microproject release of an expression vector to plant tissue or by using other standard methods, as described above, see, for example, Fromm et al., Bio / Technology 8: 833 (1990), Gordo-Kamm et al., The Plant Cell 2: 603 (1990), and Walters et al., Plant Molec. Biol. 18: 189 (1992).

Avidin gene transcription, after introduction into plant tissue, is preferably controlled by a constitutive promoter that nonspecifically stimulates gene expression in plant tissue. An example of such a constitutive promoter, suitable for this purpose, is the ubiquitin promoter, as described above.

Avidin gene transcription can also be controlled by a promoter that stimulates gene expression in a tissue-specific manner. A particularly preferred anther promoter is promoter 5126 which has been isolated from the B73 maize line resulting from selective crossings. Promoter 5126 stimulates the expression of a foreign gene in the quartet for the early uninucleated microspore stage in the anthers.

Another suitable promoter, specific for anthers, is the SGB6 promoter, which has also been isolated from line B73. The SGB6 promoter can induce the expression of a foreign gene in the tapestry cells of the anthers, from the quartet stage, to the uninucleated mid-stage of microspore development.

Alternatively, the expression of the anthers-specific gene can be ensured, using a combination of an anterior box and a core promoter. A particularly preferred antigen box is an antigen box 5126, which comprises the following nucleotide sequence:

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5 'CGGCCGCGG

ATCCGCTCAT

CTGCTCGGTA CCAACCAGCC 1

CGCCGGCGCC

TAGGCGAGTA

GACGAGCCAT GGTTGGTCGG

TTTCCTATTA AAAGGATAAT

CCATGCACAG ATCT 3 '[SEQ ID NO: 1],

GGTACGTGTC TAGA5

Another suitable anterior box is located in a 94-base7 pair DNA fragment, defined by nucleotides 583 to 490, upstream of the SGB6 transcription start site. The nucleotide sequence of the 94-base SGB6 anthers box is: 9

(-583) ACAGTTCACT AGATATGCAT GATCTTTAAC AATTGCTGCT 11 TGTCAAGTGA TCTATACGTA CTAGAAATTG TTAACGACGA GGATTGTGCG GTTTCTTTTG GCACAAATGG CATGAACAGA 13 CCTAACACGC CAAAGAAAAC CGTGTTTACC GTACTTGTCT GTAATCCGGG ACGC (-490) [SEQ ID NO: 2], IO CATTAGGCCC TGCG 17

Alternatively, a suitable anterior box is obtained from the G9 promoter from the pituitary, which stimulates gene expression, during meiosis, at the quartet stages of development. The anterior box G9 comprises the following nucleotide sequence: 21

5 'GCGGCCGCGG ATCCTGGCTG GATGAAACCG ATGCGAGAGA 23 CGCCGGCGCC TAGGACCGAC CTACTTTGGC TACGCTCTCT

AGAAAAAAAA ATTGTTGCAT GTAGTTGGCG CCTGTCACCC TCTTTTTTTT T AACAACGTA CATCAACCGC GGACAGTGGG AACCAAACCA GTAGTTGAGG CACGCCCTGT TTGCTCACGA TTGGTTTGGT CATCAACTCC GTGCGGGACA AACGAGTGCT TCACGAACGT AGATCT 3 '[SEQ ID NO: 3], AGTGCTTGCA TCTAGA

The anterior cases 5126, SGB and G9 can be obtained by synthesizing oligonucleotides, 35 as described above. Those skilled in the art will appreciate that deletion analysis can be performed to locate one or more regulatory sequences specific to the supplementary anthers from the above-described boxes. Such functional fragments of the anterior boxes may also be used to regulate avidin gene expression, in a manner specific to the anthers.

Preferred core promoters are derived from core promoter 5126, core promoter 41 SGB6, core promoter G9 and core promoter 35S of Cauliflower Mosaic virus.

A particularly preferred core promoter is the core promoter 5126, which comprises the following nucleotide sequence:

5 'AGATCTAAGT AAGGTATATA TGTGCATAGT CTCCTAATTC

TCTAGATTCA TTCCATATAT ACACGTATCA GAGGATTAAG 47

RO 120270 Β1

TTCATCTTCA AAGTAGAAGT

CACTCTTTCC GTGAGAAAGG

ACCTCTAGCT TGGAGATCGA

TTCCTTCCTT AAGGAAGGAA

GATTGATCTC CTAACTAGAG

CCTTCAATTC GGAAGTTAAG

TGGTATTTAC ACCATAAATG

TAAATACCAC ATTTATGGTG

AAATCAAAGT TTTAGTTTCA

TGCTTTGCCA ACGAAACGGT AC

TG 3 '[SEQ ID NO: 4]

The SGB6 core promoter consists of about 38 nucleotides upstream of the transcriptional site of the SGB6 gene. An SGB6 core promoter has the following nucleotide sequence:

5 'ATCTCACCCT

ATTAATACCA

TAGAGTGGGA

TAATTATGGT

TGCTGACGAG ACGACTGCTC

CCAATAGC 3 '[SEQ ID NO: 5]

GGTTATCG

A suitable core promoter, derived from the G9 gene, comprises the nucleotide sequence:

AGATCTCTAT AAAACACGCA GGGACTGGAA AGCGAGATTT TCTAGAGATA TTTTGTGCGT CCCTGACCTT TCGCTCTAAA CACAGCTGAA AGCAGCCAAA ACGCAGAAGC TGCACTGCAT GTGTCGACTT TCGTCGGTTT TGCGTCTTCG ACGTGACGTA ACATCGAGCT AACTATCTGC Hook 3 ' [SEQ ID NO: 6J. TGTAGCTCGA TTGATAGACG TCGGTAC

Also, the appropriate core promoters are provided with functional fragments of the core promoters 5126, SGB6 and G9. Methods for obtaining such functional fragments are described above.

The window of development of avidin gene expression can be expanded, using a chimeric regulatory element, comprising an anterior box from one gene and one specific promoter from one second gene. For example, SGB6 regulatory sequences stimulate quartet gene expression, at the middle uninucleate stages of development, while G9 regulatory sequences stimulate gene expression, during meiosis and during the quartet developmental stage. yet another combination of a SGB6 anterior box and a G9 promoter stimulates the transcription of a foreign gene during meiosis and during the mid-uninucleated stage of development. Thus, various combinations of the anther boxes and anthers-specific promoters are particularly useful for the present invention.

Also, a viral core promoter can be used.

Examples of suitable viral core promoters include a core promoter of Cauliflower Mosaic Virus (CaMV), a core promoter of the Mosaic Virus and other Gruber et al., Gruber et al., Supra. Preferably, the viral core promoter is the 35S CaMV promoter or a variant thereof.

For the purpose of selecting transformed cells, the expression vector contains a selectable marker gene 1, such as a herbicidal resistance gene. For example, such genes may confer resistance to phosphinothricin, glyphosate, sulfonylureas, atrazine or imidazolinone. Preferably, the 3 selectable marker gene is the bar gene or the bed gene, which encodes phosphinothricin acetyltransferase. The nucleotide sequences of the genes can be found in Leemans etal., EPA 0-242-5

246 (1987), and in White et al., Nucleic Acids Res. 18: 1062 (1990). Wohlleben et al., Gene 70:25 (1988), describes the nucleotide sequence of the pat gene. The expression of the barsau bed gene confers 7 resistance to herbicides, such as glucosinate (sold as Basta® and Ignite®, among others) and bialaphos (sold as Herbi-ace® and Liberty®). 9

The expression vector may contain DNA sequences encoding avidin under the control of a constitutive promoter or anther specific promoter, as well as the selectable marker gene 11 under the control of a constitutive promoter. Alternatively, the selectable marker gene may be released to the host cells in a selection expression vector separated by cotransformation of the embryonic tissue.

B. Restoration of male fertility in hybrid FI. 15

The methods described above can be used for the production of male sterile transgenic plants for the production of FI hybrids in large scale crossings between 17 lines obtained by selective crossings. If all egg cells of sterile male transgenic plants do not contain the recombinant avidin gene, then a proportion of Fi hybrids will have a fertile male phenotype. On the other hand, FI hybrids will have a sterile male phenotype, if the recombinant avidin gene is present in all egg cells of the sterile 21 male transgenic plants, because ethylene-induced sterility will be dominant. Thus, it may be desired to use a male fertility restoration system, to ensure the production of 23 male fertile FI hybrids. Such a system of fertility restoration has a special value in autogamous species, where the harvested product is the seed. 25

The common approach, proposed for the restoration of fertility, to a line of male sterile transgenic plants, requires the production of a second restorative line, of transgenic plants. For example, a transgenic plant, which is sterile male, due to the expression of barnase, will be crossed with a fertile male plant, which expresses barnase inhibitor, as discussed above 29, Mariani et al., Nature 357: 384 (1992).

In the present case, an analogous approach will require the production of a restorative line 31 of transgenic plants expressing ribozymes targeting avidin mRNA or expressing avidin antisense. For example, ribozymes may be designated for expressing endocrine activity, which is directed to a certain target sequence in a mRNA molecule. For example, Steinecke et al., EMBO J. 11: 1525 (1992), achieved up to 100% inhibition of neomycin phosphotransferase gene expression by ribozymes in maize protoplasts. More recently, Perriman et al., Antisense Res. & I must. 3: 253 (1993), inhibited the activity of chloramide-37 phenolic acetyl transferase in tobacco protoplasts, using a vector expressing a modified hammerhead ribozyme. In the context of the present invention, avidin mRNA provides 39 target RNA molecules corresponding to ribozymes.

In a similar approach, fertility can be restored by using a 41 expression vector containing a nucleotide sequence encoding an antisense RNA. Binding of antisense RNA molecules to mRNA target molecules results in termination of translational hybridization, 43

Paterson et al., Proc. Natl. Acad. Sci. USA, 74: 4370 (1987). In the context of the present invention, a suitable antisense RNA molecule will have a sequence that is complementary to 45 avidin mRNA. In a preferred embodiment of the invention, antisense RNA is under the control of a promoter that can be induced. Activation of this promoter then allows the restoration of 47 male fertility.

In an alternative alternative approach, vectors can be constructed, in which an expression vector encodes RNA transcripts capable of initiating PN-mediated cleavage of avidin mRNA molecules. According to this approach, an external guide sequence for targeting endogenous ribozyme, RNase P, to avidin mRNA can be constructed, which is further cleaved by cellular ribozyme, Altman et al., US Patent 5168053; Yuan et al., Science 263: 1269 (1994). Preferably, the external guide sequence comprises a sequence of 10 to 15 nucleotides complementary to avidin mRNA and a 3'-NCCA nucleotide sequence, wherein N is preferably a purine .Id. External guide sequence transcripts bind to target mRNA species by forming base pairs between mRNA and complementary external guide sequences, thus initiating mRNA cleavage by RNase P at the nucleotide located at the 5 'part of the paired base region. Id.

In an alternative method, for restoring male fertility, sterile male transgenic plants contain an expression vector that, in addition to a promoter sequence operably linked to an avidin gene, also contains a prokaryotic regulatory element. Male fertile transgenic plants are produced, which express a prokaryotic polypeptide under the control of the promoter. In FI hybrids, the prokaryotic polypeptide binds to the prokaryotic regulatory sequence and represses avidin expression.

For example, the LexA gene system / LexA operator can be used to regulate gene expression according to the present invention, see US Patent 4833080 (Ό80 patent) and Wang et al., Mol. Cell. Biol. 13: 1805 (1993). Specifically, the sterile male plant expression vector will be able to contain the LexA operator sequence, while the fertile male plant expression vector will contain the coding sequences of the LexA repressor. In the FI hybrid, the LexA repressor will bind to the LexA operator sequence and inhibit avidin gene transcription.

LexA operator DNA molecules can be obtained, for example, by synthesizing DNA fragments containing the well-known sequence of LexA operator, see, for example, Patent Ό80 and Garriga et al., Moi. Gender. Genet. 236: 125 (1992). The LexA gene can be obtained by synthesizing a DNA molecule that encodes the LexA repressor. Gene synthesis techniques are discussed above and LexA gene sequences are described, for example, by Garriga et al., Supra. Alternatively, DNA molecules encoding the LexA repressor can be obtained from plasmid pRB500, ATCC, accession number 67758.

And another method for restoring fertility uses high affinity for avidin for biotin, by spraying developing plants with a biotin solution. Also, the biotin solution comprises a minimum amount of an organic cosolvent, such as DMSO, to ensure complete solubility of the biotin. Spraying begins during the meiotic phase of pollen development and is repeated at regular intervals until pollen spread is observed. The intervals between sprays will vary from 1 to 7 days in a preferred embodiment of the invention, the spraying being repeated every 3 to 5 days.

Those skilled in the art can easily develop other strategies for restoring male fertility, using prokaryotic regulatory systems, such as the repressor / ac / lac operon system or the trp / operon trp repressor system.

The present invention, thus generally described, will be more easily understood, by reference to the following examples, which are given for illustration and are not intended to be limiting to the present invention.

Example 1. Preparation of male sterile transgenic maize by increased constitutive expression of avidin.

A method for forming transgenic maize plants has been described in EPA 0442174A1, which is incorporated herein by reference. Following is a brief description of this methodology.

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PHI5168 was used, a vector containing the avidin gene under the control of the ubiquitin promoter 1 and which also contains a PINII terminator sequence for the formation of transgenic maize plants. The structure of PHI5168 is shown in FIG. 1. A PHI610 expression vector 3, which contains the barsub gene, the control of the 35S double promoter and which also contains a PINII terminator sequence, was transformed together with the 5 avidin construct, allowing the selection of transgenic plants by the treatment of the bialophos transformation mixture. The structure of PHI610 is shown in FIG. 2. The two expression vectors 7 were transformed into suspension embryogenic cultures, derived from type II embryogenic culture, according to the method of Green et al., Molecular Genetics of Plants and Animals, 9 editors Downey et al., Academic Press, NY, 20,147 (1983). Cultures were maintained in Murashige and Skoog (MS) media, as described in Murashige et al., Physio. Plant, you guys. 15; pp. 11 453-497; (1962), supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2 mg / l and sucrose at 30 g / l. The suspension cultures were filtered through a 710 μm screen, 7 days before the experiment, 13 and the filtrate was maintained in MS medium.

Cells were harvested from suspension culture by vacuum filtration on a Buchner 15 funnel (Whatman no. 614), and 100 ml (fresh weight) of cells were placed in a Petri dish 3.3. The cells were dispersed in 0.5 ml fresh culture medium to form a sub-17 layer of cells. The uncoated Petri plate was placed in the sample chamber of the particle bombardment device, manufactured by Biolistics Inc., (Geneva, NY). A vacuum pump 19 was used to reduce the pressure in the chamber to 0.1 at, to decrease the deceleration of the microparticles by air friction. The cells were bombarded with tungsten particles having an average diameter of about 1.2 μm (GTE Sylvania Precision Materials Group, Towanda, Pennsylvania). An equal mixture of PHI5168 and PHI610 was loaded on microparticles having 23 5 µl of a DNA solution (1 µg of DNA per 100 lambda), in TE buffer at pH = 7.7 to 25 µl of a 50 mg suspension tungsten particles per ml of distilled water, in a 1.5 ml Eppendorf tube. The particles were aggregated and deposited in the tube.

Cultures of transformed plant cells containing foreign genes were grown for 27 to 4 weeks at 560R (medium) (N6 based medium with 1 mg / ml bialaphos). This medium selects cells that express the bar gene. 29

Then the formation of embryos in embryogenic cultures was induced and the cells germinated in plants. A sequence 2 of culture medium was used to germinate the somatic embryos 31 observed on the retention medium. The calf was initially transferred to a culture medium (maturing medium) containing 5.0 mg / l indolacetic acid (IAA) for 10 to 14 33 days, while continued calf proliferation, cell loading was at 50 mg per plate, to optimize the recovery per unit mass of material. 35

Then the stem was transferred from the maturing medium to a second culture medium containing a low level of IAA (1 mg / l), compared to the first culture medium. At 37 this time, the crops are brought to light. The germinated somatic embryos were characterized by a green elongation of the shoot, with a root connection access. Then, the 39 somatic embryos were transferred to the medium, in a culture tube (150 x 25 mm), for another 10 ... 14 days. At this time, the plants were about 7 ... 10 cm high and were 41 in size and vigor enough to be consolidated under greenhouse conditions.

In order to consolidate the regenerated plants, the plants to be transferred to the growth chamber 43, were removed from the sterile containers and the roots were rinsed with solidified agar medium. The seedlings were placed in a commercial mix for culture vessels, in a 45 growth chamber with a humidifying device, to maintain the relative humidity around 100%, without too much watering the roots of the plants. After 3 to 4 weeks in the humidifying room, the plants were sufficiently robust for field transfer.

RO 120270 Β1

Field plants were analyzed by observing male sterility. The selected plants were then analyzed further, for the presence of the avidin gene by PCR and Southern blot and for the expression of avidin by ELISA, by standard methods.

94 plants were analyzed by PCR, of which 53 proved to be fertile and 41 sterile. When comparing the fertility of each plant with the presence of the avidin gene by PCR, a 98% correlation was found between the presence of the gene and the sterility of the plant. 5 plants were analyzed in detail for the presence of the avidin gene by Southern blot. Three plants were found, by Southern analysis, to contain the avidin gene. All three plants have sterility problems. Two plants, which lack the avidin gene, were completely fertile. Thus, there was a 100% correlation between the presence of the avidin gene and sterility problems.

Example 2. Use of Agrobacterium strains, which contain a binary vector, including a DNA sequence, encoding avidin, for generating sterile male transgenic soybeans.

One method for forming soybean plants is that described in US patent application 07/920409, which is incorporated herein by reference. Soybean seed (Glycine max), the Pioneer 9342 variety, is sterilized on the surface by exposure to chlorine released in a glass bell. The gas is produced by adding 3.5 ml of hydrochloric acid (34 to 37% w / w) to 100 ml of sodium hypochlorite (5.25% w / w). Exposure is for 16 to 20 hours, in a container of approximately 1 cubic foot in volume. The surface sterilized seed is stored in Petri dishes at room temperature. The seed is germinated by spreading a solidified agar medium, of 1/10 strength according to Gambourg (basic medium B5 with minimum organics, Sigma Chemical, Catalog no. G5893, 0.32 g / l sucrose; 0.2% by weight / volume of 2- (Nmorfolino) ethanesulfonic acid (MES), (3.0 mM), without plant growth regulators and cultivation at 28 ° C, with a day length of 16 h and cold white fluorescent illumination of about 20 μΕπτ ² S ' ^1. After 3 or 4 days, the seed is ready for cultivation, the seed shell is removed and the root extension is removed 3 to 4 mm below the cotyledons.

Overnight cultures of Agrobacterium tumefaciens, strain LBA4404, which harbors a modified binary plasmid, containing the avidin gene, are grown at log phase in minimum A medium, containing tetracycline 1 µg / ml and the optical density is collected and measured at 550 nm. A sufficient volume of the culture is placed in 15 ml centrifuge conical tubes, so that, on sedimentation, between 1 and 2 x 10 ¹ ° cells are collected in each ejector with 10 ⁹ cells / ml. Sedimentation is by centrifugation at 6,000 xg, for 10 min. After centrifugation, the supernatant is decanted and the tubes are kept at room temperature until inoculation is required, but not more than 1 h.

The inoculations are guided in batches, so that each seed plate is treated with a new pellet resuspended by Agrobacterium. The bacterial pellets are resuspended individually in 20 ml of inoculation medium containing B5 salts (G5893), 3.2 g / l; sucrose, 2.0% w / v; 6-benzylaminopurine (BAP), 45 / pm; indolbutyric acid (IBA), 0.5 μΜ; acetosyrone (As); 100 μΜ; buffered to pH = 5.5 with 10 mM MES. Resuspension is obtained by rotation. Then the inoculum is poured into a Petri dish, which contains the prepared seed, and the cotyledon knots are macerated with a surgical scalpel. This is achieved by dividing the seed into halves by cross-section through the apex of the shoot, keeping the two cotyledons whole. The two halves of the stem apex are broken from their respective cotyledons by opening them with a surgical scalpel. The cotyledonary node is then macerated with a surgical scalpel by repeated scratches along the axis of symmetry. Care must be taken not to cut entirely by explant at the axial side. The explants are prepared at large for 5 minutes and then incubated for 30 minutes at room temperature with bacteria, but without stirring. After 30 min, the explants are transferred to plates with the same medium solidified with Gelrite (Merck & Company

RO 120270 Β1

Inc.), 0.2% w / v. The explants are imbibed with the adaxial side up and level with the surface of the medium 1 and grown at 22 ”C, for 3 days, under cold white fluorescent light, at about 20 pEm ' ² S' ¹ . 3

After 3 days, the explants are moved to a counter-selection liquid medium, which contains salts B5 (G5893) 3.2 g / l; sucrose 2% w / v; BAP 5 μΜ; IBA 0.5 μΜ; vancomycin 200 µg / ml; 5 cefotaxime 500 µg / ml, buffered at pH = 5.7 with 3 mM MES. The explants are washed in each Petri dish, with slow, constant rotating stirring, at room temperature, for 4 days. 7 The counter-selection environment is replaced 4 times.

The explants are then raised to the solidified agarose selection medium, containing salts 9 B5 (G5893) 3.2 g / l; sucrose 2% w / v; BAP 5.0 pM; IBA 0.5 pM; kanamycin sulfate 50 µg / ml; vancomycin 100 µg / ml; cefotaxime 30 µg / ml; timentin 30 µg / ml, buffered at pH = 5.7 with 3 mM MES 11. The selection medium is solidified with Seakem Agarose, 0.3% w / v. The explants are soaked in medium with the adaxial side down and grown at 28 ° C, with 16 h light period in 13 cold white fluorescent illumination, 60 to 80 pErrr ² S ' ¹ .

After 2 weeks, the explants are washed again with liquid medium on the rotary shaker 15. The wash is conducted overnight in a counter-selection medium containing kanamycin sulfate, 50 µg / ml. The next day, the explants are at the selection medium aga-17 pink / solidified. They are soaked in the environment with the adaxial face down and cultivated for another 2 weeks. 19

After one month on the selection medium, the transformed tissue is visible as green areas of regenerated tissue against a background of unhealthy whitish tissue. The explants without the green sectors are eliminated and the explants with the green sectors are transferred to the elongation medium containing salts B5 (G5893) 3.2 g / l; sucrose 2% w / v; IBA 3.3 pM; gibberellic acid 23 1.7 µM; vancomycin 100 µg / ml; cefotaxime 30 µg / ml; and timentin 30 µg / ml, buffered at pH = 5.7 with 3 mM MES. The elongation medium is solidified with Gerlite 0.2% w / v. The 25 green sectors are soaked with the adaxial side up and cultivated as before. The culture is cultivated on this medium, with transfers to fresh plates, every two weeks. When the shoots are 27

0.5 cm in length, they are excised at the base and placed in the rooting medium, in test tubes 13 x 100 ml. The rooting medium consists of salts B5 (G5893), 3.2 g / l; sucrose, 15 g / l; 29 nicotinic acid, 20 µM; pyroglutamic acid (PGA), 900 mg / l and IBA, 10 pM. The rooting medium is buffered to pH = 5.7 with MES, 3 mM and solidified with Gelrite at 0.2% w / v. 31 After 10 days, the shoots are transferred to the same environment without IBA or PGA. The shoots are rooted and held in these tubes under the same environmental conditions as before. 33

When a root system is well established, the seedling is transferred to sterile soil.

The temperature, photoperiod and intensity of light remain the same as before. 35

Avidin expression in transgenic soybean plants is confirmed and quantified using ELISA and the presence of the gene is confirmed by PCR and Southern blot. The stability of the expression can be evaluated by the same methods, on successive generations. The problems of sterility have been shown to correlate with the expression of avidin in soy. 39

Example 3. Preparation of sterile male sunflower plants by the expression of avidin.41

An expression box encoding avidin is used to generate transgenic sunflower plants and seeds. The DNA sequence encoding avidin is inserted into an expression cassette, under the control of the ubiquitin promoter.

This expression box is then subcloned into a binary vector as PHI 5765.45 using the EcoRI site. Then, the binary vector is transferred to an Agrobacterium tumefaciens helper strain.47

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Sunflower plants are transformed with Agrobacterium, strain LBA44 04, after microparticle bombardment, as described by Bidney et al., PlantMol. Bio., You. 18, p. 301, 1992. In short, Pioneer sunflower seeds, SMF-3 line, are peeled and sterilized surface. The seeds are soaked in the dark at 26 ° C, for 18 hours, on filter paper moistened with water. The cotyledons and radicles are removed and meristematic explants are grown on 374BGA medium (MS salts, Shephard vitamins, 40 mg / ml adenium sulphate, 3% sucrose, 0.8% phytoagarpH = 5.6 plus 0.5 mg / l BAP, 0 , 25 mg / l, IAA and 0.1 mg / ml GA). After 24 h, the first leaves are removed to expose the apical meristem and the explants are placed with the apical dome upwards, in a circle, in the center of a 60 x 20 mm Petri dish, which contains agar water. The explants were bombarded twice with tungsten particles suspended in TE buffer or with particles associated with an expression plasmid containing the avidin gene. The meristematic explants are cocultured on 374BGA medium, in light, at 26 ° C, for an additional 72 hours of coculture.

Merrobes treated with Agrobacterium are transferred after the coculture period of 72 hours at medium 374 (374BGA with 1% sucrose and without BAP, IAA or GA ₃ ) and supplemented with 250 pg / ml cefotaxime. The seedlings are allowed to grow for another 2 weeks, under incubation conditions 16 h a day and 26'C, in green or white. Seedlings are transferred to a medium containing kanamycin and allowed to grow. The presence of avidin in plants is confirmed and quantified, as described in Example 2. The presence of male sterility is found to correlate with the expression of avidin by plants.

Although the above refer to particularly preferred embodiments, it will be understood that the present invention is not limited in this way. Those of ordinary skill in the art will find that various modifications can be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention, which is defined by the following claims. All publications and patent applications mentioned in this specification are indicative of the level of training of those skilled in the field to which the invention belongs.

Claims (24)

claims 1. An isolated DNA molecule, comprising a nucleotide sequence, encoding avidin, operatively linked to an anther-specific promoter sequence, wherein said promoter sequence directs avidin gene transcription into the tissue of the plant anthers. An expression vector, comprising the DNA molecule isolated from claim 1. An expression vector according to claim 2, wherein said anther specific promoter sequence comprises an anterior box selected from the group consisting of: (a) a DNA molecule having the nucleotide sequence of SEQ ID NO: 1; (b) a DNA molecule having the nucleotide sequence of SEQ ID NO: 2; (c) a DNA molecule having the nucleotide sequence of SEQ ID NO: 3; and (d) a functional fragment of (a), (b) or (c). An expression vector according to claim 3, wherein said anther specific promoter further comprises a core promoter selected from the group consisting of: (a) 35S CaMV core promoter; (b) a DNA molecule having the nucleotide sequence of SEQ ID NO: 4; (c) a DNA molecule having the nucleotide sequence of SEQ ID NO: 5; (d) a DNA molecule having the nucleotide sequence of SEQ ID NO: 6 and (e) a fragment of (b), (c) or (d). RO 120270 Β1 An expression vector according to claim 2, further comprising a Lex A. gene operator sequence. 6. A transgenic plant, comprising said DNA molecule of claim 1.3 The transgenic plant according to claim 6, wherein said plant is a corn plant.5 The transgenic plant according to claim 6, wherein said anther-specific promoter sequence comprises an anterior box, selected from the group consisting of: 7 (a) a DNA molecule having the nucleotide sequence of SEQ ID NO: 1; (b) a DNA molecule having the nucleotide sequence of SEQ ID NO: 2; 9 (c) a DNA molecule having the nucleotide sequence of SEQ ID NO: 3 and (d) a fragment of (a), (b) or ( c) .11 The transgenic plant according to claim 8, wherein said anther-specific promoter further comprises a core promoter selected from the group consisting of: 13 (a) 35S CaMV core promoter; (b) a DNA molecule having the nucleotide sequence of SEQ ID NO: 4; (c) a DNA molecule having the nucleotide sequence of SEQ ID NO: 5; (d) a DNA molecule having the nucleotide sequence of SEQ ID NO: 6; and17 (e) a fragment of (b), (c) or (d). 10. Transgenic plant, comprising a nucleotide sequence encoding avidin 19 operatively linked to a Lex A promoter and operator. 11. Method of producing a transgenic plant androsteris, according to claims 21, 6 ... 10, consisting in the introduction into the plant cells of an expression vector comprising a nucleotide sequence, encoding operably linked avidin to a tissue-specific promoter 23. , wherein said promoter controls the expression of said sequence encoding avidin; and in the regeneration of a transgenic plant from the cells of the said plant, in which the expression of said sequence encoding avidin in the plant, causes androsterility. The method of claim 11, wherein said tissue-specific promoter sequence 27 is an anther-specific promoter. The method of claim 12, wherein said anther-specific promoter 29 comprises an anterior box selected from the group consisting of: (a) a DNA molecule having the nucleotide sequence of SEQ ID NO: 1; 31 (b) a DNA molecule having the nucleotide sequence of SEQ ID NO: 2; (c) a DNA molecule having the nucleotide sequence of SEQ ID NO: 3; and33 (d) a fragment of (a), (b) or (c). The method of claim 12, wherein said anther-specific promoter further comprises a core promoter selected from a group consisting of: (a) 35S CaMV core promoter; 37 (b) a DNA molecule having the nucleotide sequence of SEQ ID NO: 4 (c) a DNA molecule having the nucleotide sequence of SEQ ID NO: 5; 39 (d) a DNA molecule having the nucleotide sequence of SEQ ID NO: 6; and (e) a fragment of (b), (c) or (d) .41 The method of using an avidin gene, according to claim 11, for producing an androfert hybrid plant, comprising the steps: 43 (a) producing a first androster parent plant, comprising said DNA molecule, acting as a first foreign gene, according to claim 1, wherein the expression of avidin 45 causes androsterility; (b) production of a second transgenic parent plant expressing a second foreign gene 47; and (C) cross-fertilization of said first parent with said second parent, for the production of a hybrid plant, wherein said hybrid plant expresses said second foreign gene and wherein the product of said second foreign gene reduces avidin expression in said hybrid plant, thereby producing an androfertile hybrid plant. The method of claim 15, wherein said second foreign gene is selected from the group consisting of an antisense gene, a ribozyme gene, and an external guide sequence gene. The method of claim 15, wherein said DNA molecule of said first parent plant further comprises a LexA operator, which is operatively linked to the DNA molecule promoter and wherein said second foreign gene encodes the LexA repressor. A method for restoring androfertility in a plant, produced according to the method of claim 11, comprising applying a biotin solution to the leaves of said plant. The DNA molecule isolated from claim 1, wherein said nucleotide sequence, encoding avidin, comprises the DNA fragment of pPHI5168, which extends from the restriction site Mscl, immediately above the 5 'end of said nucleotide sequence, which encodes avidin, to the Hindll restriction site at the 3 'end of said nucleotide sequence encoding avidin. An expression vector, comprising the DNA molecule isolated from claim 19. 21. The transgenic plant according to claim 7, wherein said avidin-encoding nucleotide sequence comprises the DNA fragment of pPHI5168, which extends from the restriction site Mscl, immediately above the 5 'end of said avidin-encoding nucleotide sequence, to the Hindll restriction site at the 3 'end of said nucleotide sequence encoding avidin. The method of claim 11, wherein said nucleotide sequence, encoding avidin, comprises the DNA fragment of pPHI5168, which extends from the restriction site Mscl, immediately above the 5 'end of said nucleotide sequence, encoding avidin, to the site. Hindll restriction at the 3 'end of said nucleotide sequence encoding avidin. The method of claim 16, wherein said avidin-encoding DNA molecule comprises the DNA fragment of pPHI5168, which extends from the restriction site Mscl, immediately above the 5 'end of said nucleotide sequence encoding avidin, to the site Hindll restriction at the 3 'end of said nucleotide sequence encoding avidin. The expression vector according to claim 2, wherein said expression vector is Pphi5168.