Abstract:
A novel meiosis-specific gene sequence from  Arabidopsis thaliana  including the promoter region is described. When operably linked to a heterologous gene or to the antisense of the MEI1 gene, the normal meiotic events which give rise to viable pollen can be altered. Transgenic male sterile plants can thereby be produced by the manipulation of gene expression using a genetic regulatory element such as the one described herein.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application claims the priority of U.S. provisional application No. 60/092,277, filed Jul. 10, 1998, the entire disclosure of which is incorporated herein by reference. 
    
    
     STATEMENT OF RIGHTS UNDER FEDERALLY-SPONSORED RESEARCH 
     This invention was made with government support under grant IBN-9208337 awarded by The National Science Foundation. The government has certain rights in the invention. 
    
    
     FIELD OF THE INVENTION 
     This invention relates generally to the field of recombinant genetics, and specifically to the use of a meiosis-specific promoter for regulating the expression of genes in plants. More particularly, the invention enables the expression of desired introduced genes in transgenic plants to be controlled in a developmentally related manner. 
     BACKGROUND OF THE INVENTION 
     To optimize crop production, the agricultural industry relies on plants having certain desirable traits relating to growth characteristics, resistance to disease and pest infestation, and yield. 
     One way to attain this goal is to produce hybrid plants possessing the desirable traits of parental plants. To insure the uniformity of hybrid seed, pollination control methods must be employed which favor cross-pollination and eliminate self-pollination. Such methods of inhibiting self-pollination of plants typically include mechanical means, as well as chemical. 
     Mechanical means such as detasseling, the process currently used for plants such as corn, is labor intensive and costly to perform. For some plants, for example those whose male and female gametophytes are within the same flower, the method is even less desirable. 
     As an alternative to mechanical methods to prevent self pollination, chemicals which kill or inhibit formation of functional pollen may be used. Application of chemicals, however, may result in overall toxicity to the plant or to the environment. Another limitation of the chemical means is the possibility that one application will not be effective for plants which have an extended period of flower formation. 
     More recently, the industry has relied on pollen-control-based systems which involve genetic manipulation of the events involved in the development of functional pollen. One example is described in U.S. Pat. No. 5,689,051, the contents of which are hereby incorporated by reference in their entirety. Briefly, regulatory elements are described which alter the expression of genes involved in pollen development. The ability to interfere with the production or formation of pollen results in a plant that is male sterile and therefore, incapable of self-pollination. 
     Similarly, it is possible to control plant characteristics other than pollen development by manipulating the control of gene expression which gives rise to the desired phenotype. An example of this technology is illustrated in U.S. Pat. No. 5,723,765. It discloses a system in which the expression of genes can be controlled by external stimuli or in a developmentally related fashion. The system entails creating a transgenic plant containing a series of functionally interrelated DNA sequences, including a promoter that is active at a particular stage in plant development or in response to an external stimulus, for example, a particular environmental condition. Hence, the need for sequence information for developmental stage-specific promoters is evident. 
     Male reproductive development and ultimately, pollen formation in plants begins with meiosis. Meiosis consists of a complex set of processes involving homologous chromosome pairing, synapsis, chiasma formation and crossing over, chromosome segregation, and reductional division. In male meiosis of flowering plants, the product of meiosis is a callose-enclosed tetrad of haploid microspores, the precursors of pollen grains. Manipulation of gene expression using a genetic regulatory element such as the one described herein, whose role occurs early on in pollen development, has significant implications. 
     From the many meiotic mutants described in plants and other organisms, it is obvious that meiosis is an extremely complex event involving a large number of genes. While the dissection of meiosis by the isolation and characterization of mutants and the genes responsible has progressed in yeast and Drosophila, at present little is known of the molecular details of male meiosis and the very early development of microspores in flowering plants. Recently, however, several mutants have been described in  Arabidopsis thaliana  in which male gametophyte development is affected. Specifically, a T-DNA induced male-sterile mutant of  Arabidopsis thaliana  that is defective in meiosis and that produces tetrads that are abnormal in number and in size has been described by He et al.,  Sex. Plant Reproduction  9: 54-57 (1996). In this mutant, instead of the normal tetrad of four microspores being formed after meiosis, a “tetrad” consisting of from 5 to 8 microspores is formed. The microspores show a wide range of sizes and of DNA contents. Plants homozygous for the mutation were reported to be male sterile and not to produce filled siliques, although occasionally a few siliques containing a few seeds are produced by some homozygous mutant plants. 
     T-DNA insertional mutagenesis can be used to produce mutant plants. An  Agrobacterium tumefaciens  strain containing the cointegrate pGV3850: 1003 Ti plasmid with NOS and NPT plant markers and a bacterial NPT marker was used to create the Arabidopsis mutants described above. The disruption of the plant&#39;s normal genomic DNA by the insertion of a T-DNA gives rise to a non-functional gene and mutant phenotype. One of the resulting phenotypes is plants that are male-sterile, a desirable trait for the development of hybrid seed. 
     A common feature of Agrobacterium-mediated plant transformation is the appearance of T-DNA duplications and rearrangements in the plant genome. In other words, the insertion of the T-DNA vector is random and does not produce consistent and predictable results. To reproduce by mutagenesis the desired mutation in other plants would not be possible without undue experimentation. Information about a gene sequence involved in the meiotic events giving rise to viable pollen provides a new mechanism for manipulating those events and producing plants which are male sterile. 
     The current invention relates to a gene, MEI1, and its promoter that are involved in meiosis. Significantly, the gene seems only to function at male meiosis. A mutation in the MEI1 gene results in a plant that is male sterile, but female fertile. A mutant line of Arabidopsis, designated 6492, has a phenotype similar to plants carrying the MEI1 sequence, but is female sterile, indicating the involvement of a gene different from the MEI1 sequence described herein. The MEI1 gene is novel in that, even though there is a sequence of over 100 base pairs available beyond the 3′ stop codon TAA, there is no evidence of a 3′ end poly(A) tail of this gene. Because this gene has unique splicing signals, it may not be polyadenylated the same way that normal genes usually are. 
     Knowledge of the MEI1 gene sequence and its promoter provides new ways of producing transgenic plants possessing desired characteristics. The promoter for the MEI1 gene can be used to facilitate the expression of exogenous genes, for example, suicide genes, in a manner related directly to pollen development. The MEI1 gene itself can be used to facilitate the isolation of homologous genes in other plant species. An artificial gene coding for an antisense RNA can be introduced to prevent expression of the specific target gene to whose mRNA it is complementary, in this case, the MEI1 gene product crucial for meiosis. 
     SUMMARY OF THE INVENTION 
     The invention relates to an isolated DNA sequence comprising a nucleotide sequence capable of regulating the expression of a second DNA sequence when the DNAs are part of a recombinant DNA construct. The isolated molecule includes the nucleotide sequence of the promoter region derived from a meiosis specific gene, MEI1, of  Arabidopsis thaliana  having the sequence (SEQ ID NO.1) described in FIG.  1 . 
     In another aspect, the invention relates to a vector comprising the isolated DNA molecule and further comprising a DNA sequence encoding a gene product. The DNA sequence is operably linked to the promoter. 
     In yet another aspect, the invention relates to the expression of a gene product which disrupts the function or formation of pollen. The DNA sequence to be expressed may be heterologous with respect to the promoter. 
     In still another aspect, the invention relates to a transgenic plant comprising the isolated DNA promoter sequence of the invention. 
     In yet another related aspect, the invention provides for the production of a transformed plant cell comprising the isolated DNA sequence and subsequently the growth of transgenic plants containing the characteristic phenotype bestowed by the expression of the gene inserted into the genome of the transformed cell. 
     In another aspect, the invention relates to a method of producing a male sterile plant, comprising the steps of: (1) providing a transformed plant that contains a recombinant DNA molecule that includes (a) a DNA sequence which inhibits pollen formation or function by altering meiosis; and (b) a MEI1 or homologous promoter from other plants that is operatively linked to the DNA sequence of the invention; and (2) growing the plant under conditions such that male sterility is achieved as a result of the recombinant DNA sequence. 
     In yet another aspect, the invention relates to a plant containing a recombinant DNA molecule which includes at least two elements: (1) a DNA sequence that inhibits pollen formation or function by altering meiosis; and (2) the MEI1 promoter. The MEI1 promoter is operatively linked to the DNA sequence that inhibits pollen formation or function by altering meiosis. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 lists a nucleotide sequence comprising the coding region of the genomic clone for MEI1, including the promoter sequence (SEQ ID NO.1). The sequence for the MEI1 promoter is included in the nucleotide sequence upstream of the ATG start site of translation at position 1-3. 
     FIG. 2 represents a partial restriction map showing the location of the T-DNA insertion site, cDNAs, DNA probes used and DNA fragments used for complementation analysis. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In the description that follows certain conventions will be followed as regards the usage of terminology: The gene name appears in capital letters and is italicized (MEI1). Mutant genes are indicated by lower case letters and italics (mei1). Unitalicized capital letters denote the name of the protein gene product (MEI1). 
     The term “male sterility” in a plant refers to the absence of viable pollen. Determination of sterility can be accomplished by methods well known to those skilled in the art. The term “isolated DNA molecule”, as it is commonly understood in the art, refers to a fragment of DNA that is isolated or cloned from the genomic DNA of an organism. Isolated DNA molecules may be chemically synthesized. 
     A gene is a region of DNA that controls a discrete hereditary characteristic, usually corresponding to a single protein or RNA. This definition includes the entire functional unit, encompassing coding DNA sequences, noncoding regulatory DNA sequences and introns. The term “gene product” includes RNAs and protein products for which a gene codes. 
     As used in this specification, a developmentally-specific promoter is any promoter that is active during a particular phase of plant development and is essentially inactive at other times. A gene and a promoter are to be considered to be operably linked if they are on the same strand of DNA, and are located relative to one another such that the promoter directs transcription of the gene. The presence of intervening DNA sequences does not preclude an operable relationship. 
     The term expression refers to the production of an observable phenotype by a gene, usually by the synthesis of a protein. Expression typically involves transcription of the gene into mRNA with the subsequent translation of the mRNA into protein. Transcription is under the control of a regulatory element, a promoter. The term cloning vector refers to a plasmid or phage that is used as a vehicle into which foreign DNA is inserted for the purposes of producing more genetic material or for transformation, that is, the introduction of foreign material into a plant or plant cell. 
     A gene that results in an altered plant phenotype is any gene whose expression leads to the plant exhibiting a trait or traits that would distinguish it from a plant of the same species not expressing the gene. Examples of such altered phenotypes include, but are not limited to, a different growth habit, premature or late flowering, increased or decreased yield, sterility, mortality, susceptibility to disease and insect infestation. 
     Isolation and Characterization of the MEI1 Gene and Promoter 
     Seeds from Arabidopsis plants heterozygous for mei1 were germinated on seed germination medium (SGM) containing 50 μg/ml kanamycin (Kan). Kanamycin resistant seedlings, which include both homozygous and heterozygous plants, were transferred to soil. Plants homozygous for the mei1 mutation are male sterile whereas, heterozygous plants are fertile. In seeds obtained from plants heterozygous for mei1, approximately one-fourth of the plants were Kan sensitive and wild-type and three-fourths were Kan resistant. Of the Kan resistant plants one-third were male sterile and thus homozygous for the mutation. These data indicated the existence of a single copy of the T-DNA in the genome. 
     DNA Blot Analysis Homozygous mutant plants were used for DNA blot analysis and for the isolation of plant DNA flanking the insert T-DNA. Genomic DNA from homozygous mutant plants was isolated by the method of Dellaporta et al. ( Plant Mol. Biol. Rep . 1: 19-22 1989). The contents of this document and others containing methods used for this invention are incorporated by reference in their entirety. DNA was separated on 0.8% agarose gels and transferred to nylon membranes (Hybond-N, Amersham) in 10×SSC. DNA probes were labeled with [α- 32 P]dCTP by random oligonucleotide priming. Pre-hybridization was carried out at 65° C. in a solution containing 6×SSC, 5×Denhardt&#39;s solution, 0.5% SDS, 200 μg/ml salmon sperm DNA and hybridization was done under the same conditions but with between 2×10 5  and 1×10 6 cpm of  32 P labeled probe. The membranes were washed at 65° C. twice in 2×SSC, once in 2×SSC plus 0.1% SDS and once in 0.1×SSC. 
     Both the T-DNA left border and the right border were used as probes for DNA blot analysis. The left border T-DNA probe covered the 2.9 kb EcoRI-HindIII region of pTiC58 HindIII fragment 10. The right border T-DNA probe covered the 1.1 kb HindIII-BamHI fragment 23 containing the 3′ region of the NOS gene. 
     Cloning 
     The inverse polymerase chain reaction (PCR) technique was used to isolate plant DNA flanking the right border of the T-DNA. Genomic DNA from MEI-1 homozygous mutant plants was digested with PstI. Digestion with PstI was expected to produce a fragment larger than 5.9 kb that would consist of a part of the right T-DNA border and flanking plant DNA. The DNA was then phenol/chloroform extracted, ethanol precipitated in the presence of 0.1M sodium acetate and resuspended in water. DNA (2.0 μg) was self-ligated with 200U of T4 DNA ligase (New England Biolabs) in 500 μl reaction. Following ligation, the resulting DNA circles were used as templates for PCR. Two primers in opposite orientation to that for normal PCR within the T-DNA right border (RB) were used: primer a (TTA ATT GTT GCC GGG AAG CTA GAG)(SEQ ID NO.2); primer b (CAA CCT TCC GGC GTT CGT GG)(SEQ ID NO.3). The PCR reaction was carried out in a 30 μl reaction in the presence of 200 μM dNTP, 5 U AmpliTaq DNA polymerase, Stoffel Fragment (Perkin-Elmer) and 1 μM each of primers a and b. Amplification was carried out for 35 cycles (94° C. for 1 min; 72° C. for 2 min followed by a 10 min extension at 72° C. after the last cycle. An approximately 2 kb fragment consisting of a portion of the T-DNA right border with flanking plant DNA was isolated on a 1% agarose gel and purified by Gene Clean (Bio 101), digested with PstI and BamHI (New England Biolabs), and cloned into Bluescript plasmid (Stratagene). The resulting construct was designated pCH101. The 2 kb fragment was sequenced and found to contain 836 bp of T-DNA RB and 1107 bp of plant flanking DNA. 
     Screening a Genomic Library 
     The region of pCH101 between the BamHI and PstI sites which consists of a small region of the right border of T-DNA and plant flanking DNA was sequenced. Plasmid pCH101 was digested with PstI and HindIII (New England Biolabs), and an 889 bp plant genomic fragment flanking the T-DNA insert was produced. It was labeled with [α- 32 P]dCTP by random oligonucleotide priming and used as a probe to screen a Columbia lambda genomic library. The library was constructed from a Sau3a partial digest of genomic DNA, ligated into the XhoI sites of the lambda GEM-11 vector (Promega). Plaques were transferred to nylon membranes (Amersham) in duplicate, denatured in 0.5 M NaOH/1.5 M NaCl, neutralized in 0.5 M Tris-HCl/1.5 M NaCl (pH 7.4), and washed in 2×SSC. The membranes were prehybridized, hybridized and washed as described for DNA blot analysis. Four positive clones were isolated. Clones I and II were digested with SacI. Each clone produced one insert and the inserts were cloned into Bluescript. By sequence analysis two of the clones, numbered 1 and 2 (later referred to as Fragments I and II), were found to be overlapping and together covered a length of about 16 kb which included the site where the T-DNA was inserted in the mutant plants. These two clones were used for sequencing and a total of about 10 kb of DNA, flanking the insertion site of T-DNA in the mutant, was sequenced. 
     A fragment obtained by digesting the cloned genomic DNA with ClaI and EcoRI was used to probe genomic DNA blots of wild-type and mutant plants. The results showed that in the mutant plants an expected band larger than 4 kb was found after HindIII digestion, but in the wild type DNA a band of about 2.5 kb was present. This difference in size of fragments between wild-type and mutant plants confirmed what was expected from the sequence data, that probe 1 was in close proximity to the T-DNA insertion. 
     Characterization of the MEI1 cDNA and Genomic Sequence 
     Screening cDNA Libraries 
     Two cDNA libraries were screened. The first was a cDNA library made from Landsberg erecta inflorescences with flower buds predominantly younger than stage 11 (Smyth et al., 1990). This library was constructed by using the Stratagene UniZAP kit to clone the cDNAs between EcoRI and XhoI sites (Weigel et al., 1992). CD4-7 was the second library. Equal amounts of mRNA isolated from tissue culture grown roots, 7 day old etiolated seedlings, rosettes from staged plants of different ages and 2 light regimes, and 40 aerial tissues (stems, flowers, siliques) from the same plants as the rosettes were used to make the library in lambda-PRL2, a derivative of lambdaZipLox (D&#39;Alessio et al., 1992). Both libraries were obtained from Arabidopsis Biological Resource Center at Ohio State University. For screening the libraries, the hybridization procedure was the same as that described above for screening the genomic library. 
     A genomic fragment (2624 bp) between PstI and EcoRI (probe 3 in FIG. 2) was used to screen the cDNA library made to RNA from immature flower buds. Four cDNA clones were found. While they varied in size, sequence analysis showed that they were the same sequence (G1 in FIG.  2 ). The 3′ end of this gene is approximately 600 bp away from the site of the T-DNA insertion in the mutant. Comparison of the base sequence of the cDNA with the genomic sequence indicated the presence of two introns and three exons coding for a peptide of 200 amino acids. No obvious overall similarities to any known protein were found. Complementation analysis described later indicated that G1 was not responsible for the mutation. Nucleotide sequence comparison of the genomic DNA clone with known sequences identified a partial sequence of 304 base pairs (G3 in FIG. 2) that had almost complete identity with an Arabidopsis transcribed sequence tag (accession number Z30915). This sequence, located about 6kb upstream from MEI1 and at the terminus of the genomic clone, has similarity to ACC oxidase (ethylene-forming enzyme) from a number of plants. 
     The two cDNA libraries were then screened using a smaller DNA fragment (1739 bp in length between HindIII and EcoRI (probe 4 in FIG. 2) that included the site of T-DNA insertion but excluding the cDNA1 sequence. This screen was not successful. Because of the possibility that the MEI1 mRNA was of low abundance, a polymerase chain reaction (PCR) approach was used to screen the two cDNA libraries. Two primers were designed from the T-DNA insertion region: 5′CGGTCTCGAGATCTTCCTCTGCTGCGACCC 3′ (SEQ ID NO.4) and 5′ TTGGGATCCGCAGCCAAGACATTTTTCACAA 3′ (SEQ ID NO.5). About 5×10 6  clones were screened without success. The same primers were then used for reverse transcriptase PCR using RNA isolated from immature wild-type flowers; a cDNA fragment of about 400 bp was obtained. Nucleotide sequence comparison between the cDNA and the genomic sequence in this region showed the presence of a gene (G2 in FIG. 2) in the genomic DNA which contained at least two introns. The nucleotide sequence of the genomic DNA that includes G2 is presented in FIG.  1 . Since there is a stop codon at position 755-757 the cDNA fragment probably represents the 3′ end of the mRNA. Accordingly, another pair of primers, 5′ GAAGATCTGGTAGATGAGAAGGTCAAGATC 3′(SEQ ID NO.6) AND 5′ CGGAATTCTGATCGCAATCTTTGAAC 3′ (SEQ ID NO.7) was used to recover the 5′ end of the gene. This resulted in a fragment that allowed us to extend the sequence of the 5′ end of the mRNA by 34 nucleotides. There is a stop codon in the 5′ region of this sequence and a potential TATA box upstream of the sequence in the genomic DNA; indicating that the entire coding region of the gene has been recovered. The sequence in FIG. 1 is numbered with the A in the translation start codon as +1. 
     Sequencing 
     EcoRI digestion of two of the genomic clones isolated by screening of the genomic library generated smaller restriction fragments, which were cloned into Bluescript plasmid. Sequencing was performed by the dideoxynucleotide chain termination method (Sanger et al., 1977) using Sequenase version 2.0 (U.S. Biochemical/Amersham). T3 and −20 primers from the plasmid sequence and gene-specific primers were used to generate overlapping sequence information. The junction region extending across the genomic fragment flanking the T-DNA and the right border of the T-DNA was amplified by reverse PCR and sequenced. Sequence analysis and comparison were accomplished by using the BESTFIT program and the BLAST program in the GCG package (Program Manual for the Wisconsin package, version 9.1 UNIX, 1997, Genetics Computer Group, Madison, Wis.). 
     The gene codes for a polypeptide, 89 amino acids in length. Comparative sequence analysis did not identify any protein in the data bases that showed significant similarity to the protein product of MEI-1, although the protein exhibits 42% similarity and 20% identity over almost its entire sequence with the human acrosin-trypsin inhibitor (HUSI-II), a member of the Kazal-type serine proteinase inhibitors. The two differ, however, in that, while the basic structure of Kazal inhibitors consists of 6 cysteine residues spaced at defined intervals, the MEI-1 protein contains only 3 of the cysteine residues. 
     Intron/exon Junctions in the MEI1 Gene 
     The comparison between the nucleotide sequences of the cDNA and genomic clones indicated the presence of two introns in MEI-1. These are indicated in FIG. 1 by . . . For neither intron do the 5′ and 3′ splice sites seem to comply with the :GU . . . AG: rule. 
     GU and AG dinucleotides which flank plant introns are highly conserved. A recent analysis of 998 Arabidopsis intron sequences in the EMBL database showed that even though most introns adhere to the GU . . . AG: rule, there is a 1% exception that have GC at their 5′ ends. Both introns are strongly enriched in A+T nucleotides (68% and 73% respectively), which is the essential feature for their processing and required for efficient splicing. In the yeast,  Saccharomyces cerevisiae , an additional essential signal is the highly conserved UACUA A C (SEQ ID NO.8) branchpoint sequence (the branchpoint nucleotide is underlined), located in most instances 10-50 nucleotides upstream of the 3′ splice site. In vertebrates, a loosely conserved consensus branchpoint sequence, UNCURAC or CURAC, has been derived from direct determination of branch sites utilized during splicing of different introns. The branchpoint sequence in vertebrates is usually located 18-40 nucleotides upstream of the 3′ splice site. Plant introns were thought to contain neither conserved branchpoint sequences nor distinct 3′ proximal polypyrimidine tracts characteristic of yeast and vertebrate introns, respectively. But recent studies showed that the branchpoint sequences (YUNAN) for intron lariat formation located 21-60 nucleotides upstream of the 3′ splice site are required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences. The putative consensus branchpoint sequences (YUNAN) for intron lariat formation are also positioned upstream of the 3′ splice site in both introns. CTGAC (SEQ ID NO.9), CTAAA (SEQ ID NO.10), CTAAT (SEQ ID NO.11) are 12, 33, 41 nucleotides respectively away from the 3′ splice site in the first intron; CTCAT (SEQ ID NO.12), TTAAG (SEQ ID NO.13), TTTAT (SEQ ID NO.14), TTGAT (SEQ ID NO.15) are 6, 33, 37, 41 nucleotides respectively away from the 3′ splice site of the second intron. They may play a role in their splicing. 
     There are 7 possible sites where splicing could occur for the first intron and 5 for the second intron which would result in the same coding sequence in the mRNA. None of these possible sites, however, conforms to the :GU . . . AG: rule. The sequences common to these two introns indicate that the introns have CU at their 5′ ends and AC at their 3′ ends instead of GU and AG. 
     Complementation Aanalysis 
     Two wild-type genomic DNA fragments were used to complement the mutation. The first genomic DNA fragment, 2601 bp in length contained a gene, G1 (FIG.  2 ). The 3′-end of this fragment is approximately 600 bp away from the T-DNA insertion site in the mutant. It was generated by digesting the genomic clone I with ClaI and SpeI and then cloned into Bluescript and designated p3.12. The plasmid, pCPL951, was constructed by cloning the 2601 bp fragment from p3.12 into the plant transformation vector pCIT20 (Ma et al. 1992) between the SacI and ClaI sites at the multicloning site. The second genomic DNA fragment, 7982 bp in size, fragment II (FIG. 2) contained the entire genomic sequence of MEI1. The two fragments overlapped but fragment I did not contain the 5′ coding region of the MEI1 gene. Fragment II contained the entire gene, a transcript of which corresponds to G2 (FIG.  2 ). This gene is disrupted by T-DNA in the mutant. Fragment II was cloned into pCIT20 between two SacI sites in the multicloning site, to form pCPL961. pCPL951 and pCPL961 were mobilized into Agrobacterium EHA 101 and EHA105 and transformants were generated from hypocotyl sections by the method of Lin et al. (1994). Since the hygromycin resistance (hygr) gene is the selection marker in pCIT20, 20 μg/ml hygromycin B (Sigma) was used for selection of transformants. 
     Confirmation by Complementation, that the Isolated Sequence Represents the MEI1 Gene 
     Two wild-type DNA fragments, I and II, (2.6 Kb and 8.0 Kb in size respectively) spanning the T-DNA insertion site were introduced into the plant transformation vector pCIT20, which confers resistance to hygromycin. The resulting plasmids, pCPL951 and pCPL961, were transformed into Arabidopsis plants containing the meil mutation. 
     Hypocotyls from homozygous and heterozygous plants and one-week old seedlings were used as explants for transformation. While it is preferable to use hypocotyls from only homozygous meil plants, homozygous meil plants were not distinguishable from heterozygous plants at this stage. Selection was for both kanamycin and hygromycin resistance. The seeds from the resulting transformants were first germinated on SGM containing kanamycin for a week and kanamycin resistant seedlings were then grown on hygromycin containing medium. Seeds from individual plants of the T1, T2, or T3 generations were selected on kanamycin medium to identify plants homozygous for kanamycin resistance. Such kanamycin resistant plants were also selected for hygromycin resistance. Unless the mutation was complemented one would not expect to obtain plants homozygous for kanamycin resistance. A wild-type, that is, male fertile phenotype was observed in ten independent transformants with the 8 kb genomic fragment, but none from the 2.6kb fragment. These included 4 that were homozygous for both kanamycin and hygromycin resistance, and 6 that were homozygous for kanamycin resistance but heterozygous for hygomycin resistance. The 8.0 kb (7982 bp) genomic fragment thus complemented the mutation. That the complemented plant had the initial T-DNA insert was confirmed by DNA blot analysis using DNA from a plant homozygous for both kanamycin and hygromycin resistance. 
     The 2.6 kb fragment which did not complement the meil mutation contained the coding region of gene 1, approximately 500 bp of 5′ flanking sequence of the promoter region, and about 600 bp of the 3′ end of the MEI1 gene. The failure of this fragment to complement the mei1 mutation indicates that the G1 gene has some other function. 
     Characterization of the T-DNA Right Border and Flanking Genomic DNA 
     The last 366 bp of the T-DNA right border extending from base number 16,544 to 16,909 and another 25 bp fragment between 16,462 to 16,486 bp, were found to be missing in the mutant genome. Attempts to recover, by PCR analysis, the sequence at the junction of the left T-DNA border and the flanking plant genomic sequence were not successful. Because of rearrangements and deletions, sequencing of these DNAs is not straightforward or routine. 
     With the identification of the MEI1 gene and its promoter, it is now possible, using standard techniques such as that used in the complementation analysis described above, to introduce into plants a nucleotide construct, or vector, containing the promoter and one or more genes endogenous or exogenous to the plant. In this way the expression of genes in pollen can be regulated. The methods discussed here can also be used to interfere with the normal development of pollen or to provide pollen with new characteristics. 
     Techniques well known in the art can be used to introduce the pollen-specific MEI1 promoter and exogenous or endogenous genes into plants for the purpose of preventing pollen development. Suicide genes which would result in abortion of pollen development and hence male sterility are one example. Such genes include those that produce a toxic compound or a hydrolytic enzyme such as DNase. Another example is artificial genes such as the antisense of MEI1 which can be generated by methods well know to a person of ordinary skill in the art. An artificial gene coding for an antisense RNA can be introduced to prevent expression of the specific target gene to whose mRNA it is complementary, in this case, a gene product crucial for meiosis. 
     Transformation Methods 
     Transformation methods include a number of different well known methods for direct DNA delivery. For monocots, such as corn, delivery of DNA to the treated cells by particle bombardment is a particularly effective method, but other methods such as electroporation can also be used. For dicots, Agrobacterium delivery to explants is most commonly used. Other methods include Agrobacterium cocultivation of protoplasts; electroporation; PEG uptake or other direct DNA delivery into protoplasts and the like. 
     Cells of a plant are transformed with the foreign DNA sequence of this invention in a conventional manner. If the plant to be transformed is susceptible to Agrobacterium infections, it is preferred to use a vector containing the foreign DNA sequence which is a disarmed Ti plasmid. The transformation can be carried out using procedures described, for example, in EP 0 116 718 and EP 0 270 822. Preferred Ti plasmid vectors contain the foreign DNA sequence between the border sequences, or at least located upstream of the right border sequence. Other types of vectors can be used for transforming the plant cell, using procedures such as direct gene transfer as described in EP 0 237 356 and EP 0 275 069; in vitro protoplast transformation as described in U.S. Pat. No. 4,684,611; plant virus-mediated transformation as described in EP 0 067 553 and U.S. Pat. No. 4,407,956; and liposome-mediated transformation as described in U.S. Pat. No. 4,536,475. 
     If the plant to be transformed is corn, recently developed transformation methods are suitable such as the methods described by Fromm et al., 1990, and Gordon-Kamm et al. 1990. 
     If the plant to be transformed is wheat, a method analogous to those described above for corn or rice can be used. Preferably for the transformation of a monocotyledonous plant, particularly a cereal such as rice, corn or wheat a method of direct DNA transfer, such as a method of biolistic transformation or electroporation, is used. 
     A Method for Transformation by Particle Bombardment 
     A method for transformation by particle bombardment is given in U.S. Pat. No. 5,689,049, including a protocol for corn transformation to recover stable transgenic plants. 
     The present invention has been described with regard to preferred embodiments. However, it will be obvious to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as described in the following claims. 
     All patents, publications, or other references that are listed herein are hereby incorporated by reference.