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WO1992004454A1 - Composes et structures servant a produire des plantes steriles males - Google Patents

Composes et structures servant a produire des plantes steriles males Download PDF

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Publication number
WO1992004454A1
WO1992004454A1 PCT/US1991/006234 US9106234W WO9204454A1 WO 1992004454 A1 WO1992004454 A1 WO 1992004454A1 US 9106234 W US9106234 W US 9106234W WO 9204454 A1 WO9204454 A1 WO 9204454A1
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WIPO (PCT)
Prior art keywords
seq
alkyl
gus
plant
male
Prior art date
Application number
PCT/US1991/006234
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English (en)
Inventor
Francis C. Hsu
Joan Tellefsen Odell
Jennie Bih-Jien Shen
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E.I. Du Pont De Nemours And Company
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Publication date
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Publication of WO1992004454A1 publication Critical patent/WO1992004454A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01031—Beta-glucuronidase (3.2.1.31)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds

Definitions

  • Hybrid seed production is an important means of introducing desirable traits into agronomically valuable crop plants. For instance, quality traits such as oil content, herbicide resistance, disease resistance, adaptability to environmental conditions, and the like, can be hybridized in offspring so that the latter are invested with the most desirable traits of its parents.
  • progeny from a cross may possess new qualities resulting from the combination of the two parental types, such as yield enhancement resulting from the phenomenon known as heterosis. Controlled cross- fertilization to produce hybrid seeds has been difficult to achieve commercially due to competing self- fertilization, which occurs in most crop plants.
  • hybrid seed production is performed by one of the following means: a) mechanically removing or covering the male organs or gametes to prevent self- fertilization followed by exposing the male-disabled plants to plants with fertile male organs that contain the trait (s) desired for crossing; b) growing
  • CHOK chemical hybridizing agents
  • This invention concerns, inter alia, a method for selectively sterilizing male organs in one parent and exposing the resulting plant to plants with fertile male organs to produce seed having the desirable
  • Plants are rendered receptive to male sterility induction by introducing certain DNA
  • EPA 89-329308 discloses constructs containing antisense DNA and other genes in tobacco, tomato and
  • GUS itself has utility in producing male sterility when the plant is exposed to glucuronide conjugates of toxins.
  • GB 2197653A discloses the production of transgenic plants containing GUS and the use of constructs to produce said plants.
  • GUS is said to have utility as a reporter gene for promoter analysis and possibly as a means to select mutations in fused promoters.
  • EPA 89-344029 discloses the production of toxic proteins in the male organs of plants to produce male sterility. Also disclosed is the expression of GUS as a marker gene in male organs but without the recognition that GUS itself has utility in producing male sterility when the plant is exposed to glucuronide conjugates of toxins as disclosed herein.
  • WO 90/08828 discloses that GUS cleaves a
  • glyphosate-glucuronide is a suitable substrate for GUS or that glyphosate- glucuronide is nontoxic in plants and there is no disclosure of sulfonylurea or maleic hydrazide toxins.
  • This invention concerns a method for inducing male sterility in economically important plants which are, or can be made, capable of genetic transformation.
  • a plant of choice is transformed with a gene containing the coding region of an exogenous enzyme and a suitable male organ-specific promoter.
  • the resulting transgenic plant produces the exogenous enzyme only in its male organ (s).
  • Such transgenic plants are male-fertile when grown normally.
  • the plants of the invention can be rendered male-sterile by exposure to a protoxin that is converted to an active toxin by the exogenous enzyme in the male organ.
  • the male sterility trait is only expressed when wanted, by contacting the plant with a selected protoxin; otherwise the transgenic plant behaves normally.
  • This "silent" male sterility characteristic is advantageous in many aspects of hybrid seed production. Since the protein product coded by the engineered gene is an exogenous enzyme (preferably, GUS) which by itself does not cause male sterility in the host plant, the gene can be easily carried in any sexual propagation or hybridization schemes. The male sterility trait is only "switched on", at a desired time, by contacting the plant with a protoxin. When such a transgenic plant is chosen as the female parent for large scale hybrid seed production, large quantities of its seed can be produced by selfing.
  • GUS exogenous enzyme
  • An advantage of this invention is that the gene coding for the exogenous enzyme in the male organ can be introduced into any desirable breeding lines. The line harboring the gene can be used either as the female or male parent.
  • this invention pertains to an improved method for inducing male sterility in a plant having a male organ comprising the steps:
  • step (a) employing, in step (i), a promoter selected from the group TA29 1500 , TA29 500 (SEQ ID NO: 1), p73 (SEQ ID NO: 2), p112 (SEQ ID NO: 3), p54 (SEQ ID NO:
  • step (b) employing, in step (ii), a protoxin that comprises a toxin conjugated through a non-acyl, non-phosphoryl hydroxyl residue to glucuronic acid.
  • the male organ-specific promoter is the TA29 1500 promoter, TA29 500 promoter (SEQ ID NO: 1), or promoters from Brassica anther-specific genes
  • SEQ ID NOS: 2-7 the male organ is the anther; the exogenous enzyme is ⁇ -glucuronidase (GUS); and the protoxin is conjugated through a hydroxyl residue to glucuronic acid.
  • GUS ⁇ -glucuronidase
  • This invention also pertains to protoxins that are formed from herbicides, chemical hybridizing agents and male sterilants linked to saccharides — whose
  • protoxins are herbicidal sulfonylureas and maleic hydrazide linked to ⁇ -D- glucuronic acid.
  • protoxins Contemplated protoxins are described hereafter. It should be understood that glucuronic acid esters of these protoxins are included within the scope of this invention and that the term "protoxin" includes the following compounds and their glucuronic acid esters.
  • an interesting aspect of this invention is the use of the ester form of the protoxin, solely or in various combinations with the free acid form, to induce male sterility over a pre-targeted period of time during which the male organ is known to develop. Extensive plant esterase activity will release the free acid glucuronide (or other non-ester form of the herbicide) in a time-controlled manner to eliminate the need for more than one application of protoxin even for plants in which male fertility would otherwise normally develop over several weeks.
  • the protoxins of this invention are: (i)
  • glucuronides of chemical hybridizing agents such as maleic hydrazide.
  • the protoxin is a water-stable compound in which the glucuronide is tethered with a 0 to 6, preferably 0 to 3, atom chain wherein the chain is made up of carbon atoms or, if the tether is 2 or more atoms, one or more can be a sulfur, nitrogen or oxygen atom.
  • sulfonylureas can be prepared by one skilled in the art from the other sulfonylureas disclosed in said patents.
  • This invention employs sulfonylureas of Formula I, including their agriculturally suitable isomers, salts and derivatives thereof:
  • G is H or gluc-O(alkyl) n L;
  • n 0 or 1
  • alkyl is 1 to 3 carbon atoms optionally substituted with one or two groups selected from halogen, methyl, methoxy or methylthio;
  • L is O, S(O) m , NR 5 , SO 2 NR 4 , CO 2 , CH 2 O, or a direct bond;
  • n 0-3;
  • W is O or S
  • R, R 4 and R 5 are independently H or CH 3 ;
  • E is a single bond or CH 2 ;
  • R 1 is H, C 1 to C 3 alkyl, C 1 to C 3 haloalkyl,
  • alkylsulfinyl C 1 to C 3 alkylsulfonyl, CH 2 CN, CN, CO 2 R c , C 1 to C 3 haloalkoxy, C 1 to C 3
  • haloalkylthio C 2 to C 4 alkoxyalkyl, C 3 to C 4 alkoxyalkoxy, C 2 to C 4 alkylthioalkyl, CH 2 N 3 ,
  • R 2 is H, C 1 to C 3 alkyl, C 1 to C 3 haloalkyl,
  • halogen nitro, C 1 to C 3 alkoxy, C 1 to C 3 alkylthio, CN, C 1 to C 3 haloalkoxy, or C 2 to C 4 alkoxyalkyl;
  • R a is H, C 1 to C 4 alkyl, C 2 to C 3 cyanoalkyl,
  • R b is H, C 1 to C 4 alkyl or C 3 to C 4 alkenyl; or R a and R b can be taken together as -(CH 2 ) 3 -,
  • R c is C 1 to C 4 alkyl, C 3 to C 4 alkenyl, C 3 to C 4 alkynyl, C 2 to C 4 haloalkyl. C 2 to C 3 cyanoalkyl, C 5 to C 6 cycloalkyl, C 4 to C 7 cycloalkylalkyl or
  • R d and R e are independently H or C 1 to C 2 alkyl
  • Q is a saturated or partially saturated 5- or
  • 6-membered carbocyclic ring containing either one or two carbonyl groups, or a saturated or unsaturated 5- or 6-membered heterocyclic ring, containing 1 to 5 atoms of carbon and 1 to 4 heteroatoms selected from the group consisting of 0 to 2 oxygen, 0 to 2 sulfur and 0 to 4 nitrogen, wherein sulfur can take the form of S,
  • substituents on nitrogen can be selected from the group consisting of C 1 to C 4 alkyl, C 1 to C 4 haloalkyl, CH 2 (C 2 to C 3 alkenyl), CH 2 (C 2 to C 3 alkynyl), C 2 to C 4 alkoxycarbonyl or C 2 to C 4 alkylcarbonyl; A is
  • X is H, C 1 to C 4 alkyl, C 1 to C 4 alkoxy, C 1 to C 4 haloalkoxy, C 1 to C 4 haloalkyl, halogen, C 2 to C 5 alkoxyalkyl, C 2 to C 5 alkoxyalkoxy, amino, C 1 to C 3 alkylamino, di (C 1 to C 3 alkyl) amino, C 3 to C 5 cycloalkyl, C 1 to C 4 alkyl substituted with
  • Y is H, C 1 to C 4 alkyl, C 1 to C 4 alkoxy, C 1 to C 4 haloalkoxy, C 2 to C 5 alkoxyalkyl, C 2 to C 5 alkoxyalkoxy, amino, C 1 to C 3 alkylamino or di (C 1 to C 3 alkyl) amino;
  • R 3 is H or C 1 to C 3 alkyl
  • Z is CH or N
  • E 1 is a direct bond or CH 2 ;
  • glue is ⁇ -D-glucuronic acid
  • W is O
  • R 1 is H, halogen, C 1 to C 3 haloalkoxy, SO 2 NR a R b
  • R 2 is H, methyl, halogen, methoxy or trifluoromethyl
  • R a and R b are, independently, H or C 1 to C 4 alkyl, or R a and R b can be taken together as -(CH 2 ) 4 -, -(CH 2 ) 5 - or -CH 2 CH 2 OCH 2 CH 2 -;
  • R c is C 1 to C 4 alkyl, C 2 to C 4 haloalkyl or C 2 to C 4 alkoxyalkyl;
  • X is H, C 1 to C 4 alkyl, C 1 to C 4 alkoxy, C 1 to C 4 haloalkyl, C 1 to C 4 haloalkoxy, halogen, C 2 to C5 alkoxyalkyl, C 2 to C 5 alkoxyalkoxy, C 1 to C 3 alkylamino, C 1 to C 4 alkyl substituted with -O-gluc, C 2 to C 4 alkoxyalkyl substituted with -O-gluc or C 1 to C 4 alkoxy substituted with -O-gluc.
  • R 1 is H, halogen, SO 2 NR a R b , CONR a R b , CO 2 R c or
  • J is J-1, J-2, J-3 or J-4;
  • G is H, gluc-O- or gluc-O- (alkyl) L;
  • L is O, S(O) m , CO 2 , NR 5 or a direct bond; and m is 0 or 2.
  • R 1 is H, Cl, CONR a R b or CO 2 R c ;
  • J is J-1 or J-4;
  • G is H, gluc-O- or gluc-O-CH 2 CH 2 -L
  • L is O, S, SO 2 , NR 5 or CO 2 ;
  • R a and R b are, independently, H or C 1 to C 4 alkyl;
  • R c is C 1 to C 4 alkyl;
  • X is H, C 1 to C 3 alkyl, C 1 to C 3 alkoxy, Cl, C 1 to C 3 alkylamino or C 1 to C 3 alkyl substituted with -O-gluc, C 2 to C 3 alkoxyalkyl substituted with -O-gluc; or C 1 to C 3 alkoxy substituted with -O-gluc; and Y is C 1 to C 3 alkyl, C 1 to C 3 alkoxy, C 1 to C 3 haloalkoxy, C 1 to C 3 alkylamino or C 1 to C 3 alkyl.
  • This invention also pertains to the recited plants containing a novel DNA construct comprising a male organ-specific promoter operably linked upstream from GUS.
  • transgenic plants containing the TA29 1500 or TA29 500 promoter linked to GUS.
  • This invention also pertains to the seeds produced by the transgenic plants and all progeny that exhibit the desirable traits herein described.
  • This invention also pertains to vectors containing a novel DNA construct comprising a particular male organ-specific promoter operably linked upstream from GUS.
  • Preferred is the PZS96 Agrobacterium binary vector containing the Nptll marker gene and the TA29 1500 or TA29 500 promoter linked to GUS.
  • This invention also pertains to a method of hybrid seed production and to the transgenic plants that have been exposed to the glucuronide conjugates.
  • this invention concerns the promoter TA29 500 characterized by its ability to reliably direct anther specific expression in transgenic plants.
  • TA29 500 comprises a nucleic acid fragment derived from the TA29 gene that extends from the EcoRV restriction site, which is about 500 base pairs 5' to the transcription start site and extending to the translation initiation ATG.
  • FIG 1 shows the maps of Agrobacterium
  • pZ6ASG The 500 bp EcoRV-NcoI TA29 promoter combined with the GUS coding region and the Nos 3' region as described in Example 1 is adjacent to the NosP-Nptll-Ocs 3' selection marker gene. Both of these genes are between the T-DNA borders in the binary vector pZS96, which includes a gene for ampicillin resistance (Amp R ) and an origin of replication from pBR322 (ori), and the replication (rep pVS1) and
  • pZ6ALG As in A) except that the 1500 bp Clal-Ncol TA29 promoter is combined with GUS-Nos 3' as described in Example 2.
  • Figure 2 shows the GUS enzyme activity assayed in anthers of transgenic plants containing the
  • Figure 2A represents a wild type tobacco plant, which shows virtually no endogenous GUS activity.
  • Figures 2B and 2C show the anther-specific enzyme activity in two independent transformants that demonstrate anther- specific expression of GUS. Substantially no activity was observed in wild-type or transformed plant ovules or leaves (old or new).
  • “Male sterility" of a plant refers to the inability of the plant to produce fertile, functional pollen to germinate on the stigma and to effect the fertilization of the egg.
  • “Male organ” of the plant refers to the part of the flower that physically contains pollen. Pollen grains, at all stages of development, are considered a part of the male organ. In general, male organ denotes “stamen” which includes the filament, anther and pollen in the anther. For the sake of simplicity, the term is
  • gamete is meant a mature germ cell capable of forming a new individual by fusion with another gamete.
  • Tapetum refers to the innermost layer of anther wall which surrounds the developing pollen. At a late stage of pollen maturation, the tapetum layer
  • DNA-construct refers to a linear or circular molecule of deoxyribonucleic acid (DNA), that is a composition of DNA fragments derived from any source.
  • exogenous enzyme refers to an enzyme produced according to the information in the coding region of a gene which is introduced by transformation into the plant.
  • a “chemical hybridizing agent” means a chemical that can render a plant partially or fully male sterile when it is applied to the plant.
  • protoxin refers to a chemical that releases a toxin upon reaction with the exogenous enzyme. Without interacting with the exogenous enzyme, the protoxin can also be converted into the toxin under certain chemical conditions.
  • An agent that is "toxic" to the male organ refers to an agent that kills or renders the male organ non-functional. It may cause the production of dead pollen, or living but non-functional pollen, or both. The living but non-functional pollen cannot effect egg fertilization when applied to the stigma of the pistil.
  • hybridization means the bonding of complementary segments of DNA to DNA or RNA to DNA.
  • hybridization in the biological sense, refers to the production of offspring by crossbreeding of two plants that are genetically different.
  • the "female parent" in hybridization means the plant whose eggs in the ovary of the flower are
  • Beta-glucuronidase is any enzyme that catalyzes the hydrolysis of ⁇ -O-glucuronide conjugates into glucuronic acid and the aglycone.
  • GUS catalyzes the hydrolysis of a glucuronide conjugate protoxin into the toxin and glucuronic acid.
  • glucuronide is used interchangeably with “beta-glucuronide”. It denotes a derivative of
  • glucuronic acid in which a compound (the aglycone) is conjugated through the beta linkage to the oxygen atom on carbon number 1 of the glucuronic acid.
  • the "aglycone” of the glucuronide refers to any compound that is conjugated to the glucuronic acid.
  • the chemical nature of the aglycone is limited only by the requirement that it possess a conjugatable hydroxyl group.
  • nucleotide sequence refers to a polymer of DNA or RNA, which can be single- or double- stranded, optionally containing synthetic, non-natural, or altered nucleotides capable of incorporation into DNA or RNA polymers.
  • DNA segment refers to a linear fragment of single- or double-stranded DNA derived from any source.
  • the expression "DNA in plant cells” includes all DNA present in plant cells.
  • a “gene” is intended to mean a DNA segment which includes a 5' regulatory region, a coding region, and a 3'
  • Codon region refers to a DNA segment which encodes a regulatory molecule or any polypeptide.
  • Gene product refers to a polypeptide resulting from transcription, translation, and, optionally, post- translational processing of a selected DNA segment.
  • expression is intended to mean the translation to gene product from a gene coding for the sequence of the gene product.
  • a DNA chain coding for the sequence of gene product is first transcribed to a complementary RNA which is called a messenger RNA and then, the thus transcribed messenger RNA is translated into the above-mentioned gene product.
  • promoter refers to a sequence of DNA, usually upstream (5') of the coding sequence, which controls the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at the correct site.
  • a “promoter fragment” constitutes a DNA sequence consisting of the promoter region.
  • a promoter region can include one or more regions which control the effectiveness of transcription initiation in response to physiological conditions, and a
  • tissue specific promoters are those that direct gene expression primarily in specific tissues.
  • male organ-specific promoter refers to a promoter that directs gene expression primarily in the male organ, i.e., pollen, anther tissues, filament of the anther, and gamete. Transcription stimulators, enhancers or activators can be integrated into these promoters to create a promoter with a high level of activity that retains its specificity.
  • regulatory nucleotide sequence refers to a nucleotide sequence located proximate to a coding region whose transcription is controlled by the regulatory nucleotide sequence in conjunction with the gene
  • the regulatory nucleotide sequence is located 5' to the coding region.
  • a promoter can include one or more regulatory nucleotide sequences.
  • Polyadenylation nucleotide sequence (or “region”) refers to a nucleotide sequence located 3' to a coding region which controls the addition of polyadenylic acid to the RNA transcribed from the coding region in
  • Transferring refers to methods to transfer DNA into cells including, but not limited to, microinjection, permeabilizing the cell membrane with various physical (e.g., electroporation) or chemical (e.g., polyethylene glycol, PEG) treatments, high- velocity microprojectile bombardment also termed
  • biolistics or infection with Agrobacterium tumefaciens or A. rhizogenes.
  • Transformant or “transgenic plant” means a plant which has acquired properties encoded on a nucleic acid molecule that has been transferred to cells during the process known as transformation.
  • Integrated means that the DNA is incorporated into the plant genome.
  • the ⁇ -glucuronidase enzyme is specifically produced in anther or pollen cells.
  • GUS catalyzes the hydrolysis of a wide variety of glucuronides. Most any aglycone conjugated to D- glucuronic acid through a ⁇ -O-glycosidic linkage is a suitable substrate.
  • GUS enzyme activity can be easily assayed using a number of methods including spectrophotometrically using p-nitrophenyl-glucuronide as the substrate, fluorometrically using 4-methyl umbelliferyl-glucuronide (MUG) as the substrate, or histochemically using 5-bromo-4-chloro-3-indolyl- glucuronide (X-Gluc) as the substrate.
  • spectrophotometrically using p-nitrophenyl-glucuronide as the substrate
  • fluorometrically using 4-methyl umbelliferyl-glucuronide (MUG) as the substrate
  • X-Gluc 5-bromo-4-chloro-3-indolyl- glucuronide
  • the GUS enzyme is naturally encoded by the uidA gene of Escherichia coli. The cloning and
  • the GUS enzyme is expressed as the product of a chimeric gene that is transferred into plants, that includes a regulatory nucleotide sequence which directs expression specifically in cells located in the anther or in pollen. Suitable regulatory nucleotide sequences are known in the art.
  • the particular anther or pollenspecific promoter which is employed with a selected plant species is not critical to the method of the invention.
  • a partial list of suitable promoters includes those from the TA29 and TA13 tobacco genes described by Goldberg, Science, 240: 1460-1467 (1988), the LAT52 tomato gene described by Twell et al, Mol. Gen. Genet.
  • Novel anther promoters are derived from Brassica napus genes corresponding to the newly-identified anther- specific cDNAs with SEQ ID NOS: 2-7. Most preferably, the regulatory nucleotide sequence is a TA29 promoter.
  • Nucleic acids can generally be introduced into plant protoplasts, with or without the aid of electroporation, polyethylene glycol, or other processes known to alter membrane permeability. Nucleic acid constructs can also be introduced into plants using vectors comprising part of the Ti- or Ri- plasmid, a plant virus, or an autonomously replicating sequence. Nucleic acid constructs can also be
  • microprojectiles introduced into plants by microinjection or by high- velocity microprojectiles, also termed "particle
  • Agrobacterium tumefaciens containing the nucleic acid fragment between T-DNA borders in a binary vector in trans to a disarmed Ti-plasmid.
  • the Agrobacterium can be used to transform plants by inoculation of tissue explants, such as stems, roots, or leaf discs, or by co-cultivation with plant protoplasts.
  • tissue explants such as stems, roots, or leaf discs
  • co-cultivation with plant protoplasts The range of crop species in which foreign genes can be introduced is increasing rapidly as tissue culture and transformation methods improve and as appropriate selectable markers become available. Thus, this invention is applicable to a broad range of
  • DNA sequences are introduced into plant cells by co-cultivation of leaf disks or plant tissue explants with Agrobacterium tumefaciens.
  • a nopaline synthase gene polyadenylation nucleotide sequence is pZ6ASG or pZ6ALG or derivatives thereof. These plasmids can be used to generate plants that express the GUS enzyme in their anthers by those skilled in the art or as taught in this application.
  • plants expressing GUS in the anthers or pollen are contacted with a protoxin, such as a general cytotoxic agent or a herbicide that has been conjugated through an oxygen atom to glucuronic acid.
  • the protoxin is transported to all regions of the plant, but is efficiently cleaved to toxin only in the male organs and exhibits cytotoxicity only to the male organs.
  • the plant is thus rendered male-sterile upon exposure to the protoxin.
  • protoxins will be equally effective as male-sterilants in all plants that have anther-specific expression of GUS. Some of them may be toxic to certain plants. Others may have relatively low toxicity to the male organ. Nevertheless, given the disclosure presented herein, and with a minimum of experimentation, one skilled in the art will be able to easily determine which protoxin (s) to employ with a specific plant.
  • a chimeric gene was constructed to obtain
  • the promoter was derived from the tobacco TA29 gene, a gene that is expressed naturally only in the tapetal tissue of the tobacco anther.
  • a clone containing the TA29 gene was obtained from Dr. Goldberg at the University of
  • the TA29 gene can also be obtained by methods taught in EPA 84-344,029.
  • One skilled in the art can prepare a probe to the TA29 cDNA sequence given in Figure 2 of '029 and isolate a TA29 gene-containing clone from a tobacco genomic library using that probe.
  • the TA29 gene sequence is given in Fig. 3 of EPA 89-344029.
  • a TA29 promoter fragment was prepared from the TA29 gene by first cloning an Sstl-Hindlll fragment, which was expected to contain the TA29 promoter region based on the location of the 5' end of the messenger RNA, from the Goldberg clone into the Sstl and Hindlll digested vector M13mp18. This fragment proved too large to carry out further steps so an approximately 500 base pair (bp) EcoRV-Hindlll fragment was isolated and cloned into Smal and Hindlll digested M13mp19.
  • AGAAATTAGCTACCATGGTAGCTCCAAAAT (SEQ ID NO: 8) was synthesized using an Applied Biosystems DNA synthesizer and following the manufacturer's procedure. This oligonucleotide was used in a site-directed mutagenesis procedure as described in Viitanen et al., J. Biol. Chem., 263:15000-15007 (1988), to create an Ncol site surrounding the translation initiation ATG. The approximately 500 bp TA29 promoter fragment containing the new Ncol site was then moved as an Sstl-Hindlll fragment, the Sstl site being derived from the M13mp19 polylinker, into Sstl and Hindlll digested pTZ19
  • Ncol-Hindlll fragment that includes the GUS coding region and a nopaline synthase gene polyadenylation nucleotide sequence (NOS 3') was prepared from the plasmid pTZCGNC.
  • the pTZCGNC vector contains pTZ19 and a chimeric NOS/P-GUS-NOS 3' gene and was constructed in the following manner.
  • a GUS coding region fragment was prepared from pRAJ275, which is described in Jefferson et al., Proc. Natl. Acad. Sci. USA 83, 8447-8451 (1986) and is available from Clontech Laboratories.
  • the plasmid, pRAJ275 was digested with EcoRI, the end made blunt, and a BamHI linker was added. It was then digested with Hindlll to prepare a Hindlll-BamHI.
  • the plasmid, p.KNK bears ATCC deposit accession number 67284. It is a pBR322 based vector which contains a neomycin phosphotransferase II (Nptll) promoter fragment, a nopaline synthase (NOS) promoter fragment, the coding region of Nptll and the polyadenylation nucleotide sequence from the NOS gene.
  • Nptll neomycin phosphotransferase II
  • NOS nopaline synthase
  • the 320 bp Clal-Bglll fragment in pKNK that contains the Nptll promoter was obtained as a Hindlll-Bglll fragment from the Nptll gene of the transposon Tn5 described by Beck et al., Qsns. 19: 327-336 (1982).
  • the Hindlll site was
  • Nptll promoter fragment is followed by a 296 bp Sau3A-PstI NOS promoter (NOS/P) fragment corresponding to
  • the NOS/P is followed by a 998 bp Hindlll-BamHI sequence containing the Nptll coding region obtained from the transposon Tn5 [Beck et al., Gene 19: 327-336 (1982)] by the creation of Hindlll and BamHI sites at
  • nucleotides 1540 and 2518 respectively.
  • the Nptll coding region is then followed by a 702 bp BamHI-Clal fragment containing the 3' end of the nopaline synthase gene including nucleotides 848 to 1550 [Depicker et al., J. ADPI. Genet. 1: 561-574 (1982)].
  • the remainder of pKNK consists of pBR322 sequences from 29 to 4361.
  • the resulting plasmid contains a chimeric gene that has a 500 bp TA29 promoter and a NOS 3' as the regulatory signals surrounding the GUS coding region in the vector pTZ19.
  • This chimeric gene was transferred into pZS96, a binary vector used in Agrobacterium tumefaciens
  • the plasmid, pZS96 contains the origin of replication and ampicillin resistance gene from pBR322 for maintenance and selection in E. coli. It contains the replication and stability regions of the Pseudomonas aeruginosa plasmid pVS1, described by Itoh et al., Plasmid 11: 206-220 (1984), which are required for replication and
  • pZS96 was digested with Smal and Hindlll and ligated to a Scal-Hindlll fragment containing the TA29 500 /P-GUS-NOS 3' gene isolated from pTZAS, the Seal site being located in the pTZ sequence. (Seal and Smal digests both leave blunt ends.)
  • the resulting plasmid called pZ6ASG ( Figure 1A) contains a chimeric gene that has a 549 op TA29 promoter (SEQ ID NO: 1) and a NOS 3' as the regulatory signals surrounding the GUS coding region in the vector pZS96.
  • a second promoter fragment from the TA29 gene was prepared by first isolating an approximately 1500 bp Clal-Hindlll fragment from the Sstl-Hindlll fragment that was subcloned from the Goldberg TA29 gene clone described above. During the isolation, the Clal end was filled in so that this fragment could be cloned into the Hindi (blunt end) and Hindlll sites of
  • the TA29 promoter fragment containing the new Ncol site was then moved as a Smal-Hindlll fragment, the Smal site being derived from the M13mp19 polylinker, into Smal and Hindlll digested pTZ19 creating pTZAL.
  • the Clal fragment containing the NOS/P-GUS-NOS 3' gene in pTZCGNC was isolated, the ends made blunt, and it was cloned into Sphl digested and blunted pTZ19. This step was carried out to eliminate the polylinker sites that are located between the NOS 3' and the Hindlll site in pTZASG.
  • Ncol- Hindlll fragment that includes the GUS-NOS 3' was prepared from the resulting plasmid and it was cloned into Ncol and Hindlll digested pTZ.AL creating pTZALG.
  • An Asp718-HindIII fragment containing the entire chimeric TA29 1500 /P-GUS-NOS 3' gene was ligated into Asp718 and Hindlll digested pZS96, described above, creating pZ6ALG ( Figure 1B).
  • the resulting plasmid is unique in that it contains a chimeric gene that has with the 1500 bp TA29 promoter and GUS coding region, a NOS 3' as the polyadenylation regulatory signal, and this chimeric gene is present in the pZS96 vector.
  • transformants were analyzed to demonstrate anther specific expression of this gene.
  • the plasmid pZ6ASG was transferred into
  • Agrobacterium tumefaciens by a method involving a three-way mating that was essentially as described by Fraley et al., Proc. Natl. Acad. Sci. USA, 80: 4803- 4807 (1983) except for the following points.
  • the plasmid, pZ6ASG was mated into Agrobacterium strain LBA4404 described by Hoekema et al., Nature 303: 179- 180 (1983). Colonies from the pZ6ASG mating were selected on MinA (Table 1) plates containing 100 ⁇ g/mL rifampicin, 25 ⁇ g/mL carbanicillin, and 10 ⁇ g/mL tetracycline.
  • Leaf disks 8 mm in diameter, were prepared from whole leaves using a sterile paper punch.
  • Leaf disks were inoculated by submerging them for several minutes in 20-30 mL of a 1:10 dilution of the overnight culture of Agrobacterium harboring the pZ9ASG plasmid. After inoculation, the leaf disks were placed on CN agar medium (Table 1) in petri dishes which were then sealed with parafilm. The petri dishes were incubated under mixed fluorescent and grow lights for 2-3 days in a culture room maintained at approximately 25°C.
  • leaf disks were transferred to fresh CN agar medium containing 500 mg/L cefotaxime and 100 mg/L kanamycin. Leaf disks were incubated under the growth conditions described above for 3 weeks and then transferred to fresh media of the same composition.
  • Root Induction medium 'A' (Table 1) containing 500 mg/L cefotaxime and 100 mg/L
  • Root formation was recorded within 3 weeks. Rooted shoots were then transplanted to wet Metro-Mix in 8-inch pots, moved to a growth chamber set at the conditions described above, and covered with plastic bags for 7-10 days. After 4-6 weeks, plants were transferred to the greenhouse and allowed to grow to maturity.
  • tissue sample was ground on ice in a microfuge tube containing 125-200 ⁇ L of GUS extraction buffer (50 mM sodium phosphate pH 7.0, 10 mM EDTA, 0.1% triton X-100, 0.1% sarkosyl, 10 mM ⁇ -mercaptoethanol) with a GUS extraction buffer (50 mM sodium phosphate pH 7.0, 10 mM EDTA, 0.1% triton X-100, 0.1% sarkosyl, 10 mM ⁇ -mercaptoethanol) with a GUS extraction buffer (50 mM sodium phosphate pH 7.0, 10 mM EDTA, 0.1% triton X-100, 0.1% sarkosyl, 10 mM ⁇ -mercaptoethanol) with a GUS extraction buffer (50 mM sodium phosphate pH 7.0, 10 mM EDTA, 0.1% triton X-100, 0.1% sarkosyl, 10 mM ⁇ -mercap
  • Seeds were collected following self fertilization of individual transformants. To determine the number of genetic loci for the TA29 500 /P-GUS-NOS 3' gene, and linked kanamycin resistance marker, seeds were
  • Root Induction medium 'A' containing 100 or 200 ⁇ g/mL kanamycin. Seeds were sterilized by incubation for 30 minutes in 10% Clorox ® and 0.1% SDS and sewn at 60 seeds per plate. Sensitive seeds germinate, but the seedlings bleach after 2-3 weeks. A segregation ratio of 3 resistant to 1
  • transformants exhibited a ratio which was greater than 3:1, indicating the presence of more than one
  • kanamycin resistant plants from lines showing 3:1 segregation in the seed germination test were potted in soil and grown to maturity in the greenhouse. Seed were collected after self fertilization and germinated on kanamycin medium as described above. Populations of seed that were 100% kanamycin resistant representing homozygous lines were identified.
  • the chimeric TA29 1500 /P-GUS-NOS 3' gene was introduced into tobacco by Agrobacterium tumefaciens infection of leaf disks as described above using
  • Agrobacterium harboring the pZ6ALG plasmid harboring the pZ6ALG plasmid. Primary transformants were analyzed to demonstrate anther specific expression of the introduced chimeric GUS gene using the enzyme fluorescence assay as described above.
  • the twenty-four independent tobacco transformants that were assayed were placed in the same categories described for the TA29 500 /P-GUS-NOS 3' transformants.
  • the anther specific GUS expression was further characterized as being limited to tapetal cells by incubating anthers in 0.1 mM NaP ⁇ 4 buffer, pH 7.0, 0.5 mM K 3 [Fe(CN) 6 ], 0.5 mM K 4 [Fe(CN) 6 ] -3H 2 O, 10 mM Na 2 EDTA containing 1 mg/mL X-Gluc (5-bromo-4-chloro-3-indolyl- ⁇ -glucuronide).
  • This GUS substrate releases a blue product upon GUS hydrolysis. Microscopic observation showed that the blue staining was limited to the tapetal cells in the anther. Wild-type control tobacco anthers showed no blue staining.
  • Seeds were collected following self-fertilization of individual transformants. To determine the number of genetic loci for the TA29 1500 /P-GUS-NOS 3' gene, and linked kanamycin resistance marker, seeds were
  • kanamycin resistant plants from lines showing 3:1 segregation in the seed germination test were potted in soil and grown to maturity in the greenhouse. Seed was collected after self fertilization, and seeds from the progeny of two lines were germinated on kanamycin containing medium. A population of seed that is 100% resistant was identified for each line thereby establishing these as homozygous populations.
  • the chimeric TA29 1500 /P-GUS-NOS 3' gene was introduced into tomato (Lycopersicum esculentum. var. Herbs red cherry) by Agrobacterium tumefaciens
  • a single colony of A ⁇ robacterium harboring pZ6ALG was used to inoculate 3 mL of MinA (Table 1) broth and it was grown at 25°C with shaking over two nights.
  • the culture was diluted to an OD 650 of 0.1 in a flask containing 30 mL of MinA broth and grown with shaking at 28°C to a density equal to an OD 650 of 0.3.
  • a sterile scalpel cotyledons were excised from seedlings and approximately 2 mm of tissue was removed from each end. These explants were planted underside down at 10 per plate on CTM agar (Table 2) containing 75 ⁇ M acetosyringone in dimethylsulfoxide (DMSO). Then, 5 mL of the Agrobacterium solution was added to each plate, making sure to wet all of the explants, and then
  • TA29 1500 /P-GUS-NOS 3' gene was assayed using the X-Gluc GUS enzyme assay described above. Immature buds were excised, dissected into pieces, and incubated in the X-Gluc solution overnight. Blue staining was observed only in the anthers of buds from all 6 of the plants tested. Seeds were collected following self- fertilization of each transformant. Single locus homozygous lines were identified as described in Example 3.
  • B5 vitamins Img/mL nicotinic acid, 10 mg/mL thiamine hydrochloride, Img/mL pyridoxine hydrochloride, 100 mg/mL M-inositol
  • the chimeric TA29 1500 /P-GUS-NOS 3' gene was introduced into Brassica napus (cv. Westar) by
  • a single colony of Agrobacterium harboring pZ6ALG was used to inoculate 3 mL of MinA (Table 1) broth and it was grown at 28°C with shaking for 18-20 hours. The culture was grown to an OD 650 of 1.0-2.0. Then 22.5 mL of bacterial dilution medium (1 package MS minimal organic medium and 30 g sucrose per liter) containing 100 ⁇ M acetosyringone was placed in a sterile dish. Using a sterile scalpel, the seedling hypocotyls were cut into 1 cm segments and placed immediately into the bacterial dilution medium.
  • a cDNA library of Brassica napus (cv. Westar) was constructed using poly(A) + -RNA isolated from developing anthers dissected from 2-3 mm flower buds. Total and poly (A) + -RNAs were purified using RNA Extraction and mRNA Purification Kits (Pharmacia) according to the manufacturer's specifications.
  • cDNA was synthesized using the RiboCloneTM cDNA Synthesis System (Promega), ligated with EcoRI adaptors (Promega), inserted into the EcoRI site of the Lambda ZAP II vector
  • RNA blots [Maniatis et al.. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982)] con9aining total RNA purified from seedlings, petals, filaments, different stages of anthers and pistils were hybridized with the 32 P-labeled cDNA insert prepared from each clone.
  • the mRNAs encoded by p112 and p158 accumulated very early in the development of anthers, in flower buds less than 2 mm in length, and the levels of message declined and disappeared during anther maturation. In contrast, the levels of mRNA encoded by p54 and p73 cDNAs increased during anther development and reached the peak level at anthesis.
  • Tissue sections were prepared from Brassica napus anthers dissected from different stages of flower buds.
  • Sense and antisense probes were prepared from the cDNA clones using the polymerase chain reaction (GeneAmp kit, Perkin-Elmer Cetus) with universal and reverse primers (Pharmacia), followed by preparation of
  • the antisense probe hybridized only to the tapetal cell layer surrounding the developing pollen grains and not to any other cells in the section indicating that the p112 mRNA is tapetal-cell specific.
  • the control p112 sense probe did not show any specific hybridization.
  • Hybridization of the p158 antisense probe also showed the p158 mRNA to be tapetum-specific, but the signal level was lower than that of p112.
  • Hybridization of the p54 antisense probe showed pollen-specific expression of p54 mRNA, which accumulated to a maximum level in mature pollen.
  • the in situ hybridization result for p42 showed that the mRNA was localized in the outer parenchymatic cells of the vascular bundle, the tapetal cell layer and pollen grains, but not in the connective tissue or the outer cells of the exotecium.
  • the in situ localization of p73 mRNA was not examined, but its message accumulated at the maximum level in the mature anther indicating it could be another pollen-specific gene.
  • DNA from cDNA clones p42, p42W, p54, p73, p112 and p158 was made for sequence analysis by purifying double-stranded plasmid using standard procedures
  • nucleotide sequences of the cDNAs were determined by the dideoxy method using a T7 SequencingTM kit (Pharmacia). Adjacent regions of the clones were sequenced by priming with synthetic oligonucleotides designed from sequences obtained from previous gel readings.
  • the cDNA and deduced amino acid sequences of p42 showed significant homology with chalcone synthase from mustard [Ehmann and Schaefer, Plant Mol. Biol. 11:869- 870 (1988)], parsley [Reimold et al. EMBO J. 2: 1801- 1805 (1983)] and soybean [Akada et al. Nucleic Acids Res. 18:3398 and 5899 (1990)].
  • the cDNA sequences of p42W and p112 share 98% identity, and p42W has 18 extra nucleotides at the 5'-terminus and 399 extra
  • the anther- specific promoter is then isolated as the DNA segment located 5' to the transcription start site.
  • novel Brassica napus tapetum and pollen specific promoters are prepared and used to construct chimeric genes for anther-specific expression of GUS.
  • the promoter fragment is placed adjacent to the GUS-NOS 3'construction in pTZALG, after removing the TA29 promoter fragment.
  • the resulting chimeric genes: 42/P- GUS-NOS 3', 42W/P-GUS-NOS 3', 54/P-GUS-NOS 3', 73/P- GUS-NOS 3', 112/P-GUS-NOS 3', and/or 158/P-GUS-NOS3' are each cloned into pZS96.
  • the genes are then introduced into tobacco, tomato, and/or Brassica as described previously, and GUS expression is analyzed in transformants as described in Example 9.
  • dVB vascular bundle
  • Chimeric genes for expression of GUS specifically in pollen can be constructed by making use of other pollen specific promoters known to one skilled in the art.
  • LAT52 is a gene from tomato that is expressed preferentially in the pollen. The identification, cloning, and characterization of this gene is described by Twell et al., Mol. Gen. Genet. 217: 240-245 (1989). Genes that are expressed specifically in pollen have also been studied in Zea mays and Tradescantia paludosa by Stinson et al., Plant Physiol. 83: 442-447 (1987). In Hanson et al., The Plant Cell 1: 173-179 (1989) the isolation of the pollen specific Zmc13 gene is
  • a fragment containing the LAT52 or Zmc13 promoter region is prepared from the clone of the gene and placed adjacent to the GUS-NOS 3' construction in pTZALG, after
  • the chimeric LAT52/P-GUS-NOS 3' and Zmc13/P-GUS- NOS 3' genes, described above, are introduced into tobacco by infection of leaf disks with Agrobacterium tumefaciens harboring pZ6TGN and pZ6CGN, respectively. The procedure is carried out as described in Example 3. Kanamycin-resistant transformants are tested for pollen specific expression of GUS by the enzyme assays
  • a protein extract is prepared from collected pollen and assayed using the MUG substrate. Samples of ovule and leaf tissue are also tested for GUS activity as described above to determine the specificity of expression in the pollen. Seed is collected from plants showing anther specific GUS activity, and homozygous lines are established as described in Example 3.
  • the chimeric LAT52/P-GUS-NOS 3' and Zmc13/P-GUS- NOS 3' genes are introduced into tomato by infection of cotyledon explants with Agrobacterium tumefaciens harboring pZ6TGN and pZ6CGN, respectively. The procedure is carried out as described in Example 5. Kanamycin-resistant transformants are tested for pollen specific expression of GUS by the enzyme assays described in Examples 4 and 5. Pollen is collected from mature anthers, incubated in X-Gluc solution, and blue staining of the pollen is assessed
  • a protein extract is prepared from collected pollen and assayed using the MUG substrate. Samples of ovule and leaf tissue are also tested for GUS activity as described above to determine the specificity of expression in the pollen. Seed is collected from plants showing anther-specific GUS activity, and homozygous lines are established as described in Example 3.
  • the chimeric LAT52/P-GUS-NOS 3' and Zmc13/P-GUS- NOS 3' genes are introduced into Brassica by infection of hypocotyl explants with Agrobacterium tumefaciens harboring pZ6TGN and pZ6CGN, respectively. The procedure is carried out as described in Example 6. Kanamycin-resistant transformants are tested for pollen-specific expression of GUS by the enzyme assays described in Examples 4 and 5. Pollen is collected from mature anthers, incubated in X-Gluc solution, and blue staining of the pollen is assessed
  • a protein extract is prepared from collected pollen and assayed using the MUG substrate. Samples of ovule and leaf tissue are also tested for GUS activity as described above to determine the specificity of expression in the pollen. Seed is collected from plants showing anther-specific GUS activity, and homozygous lines are established.
  • aglycone the only structural requirement on the aglycone is a hydroxyl or oxyanion residue.
  • conjugation can be performed by one or more of the means outlined below.
  • suitable substrates include, but are not limited to, phenols, salts of phenols, organic alcohols, salts of organic alcohols, suitably activated carbonyl compounds such as 1,3- dicarbonyls, imides, and activated secondary amides, and so forth.
  • a variety of compounds that are phytotoxic contain hydroxyl groups. Some of these compounds have been made into glucuronides and tested on transgenic plants expressing male organ-specific GUS.
  • the compounds of Formula I can be prepared by either of two methods, differing by choice of
  • Method A utilizes the reagent of Formula VII, whose synthesis is
  • the reaction of Scheme 1 (a) can be carried out by contacting a glucuronide methyl ester of Formula II with 2-5 equivalents of sodium hydroxide in methanol at a temperature between 0 and 40°C for 0.1 to 24 hours.
  • the product can be isolated by adding a sufficient amount of a cationic ion-exchange resin to protonate the glucuronic acid sodium salt, filtering the resin, and evaporating the solvent.
  • the reaction of Scheme 1 (b) can be carried out by the hydrogenolysis of a methanolic solution of a 2,3,4- tri-O-benzyl glucuronide methyl ester of Formula III with a palladium catalyst, such as 20% palladium hydroxide on carbon (Pearlman's catalyst) at 0 to 30°C for 0.1 to 24 hours under a hydrogen pressure of 1-10 atmospheres.
  • a palladium catalyst such as 20% palladium hydroxide on carbon (Pearlman's catalyst) at 0 to 30°C for 0.1 to 24 hours under a hydrogen pressure of 1-10 atmospheres.
  • the product can be isolated by filtration and removal of solvent.
  • reaction of Scheme 1 (c) can be carried out by contacting a protected sulfonamide glucuronide of
  • Formula IV with an N-heterocyclyl carbamic acid phenyl ester of Formula V examples of which are well-known in the art and which are known to form highly herbicidal sulfonylureas with suitable sulfonamides.
  • an inert solvent such as acetonitrile
  • an amidine base such as 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) are added at -10 to 40°C, and the mixture is allowed to react for 0.1 to 2 hours, after which time the product is isolated by acidification with aqueous acid and filtration or extraction into a suitable organic solvent.
  • DBU 1,8- diazabicyclo[5.4.0]undec-7-ene
  • Purification can be accomplished by recrystallization or chromatography on silica gel.
  • the compound of Formula IV can be prepared by the selective O- glycosylation of a hydroxy-substituted sulfonamide with the aforementioned reagent VII in the presence of an acidic catalyst, such as boron trifluoride etherate in a suitable solvent, such as dichloromethane, at -40 to 0°C for 0.1 to 2 hours.
  • an acidic catalyst such as boron trifluoride etherate in a suitable solvent, such as dichloromethane, at -40 to 0°C for 0.1 to 2 hours.
  • the product can be isolated by washing the organic solution with aqueous NaHCO 3 , removing the solvent, and by chromatography on silica gel and/or recrystallization.
  • hydroxylated sulfonamides can be accomplished by a wide variety of methods well-known to one skilled in the art.
  • Method B utilizes a commercially-available glucosylating reagent
  • n, R, R 1 , are as previously defined and Ac is
  • the reaction of Scheme 2 (a) can be carried out by contacting a solution of a compound of Formula VIII in methanol with a catalytic amount of sodium methoxide (0.01 to 0.1 equivalents) for 0.1 to 10 hours at 0 to 30°C.
  • the product can be isolated by neutralization of the catalyst and concentration of the reaction mixture and may optionally be purified by chromatography.
  • reaction of Scheme 2 (b) can be carried out exactly as described above for the reaction of Scheme 1(c).
  • the reaction of Scheme 2(c) can be carried out by contacting a compound of Formula X with
  • trifluoroacetic acid which can be used as a solvent at 0-40°C for 0.1 to 10 hours.
  • the product of Formula IX can be isolated by removal of excess trifluoroacetic acid under vacuum and chromatography on silica gel.
  • the reaction of Scheme 2 (d), can be accomplished by contacting a hydroxylated t-butyl sulfonamide of Formula XI with 1-2 equivalents of methyl 2,3,4- triacetyl-1-bromo- ⁇ -D-glucuronate and 1-3 equivalents of a silver salt such as silver carbonate in an inert anhydrous solvent such as benzene or dichloromethane for 1-100 hours.
  • a silver salt such as silver carbonate
  • an inert anhydrous solvent such as benzene or dichloromethane for 1-100 hours.
  • the product can be isolated by filtration followed by chromatography of the filtrate on silica gel.
  • Method C can be employed utilizing the commercially-available reagent acetobromo- ⁇ -O-glucuronic acid, methyl ester (XII) and is outlined in Scheme 3.
  • N- iodosuccinimide N- iodosuccinimide
  • a strong acid such as trifluoromethanesulfonic acid
  • an inert atmosphere such as nitrogen
  • an inert anhydrous solvent such as dichloromethane for 0.1-10 h
  • reaction of Scheme 3(c) can be carried out by contacting XII with 1-10 equivalents of 4-penten-1-ol in the presence of 1-2 equivalents of an inorganic metal salt such as silver carbonate, at 0 to 40°C in an anhydrous inert solvent such as benzene for 1-40 h.
  • an inorganic metal salt such as silver carbonate
  • an anhydrous inert solvent such as benzene for 1-40 h.
  • G represents an aglycone toxin
  • inorganic metal salt such as mercuric cyanide at
  • G or HOG represent an aglycone toxin.
  • step C To a solution of 0.30 g of the product of step B in 15 mL of methanol was added 0.15 g of 20% palladium hydroxide-on-carbon (Pearlman's catalyst) and the mixture was stirred under a hydrogen atmosphere for 2 hours at ambient temperature. The catalyst was removed by filtration and the solution of the product was concentrated and partially purified by chromatography on silica gel, eluting with a gradient of methanol in dichloromethane.
  • Pearlman's catalyst palladium hydroxide-on-carbon
  • the intermediate methyl ester was saponified by dissolving it in 10 ml of methanol and adding 1 mL of a solution of 1N NaOH in 9 ml MeOH. After allowing the solution to stand for 2 1/2 hours at ambient
  • step B The product from step A (200 mg, 0.35 mmole) in trifluoroacetic acid (5 mL) was stirred at room temperature
  • Trifluoroacetic acid was removed under reduced pressure at room temperature.
  • step D The product from step C (0.1 g, 0.14 mmole) was dissolved in methanol (10 ml). A catalytic amount of NaOMe in MeOH was added. The reaction solution was stirred at room temperature for 1 hour. A drop of acetic acid was added. The solvent was removed under reduced pressure at room temperature. The residue thus obtained was purified on a silica gel column (eluted with 10% MeOH in CH 2 CI 2 ) to give the desired product (a foam, 74 mg, 90%).
  • step D The product from step D (70 mg, 0.1 mmole) was dissolved in MeOH (2 mL), dioxane (1 mL) and water (1 mL). NaOH aqueous solution (1N, 1 mL) was added. The mixture was stirred for 2 1/2 hours. The water was evaporated with a stream of air to give the product as an oil (70 mg).
  • Tables 5-12 contain representative protoxins that can be made and used by procedures described
  • Some aglycones that are difficult to make into glucuronides by conventional chemical synthesis means can be glucuronidated by enzymatic procedures.
  • Glucuronidation is a major means of detoxification and elimination of xenobiotics in mammals.
  • UDP-glucuronosyl transferase UDP-glucuronosyl transferase
  • UDP-glucuronosyl transferase has been shown to perform conjugation in vitro. Some glucuronide protoxins were prepared this way.
  • Magnesium chloride at 4 mM was used to facilitate the reaction. If the aglycone was difficult to solubilize in aqueous solution, up to 5% ethanol was used to achieve 2 mM concentration. Due to the relatively high ⁇ -glucuronidase (GUS) activity in the crude rabbit liver microsome enzyme preparation, 150 mM glucaro-1,4-lactone was included in the reaction to suppress GUS activity. If the reaction was run for more than one day, UDP- glucuronic acid was replenished daily. The reaction vessel was shaken gently at 37°C for the entire period.
  • GUS ⁇ -glucuronidase
  • chloride phase (contains aglycone) and an aqueous phase/partition 3x with n-butanol/obtain a butanol phase (glucuronide) and an aqueous phase.
  • glucuronide a small amount of the aglycone and UDP- glucuronic acid was evaporated to dryness in a rotary evaporator. It was then further purified by a
  • Glucuronides of the following compounds were prepared by enzymatic techniques:
  • ⁇ -glucuronidase enzyme activity is usually done in vitro.
  • the transgenic plant material harbouring the GUS gene product, ⁇ -glucuronidase is disrupted for color staining or other assays for the enzyme activity. It is generally assumed that the activity of ⁇ -glucuronidase detected in vitro is well correlated with the in vivo activity.
  • 4-methylumbelliferone glucuronide was fed to tobacco plants described in Examples 3 and 4, which exhibited anther-specific GUS expression by in vitro assays, using the cotton wick method as described below. Following different times after the feeding, anthers of young flowers were collected and assayed for the
  • MU 4-methylumbelliferone
  • MU- glucoside The phosphate buffer extract of anthers was divided into three aliquots. One aliquot was assayed for MU by fluorescence measurement. The second aliquot was treated with ⁇ -glucosidase, followed by MU
  • the protoxin solution was locally applied to the inflorescence stalk of the transgenic plant through a fine cotton wick.
  • the wick pulled through the stalk by a sewing needle, was usually embedded at a point about one to two centimeters from flower clusters.
  • the concentration of protoxin varied depending on the toxicity of the aglycone and the size of the inflorescence.
  • One properly embedded wick can deliver up to 1.5 mL of solution per day into the inflorescence stalk, and it can function well for over a week.
  • Freshly shed pollen from treated flowers was assayed for germinability in Brewbaker and Kwak medium (15% sucrose, 300 ppm calcium nitrate, 100 ppm boric acid). To test the degree of male sterility more definitively, the same treated pollen was also used to pollinate the stigma of untreated flowers. If a fruit resulted from such pollination, it indicated that the treatment did not cause complete male sterility.
  • the concentrations of the glucuronides were between 1 - 3 parts per million (ppm), depending on the size of the infloresence.
  • the chemical solution uptake rate was about 0.5 to 1.0 mL per day. More solution was added to the small vial in which the cotton wick was bathed. The solution addition was calibrated according to the growth of flowers on the inflorescence. All treated
  • Transgenic tobacco plants as described in Example 4 were sprayed with the methyl ester of maleic hydrazide glucuronide.
  • the pod-forming ability of said treated plants was adversely affected versus control plants.
  • NAME COSTELLO, JAMES A.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CAAGAAGCTC GTGGCAAAGG CTGGCTCTGG TTCATTGGAG TTGAATGACC TGTTCTGGGC 420
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention décrit un procédé servant à générer une stérilité mâle dans des plantes pouvant être transformées génétiquement. La plante est transformée avec une structure d'ADN combinant un promoteur spécifique d'organe mâle avec un enzyme qui réagit à une protoxine pour libérer, dans l'organe mâle ou gamète, une toxine de l'organe mâle ou gamète. L'invention décrit également la structure d'ADN, la protoxine, la plante transgénique contenant la structure, les semences produites par la plante transgénique, un procédé de production de semence hybride, la progéniture de la semence et les plantes transgéniques mises en contact avec la protoxine.
PCT/US1991/006234 1990-09-06 1991-09-05 Composes et structures servant a produire des plantes steriles males WO1992004454A1 (fr)

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TR24199A (tr) * 1986-05-02 1991-07-01 Vickers Plc Zirhli araclar icin (dozer bicagi gibi) bicak gibi atasman.
EP0589841A2 (fr) * 1992-09-24 1994-03-30 Ciba-Geigy Ag Procédés de production de semences hybrides
WO1994029465A1 (fr) * 1993-06-08 1994-12-22 Nunhems Zaden B.V. Procede de generation de plantes males steriles
WO1996040950A1 (fr) * 1995-06-07 1996-12-19 Pioneer Hi-Bred International, Inc. Procedes et produits d'assemblage de production de plantes males steriles
WO1997004116A1 (fr) * 1995-07-24 1997-02-06 Zeneca Limited Inhibition de la respiration cellulaire et production de plantes males steriles
US5689051A (en) * 1994-12-08 1997-11-18 Pioneer Hi-Bred International, Inc. Transgenic plants and DNA comprising anther specific promoter 5126 and gene to achieve male sterility
US5728926A (en) * 1988-02-03 1998-03-17 Pioneer Hi-Bred International, Inc. Antisense gene systems of pollination control for hybrid seed production
US5728558A (en) * 1988-02-03 1998-03-17 Pioneer Hi-Bred International, Inc. Molecular methods of hybrid seed production
US5763243A (en) * 1994-12-08 1998-06-09 Pioneer Hi-Bred International, Inc. Reversible nuclear genetic system for male sterility in transgenic plants
WO1998039462A1 (fr) * 1997-03-03 1998-09-11 Novartis Ag Procede de production de graines hybrides utilisant la fertilite femelle conditionnelle
US6162964A (en) * 1988-02-03 2000-12-19 Pioneer Hi-Bred International, Inc. Molecular methods of hybrid seed production
US6184439B1 (en) 1988-02-03 2001-02-06 Pioneer Hi Bred International, Inc. Antisense gene systems of pollination control for hybrid seed production
US6262339B1 (en) * 1993-06-08 2001-07-17 Hoechst Schering Agrevo Gmbg Process for generating male sterile plants
US6392123B1 (en) 1997-03-03 2002-05-21 Syngenta Participations Ag Female-preferential promoters isolated from maize and wheat
EP1213355A2 (fr) * 1990-12-24 2002-06-12 Biogemma Uk Limited Promoteurs spécifiques de tapetum provenant des brassicaceae spp
WO2003003816A2 (fr) 2001-07-06 2003-01-16 Monsanto Technology Llc Compositions et procedes destines a augmenter la segregation de transgenes dans les plantes
WO2003072792A3 (fr) * 2002-02-26 2004-03-18 Syngenta Ltd Procede de production selective de plantes steriles males ou femelles
EP0975778B1 (fr) * 1997-04-03 2007-06-13 DeKalb Genetics Corporation Utilisation de lignees de mais resistantes aux glyphosates
US7262055B2 (en) 1998-08-25 2007-08-28 Gendaq Limited Regulated gene expression in plants
US7285416B2 (en) 2000-01-24 2007-10-23 Gendaq Limited Regulated gene expression in plants
US8158850B2 (en) 2007-12-19 2012-04-17 Monsanto Technology Llc Method to enhance yield and purity of hybrid crops
US8716558B2 (en) 1999-06-30 2014-05-06 Marker Gene Technologies, Inc. Method of altering glycosylation of proteins in response to nojirimycin glucuronide in a plant cell expressing glucuronidase

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Cited By (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TR24199A (tr) * 1986-05-02 1991-07-01 Vickers Plc Zirhli araclar icin (dozer bicagi gibi) bicak gibi atasman.
US5741684A (en) * 1988-02-03 1998-04-21 Pioneer Hi-Bred International, Inc. Molecular methods of hybrid seed production
US6191343B1 (en) 1988-02-03 2001-02-20 Pioneer Hi-Bred International Molecular methods of hybrid seed production
US6184439B1 (en) 1988-02-03 2001-02-06 Pioneer Hi Bred International, Inc. Antisense gene systems of pollination control for hybrid seed production
US6737560B1 (en) 1988-02-03 2004-05-18 Pioneer Hi-Bred International, Inc. Molecular methods of hybrid seed production
US5728558A (en) * 1988-02-03 1998-03-17 Pioneer Hi-Bred International, Inc. Molecular methods of hybrid seed production
US5728926A (en) * 1988-02-03 1998-03-17 Pioneer Hi-Bred International, Inc. Antisense gene systems of pollination control for hybrid seed production
US6198026B1 (en) 1988-02-03 2001-03-06 Pioneer Hi-Bred International, Inc. Molecular methods of hybrid seed production
US6162964A (en) * 1988-02-03 2000-12-19 Pioneer Hi-Bred International, Inc. Molecular methods of hybrid seed production
US6198023B1 (en) 1988-02-03 2001-03-06 Pioneer Hi-Bred International, Inc. Molecular methods of hybrid seed production
US6255564B1 (en) 1988-02-03 2001-07-03 Paladin Hybrids, Inc. Molecular methods of hybrid seed production
EP1213355A2 (fr) * 1990-12-24 2002-06-12 Biogemma Uk Limited Promoteurs spécifiques de tapetum provenant des brassicaceae spp
EP1213355A3 (fr) * 1990-12-24 2002-11-20 Biogemma Uk Limited Promoteurs spécifiques de tapetum provenant des brassicaceae spp
US5659124A (en) * 1992-09-24 1997-08-19 Novartis Corporation Transgenic male sterile plants for the production of hybrid seeds
EP0589841A2 (fr) * 1992-09-24 1994-03-30 Ciba-Geigy Ag Procédés de production de semences hybrides
US5824542A (en) * 1992-09-24 1998-10-20 Novartis Finance Corporation Methods for the production of hybrid seed
EP0589841A3 (fr) * 1992-09-24 1995-04-26 Ciba Geigy Ag Procédés de production de semences hybrides.
US6262339B1 (en) * 1993-06-08 2001-07-17 Hoechst Schering Agrevo Gmbg Process for generating male sterile plants
WO1994029465A1 (fr) * 1993-06-08 1994-12-22 Nunhems Zaden B.V. Procede de generation de plantes males steriles
US6013859A (en) * 1994-07-14 2000-01-11 Pioneer Hi-Bred International, Inc. Molecular methods of hybrid seed production
US7521241B2 (en) 1994-08-20 2009-04-21 Gendaq Limited Regulated gene expression in plants
US5792853A (en) * 1994-12-08 1998-08-11 Pioneer Hi-Bred International, Inc. Reversible nuclear genetic system for male sterility in transgenic plants
US5837851A (en) * 1994-12-08 1998-11-17 Pioneer Hi-Bred International, Inc. DNA promoter 5126 and constructs useful in a reversible nuclear genetic system for male sterility in transgenic plants
US5795753A (en) * 1994-12-08 1998-08-18 Pioneer Hi-Bred International Inc. Reversible nuclear genetic system for male sterility in transgenic plants
US5763243A (en) * 1994-12-08 1998-06-09 Pioneer Hi-Bred International, Inc. Reversible nuclear genetic system for male sterility in transgenic plants
US5689051A (en) * 1994-12-08 1997-11-18 Pioneer Hi-Bred International, Inc. Transgenic plants and DNA comprising anther specific promoter 5126 and gene to achieve male sterility
WO1996040950A1 (fr) * 1995-06-07 1996-12-19 Pioneer Hi-Bred International, Inc. Procedes et produits d'assemblage de production de plantes males steriles
WO1997004116A1 (fr) * 1995-07-24 1997-02-06 Zeneca Limited Inhibition de la respiration cellulaire et production de plantes males steriles
US6815577B1 (en) 1997-03-03 2004-11-09 Syngenta Participations Ag Method of hybrid seed production using conditional female sterility
US6392123B1 (en) 1997-03-03 2002-05-21 Syngenta Participations Ag Female-preferential promoters isolated from maize and wheat
WO1998039462A1 (fr) * 1997-03-03 1998-09-11 Novartis Ag Procede de production de graines hybrides utilisant la fertilite femelle conditionnelle
EP0975778B1 (fr) * 1997-04-03 2007-06-13 DeKalb Genetics Corporation Utilisation de lignees de mais resistantes aux glyphosates
US7262055B2 (en) 1998-08-25 2007-08-28 Gendaq Limited Regulated gene expression in plants
US8716558B2 (en) 1999-06-30 2014-05-06 Marker Gene Technologies, Inc. Method of altering glycosylation of proteins in response to nojirimycin glucuronide in a plant cell expressing glucuronidase
US7285416B2 (en) 2000-01-24 2007-10-23 Gendaq Limited Regulated gene expression in plants
US7960610B2 (en) 2001-07-06 2011-06-14 Monsanto Technology Llc Methods for enhancing segregation of transgenes in plants
EP1404850A4 (fr) * 2001-07-06 2006-05-10 Monsanto Technology Llc Compositions et procedes destines a augmenter la segregation de transgenes dans les plantes
US9175298B2 (en) 2001-07-06 2015-11-03 Monsanto Technology Llc Compositions for enhancing segregation of transgenes in plants
US7288694B2 (en) 2001-07-06 2007-10-30 Monsanto Technology Llc Methods for enhancing segregation of transgenes in plants and compositions thereof
WO2003003816A2 (fr) 2001-07-06 2003-01-16 Monsanto Technology Llc Compositions et procedes destines a augmenter la segregation de transgenes dans les plantes
EP2239332A2 (fr) 2001-07-06 2010-10-13 Monsanto Technology LLC Compositions et procédés destinés à augmenter la ségrégation de transgènes dans les plantes
US7858370B2 (en) 2001-07-06 2010-12-28 Monsanto Technology Llc Compositions for enhancing segregation of transgenes in plants
EP2272965A2 (fr) 2001-07-06 2011-01-12 Monsanto Technology LLC Procédés pour améliorer la ségrégation de transgènes dans les plantes et compositions correspondantes
EP1404850A2 (fr) * 2001-07-06 2004-04-07 Monsanto Technology LLC Compositions et procedes destines a augmenter la segregation de transgenes dans les plantes
WO2003072792A3 (fr) * 2002-02-26 2004-03-18 Syngenta Ltd Procede de production selective de plantes steriles males ou femelles
US7939709B2 (en) 2002-02-26 2011-05-10 Syngenta Limited Method for selectively producing male or female sterile plants
US8642836B2 (en) 2002-02-26 2014-02-04 Syngenta Limited Method of selectively producing male or female sterile plants
EP2278017A1 (fr) * 2002-02-26 2011-01-26 Syngenta Limited Procédé de production séléctive des plantes stérile male ou femelle
US8946507B2 (en) 2002-02-26 2015-02-03 Syngenta Limited Method of selectively producing male or female sterile plants
EP1481068A2 (fr) * 2002-02-26 2004-12-01 Syngenta Limited Procede de production selective de plantes steriles males ou femelles
US8158850B2 (en) 2007-12-19 2012-04-17 Monsanto Technology Llc Method to enhance yield and purity of hybrid crops

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