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WO1993009237A1 - Mais tres sucre et ameliore - Google Patents

Mais tres sucre et ameliore Download PDF

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Publication number
WO1993009237A1
WO1993009237A1 PCT/EP1992/002531 EP9202531W WO9309237A1 WO 1993009237 A1 WO1993009237 A1 WO 1993009237A1 EP 9202531 W EP9202531 W EP 9202531W WO 9309237 A1 WO9309237 A1 WO 9309237A1
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WIPO (PCT)
Prior art keywords
corn
promoter
supersweet
dna
adp
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PCT/EP1992/002531
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English (en)
Inventor
Scott E. Nichols
Michael H. Pauly
Ralph M. Sinibaldi
Donald P. Weeks
Frederick Charles Baker
Marian L. Duncan
Original Assignee
Sandoz Ltd.
Sandoz-Patent-Gmbh
Sandoz-Erfindungen Verwaltungsgesellschaft M.B.H.
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Application filed by Sandoz Ltd., Sandoz-Patent-Gmbh, Sandoz-Erfindungen Verwaltungsgesellschaft M.B.H. filed Critical Sandoz Ltd.
Priority to HU9401302A priority Critical patent/HU219313B/hu
Priority to EP92923267A priority patent/EP0611395A1/fr
Priority to JP5508166A priority patent/JPH07500965A/ja
Priority to AU29203/92A priority patent/AU670417B2/en
Publication of WO1993009237A1 publication Critical patent/WO1993009237A1/fr

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    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis

Definitions

  • This invention relates to plant biotechnology and specifically to corn, alternatively novn as maize, Zea mays or Indian corn.
  • the invention relates to supersweet corn with improved qualities, methods of producing the improved supersweet corn, vectors for the genetic engineering of the corn, DNA used to transform corn, and genomic clones containing the desired DNA.
  • the endosperm is a major site of starch deposition during maize kernel development. Many genetic loci affecting carbohydrate metabolism are known and some have been biochemically characterized. When some of these genes are not expressed, the result is generally a decrease in starch biosynthesis and a concomitant accumulation of sucrose to give a seed product commonly referred to as sweet corn and used as a human food product. Corn in which the levels of starch biosynthesis are normal is commonly referred to as field corn, and is often used for animal feed.
  • Su Sud-1
  • Sh-2 shrunken-2
  • Bt-2 brittle-2
  • Sweet corn results from the presence of a homozygous recessive allele at the sugary-1 locus on chromosome 4, designated su. When su is present, there is a 3-5 fold increase in the percentage of sucrose (dry weight) relative to wild-type Su lines.
  • the Sh-2 and Bt-2 genes, both involved in starch production, encode subunits of the enzyme ADP-glucose pyrophosphorylase (ADP-GPP) ( ⁇ _D-glucose-l-phosphate adenyl transferase, EC 2.7.7.27).
  • ADP-GPP ADP-glucose pyrophosphorylase
  • ADP-GPP catalyzes the reversible synthesis of ADP-glucose and pyrophosphate from ATP and glucose-1-phosphate. The equilibrium constant for this reaction is unity and the reaction is driven by pyrophosphate hydrolysis.
  • ADP-glucose is the glucosyl donor for starch chain elongation catalysed by starch synthetase.
  • ADP-GPP is made up of four subunits: two subunits of a 60 kDa polypeptide encoded by the Sh-2 gene (hereinafter referred to as the Sh-2 gene), and two subunits of a 55 kDa polypeptide encoded by the Bt-2 gene (hereinafter referred to as the Bt-2 protein).
  • the 60 kDa or the 55 kDa subunits are not properly synthesized, and little or no functional ADP-GPP is made.
  • starch synthesis is impaired, sucrose accumulates to 2-4 times the levels of normal sweet corn, and the corn is "supersweet".
  • supersweet corn which, when harvested for food at approximately 77% moisture content, typically about 19-21 days after pollination, has high sugar and low starch for flavour but has mature kernels at approximately 30-35% moisture content, about 55 days after pollination, with a starchy field corn like endosperm for ease of seed processing, improved germination and disease resistance. No such supersweet corn is currently available.
  • European patent application EP 0 368 506 discloses a plant having enhanced ability to produce starch comprising a starch synthesizing plant having stably incorporated within its genome by transformation of one or more than one additional copy of a gene encoding ADP-glucose pyrophosphorylase.
  • International patent application WO 91/19806 concerns a method for increasing the starch content of a plant which comprises altering said plant to increase the ADP glucose pyrophosphorylase activity in said plant.
  • EP 0455 316 concerns plasmids which may be used to increase or decrease protein concentration or to both reduce starch concentration and increase saccharide concentration in plant cells, for instance by decreasing ADP-GPP activity.
  • IGFB European patent application EP 0455 316
  • This invention provides corn with improved kernel characteristics such that when harvested for food it has "supersweet” taste, but when harvested for seed, it has starchy kernels similar to those of field corn.
  • this invention provides supersweet corn which has been genetically modified to express ADP-glucose pyrophophorylase activity at approximately 25-30 days post pollination.
  • the invention includes genetically modified corn plants, and propagatable parts thereof including cells and tissue, and seeds, and progeny thereof including hybrid progeny thereof.
  • this aspect of the invention includes supersweet corn which is homozygous recessive sh-2 and contains within its genome a functional Sh-2 structural gene under the control of a heterologous promoter.
  • this aspect also includes supersweet corn which is homozygous recessive bt-2 and contains within its genome a functional Bt-2 structural gene under the control of a heterologous promoter.
  • this aspect also includes supersweet corn which is sh-2 and/or bt-2 homozygous recessive and contains within its genome a functional Sh-2 gene under the control of a heterologous promoter and a functional Bt-2 gene under the control of a heterologous promoter.
  • the heterologous promoters used in the above embodiments of the invention may comprise developme tally delayed promoters such that the Sh-2 gene and/or Bt-2 gene does not begin to be expressed until 20 to 25 days post pollination.
  • Such corn plants are phenotypically characterized by having supersweet corn kernels at the time at harvest for food but have kernels which resemble the starchy kernels of field corn when harvested later for seed production.
  • heterologous promoters used in this aspect of the invention may comprise inducible promoters.
  • inducible promoters if the plant is grown for its seed crop, it is exposed to the inducer and starch synthesis takes place. If grown for a food crop, the plant is not exposed to the inducer, so very little, if any starch synthesis takes place and the kernels are supersweet.
  • the invention also provides sweet corn which has been genetically modified by inclusion of anti-sense DNA the transcription of which is regulated or regulatable such that expression of ADP-glucose pyrophosphorylase activity is or can be inhibited until at least 25 to 30 days post pollination.
  • the sweet corn contains within its genome anti-sense Sh-2 DNA under the control of a heterologous promoter or/and anti-sense Bt-2 DNA under the control of a heterologous promoter.
  • the heterologous promoters used may comprise inducible promoters, such that when the plant is exposed to the inducer anti-sense DNA is transcribed and blocks expression of ADP-GPP activity resulting in supersweet corn kernels. If no inducer is used with such inducible promoters, normal sweet corn results which may be harvested for food or seed as is customary for normal sweet corn. When an inducer is used the resulting supersweet corn is preferably harversted for food and is not grown on for seed harvest.
  • the promoters used in this further aspect may comprise a promoter which is active in early endosperm development, i.e. a promoter which is active during the period up to 25 to 30 days post pollination.
  • promoters include natural endosperm promoters which are active early in endosperm development.
  • synthetic promoters which function as early active endosperm promoters may be used.
  • a natural or synthetic constitutive promoter element which has been manipulated to contain an early active endosperm promoter element and which is operatively linked to an element which confers endosperm activity may be used.
  • the invention provides a DNA expression cassette comprising a protein coding DNA sequence and a promoter wherein the DNA sequence comprises DNA coding for a corn ADP-GPP subunit (e.g. Sh-2 or Bt-2) or a functional part thereof or a DNA sequence which hybridises therewith under stringent hybridisation conditions and which codes for a protein, having the activity of an ADP-GPP subunit i.e. a protein, having ADP-GPP subunit activity, and the promoter comprises a developmentally delayed promoter or an inducible promoter.
  • a corn ADP-GPP subunit e.g. Sh-2 or Bt-2
  • the promoter comprises a developmentally delayed promoter or an inducible promoter.
  • the invention also provides a DNA transcription cassette comprising an anti-sense DNA sequence complementary to a DNA sequence which codes for a peptide having ADP-GPP subunit activity, and an inducible promoter or a promoter which is active in early endosperm development.
  • the invention includes vectors containing the DNA expression or transcription cassettes, processes for transforming corn with the cassettes or vectors and corn plants and parts thereof (tissue and cells) transformed with the cassettes or vectors.
  • the invention also includes cultivation processes in which genetically modified supersweet corn or sweet corn according to the invention in which inducible promoters are used, is cultivated and exposed to a corresponding inducer as required for food or seed harvest.
  • the invention includes genomic clones of Sh-2 and Bt-2.
  • the invention includes the genomic clone of Sh-2 contained in plasmid pZ01300 and the genomic clone of Bt-2 contained in plasmid pZ01301, which were deposited at the American Type Culture Collection on 2 October 1991 under accession numbers 75129 and 75130 respectively, and parts variants and analogues thereof.
  • the invention includes the Sh-2 genomic DNA sequence as set out in the Sequence listing as SEQ ID N0:1 and parts, variants and analogues thereof. Applicants hereby indicate that they elect the "expert solution" as regards availability of the above deposits during pendency of the EPC patent application designated herein, under the provisions of Rule 28 (4) EPC.
  • “Sweet corn” means Zea mays in which the genes at the sugary-1 locus are in the homozygous recessive condition.
  • Supersweet corn means Zea mays in which the shrunken-2 and/or brittle-2 genes are in the homozygous recessive condition.
  • Heterologous promoter means a promoter which does not naturally control expression of its associated structural gene, although the promoter may be of Zea mays origin.
  • sh-2 refers to a recessive shrunken-2 gene.
  • sh-2 When both copies of the shrunken-2 gene are recessive (sh-2), the result is supersweet corn.
  • B-2 refers to the wild-type brittle-2 gene.
  • bt-2 refers to the recessive brittle-2 gene.
  • bt-2 recessive
  • Dpp days post pollination
  • the time of "harvest for food” is generally that time when kernel moisture content is approximately 77%. Under typical environmental growing conditions, this is generally 19-21 dpp.
  • the time of "harvest for seed crop” is generally that time when the kernel moisture content is approximately 30-35%. Under optimal environmental growing conditions, this is approxmately 55 dpp.
  • “Stringent hybridisation conditions” as used throughout the specification and claims are those in which hybridisation is effected in a standard manner at 65°C in 4X buffered saline (a.k.a. SSPE buffer) followed by merely washing at 57°C in 0.2X SSPE, which will not affect true duplexes which have formed.
  • Figure 1 shows the Northern blot for Sh-2 mRNA in two corn lines, a regular sweet corn hybrid "201 X 202" (su Sh-2) and a supersweet inbred "101" (Su sh-2).
  • the probe used is [32P]-Sh-2 cDNA.
  • Lanes 1-5 are “201 X 202" and lane 6 is "101”.
  • Lane 1 shows 2.5 ⁇ g endosperm bound polysomal RNA at 25 dpp; Lane 2 shows 2.5 ⁇ g free polysomal RNA at 25 dpp; Lane 3 shows 10 ⁇ g total endosperm RNA at 25 dpp; Lane 4 is 10 ⁇ g leaf total RNA; Lane 5 is 10 ⁇ g root RNA; Lane 6 is 10 ⁇ g total endosperm RNA 21 dpp.
  • Figure 2 is the same blot as in Figure 1 after it is stripped and subsequently probed with [32P]-labelled Bt-2 cDNA.
  • Figure 3 is a graph showing the developmental profile of both polysomal and total Bt-2 RNA.
  • Figure 4 is a graph showing the developmental profile of both polysomal and total Sh-2 RNA.
  • Figure 5 is a composite restriction map of the Sh-2 genomic clone.
  • Figure 6 is a restriction map of the Bt-2 genomic clone.
  • Figure 7 is a graph of ADP glucose pyrophosphorylase activity of anti-sense transformants. 17.5 ⁇ g of total soluble cellular protein was added to each assay. The assay is coupled to NAPDH reduction and absorbance at 340 n is measured. The various aspects of the invention identified above are now described in greater detail.
  • One approach to modifying supersweet corn so that it retains a high sucrose concentration when harvested for food, but whose seed crop is starchy is to transfer either the Sh-2 structural gene into a homozygous sh-2 plant and/or a Bt-2 structural gene into a homozygous bt-2 plant under the control of a promoter which does not engender transcription until after harvest of the food crop.
  • These promoters collectively referred to as “developmentally delayed " " promoters, normally control synthesis of mRNAs that appear late in the development of endosperm.
  • Such promoters may be detected in an assay utilizing identifiable markers (such as GUS) operably linked to the putative promoter and assayed in endosperm tissue.
  • the developmentally delayed promoter does not become active in endosperm tissue until after about 20 days post pollination (dpp).
  • dpp days post pollination
  • the optimum promoter for a given heterologous gene may depend on characteristics of the given gene, but should be chosen so that maximum expression and/or gene product accumulation occurs at 25-30 dpp.
  • Developmentally delayed promoters may be obtained using the procedures described herein.
  • Sh-2 and Bt-2 protein accumulations are also measured as a confirmation of the above finding.
  • Anti-sera are made to both Sh-2 and Bt-2 proteins. Protein is isolated from the endosperm at different times post-pollination and is electrophoretically separated and blotted onto a nitrocellulose filter. The filters are probed using the appropriate anti-serum and an antibody sandwich assay is used to visualize the band. The binding is quantified, and is found to confirm the above findings.
  • this invention also includes plants transformed with both the Sh-2 and Bt-2 genes, said genes each under the control of a heterologous promoter, and preferably each under the control of a different heterologous promoter.
  • a modified subtractive technique strategy may also be used.
  • double stranded cDNA from a particular time point is made blunt-ended and linkers are added.
  • the linkers serve as a PCR oligomer binding site.
  • the cDNA is then amplified prior to cloning and can be used as a probe as well.
  • This technique allows identification of clones which have either qualitative or quantitative differences between two different mRNA populations.
  • Two developmentally delayed promoters are isolated, one which directs transcription beginning at approximately 20 dpp, hereinafter referred to as Promoter 20, and one which initiates transcription at approximately 25 dpp, hereinafter referred to as Promoter 25.
  • Genomic clones of Sh-2 and Bt-2 are made using a genomic library of maize inbred line W22 and Lambda EMBL3.
  • W22 is a publicly available line which can be obtained from Maize Cooperative, Stock Center, Univ. Missouri, Columbia, Missouri).
  • the genomic clones are screened using cDNA of Sh-2 or Bt-2. Two clones are isolated for Sh-2, shown in Figure 5. A single clone is isolated for Bt-2, and this appears in Figure 6.
  • the genomic clones for Bt-2 and for Sh-2 comprise another aspect of this invention.
  • Sh-2 genomic clones were difficult, as standard sub-cloning techniques did not readily produce the desired subclones. Surprisingly it was found that the Sh-2 gene contains a sequence which interferes with plasmid replication.
  • One technique which successfully produced clones, albeit at a low level (approximately 1% of the expected level) was a three-way ligation.
  • One aliquot of vector carrying Sh-2 sequences was cut in its ampicillin resistance (ampr) gene with Seal and in its polylinker with BamHI. A second aliquot was cut with Seal and Sail.
  • constructs may be assembled: Promoter 20-Sh-2 and Promoter 25-Bt-2.
  • constructs and other constructs which include other preferred developmentally delayed promoters operably linked to either the Sh-2 or the Bt-2 structural gene are specific embodiments of this invention.
  • an alternative approach is to place the Sh-2 and/or Bt-2 genes under the control of a promoter which is not developmentally regulated, but is inducible chemically.
  • a preferred class of inducible promoters are steroid responsive promoters. Gene expression activation by steroids such as ecdysteroid-like molecules occurs via ligand binding to protein receptors which are members of the steroid receptor superfamily. The steroid ligand binds to its cognate receptor altering receptor conformation, and the resultant binary complex recognises and binds to a steroid response element (SRE) present in the promoter to modulate transcription.
  • SRE steroid response element
  • the plant cell is transformed with genes encoding both the receptor and the the desired gene operably linked with a steroid responsive promoter, i.e. a natural or constructed promoter comprising an SRE.
  • a steroid responsive promoter i.e. a natural or constructed promoter comprising an SRE.
  • promoters include those which are responsive to the estrogen/ estrogen receptor, progesterone/ progesterone receptor, vitamin D/ vitamin D receptor and dexamethasone/ glucocortocoid receptor complexes.
  • Preferred promoter are those which are responsive to the invertebrate molting hormone, 20-hydroxyecdysone (20-0HE) / receptor complex, which can be induced by the agonist compound l,2-dibenzoyl,l-tert-butyl hydrazine.
  • the corn plant is transformed with two constructs: 1) the inducible promoter Sh-2 and/or Bt-2 construct and 2) a receptor for the inducer. Shortly before or after pollination the transformed corn is exposed to the chemical inducer. Transcription of the Sh-2 and/or Bt-2 gene is then initiated, leading to kernels with starchy endosperms. If the plant is transformed with both the Sh-2 and Bt-2 structural genes, both genes may be under the control of an inducible promoter, preferably a part of a ecdysteroid system.
  • EcRB ecdysteroid binding protein
  • Plants which are transformed with an inducible promoter do not exhibit substantial ADP-GPP activity until presented with the inducer, . after which starch synthesis and starch accumulation occurs.
  • the progeny of these plants (either inbred or the result of a cross with a non-related, transformed or non-transformed plant) inducibly express the starchy phenotype. All of these progeny plants are included within the invention.
  • an anti-sense transcription construct can be used to regulate starch synthesis.
  • the Sh-2 gene or the Sh-2 cDNA in its anti-sense configuration is placed under the control of an inducible promoter, such as those described above or an endosperm promoter active early in endosperm development.
  • an inducible promoter such as those described above or an endosperm promoter active early in endosperm development.
  • a synthetic promoter can be made which contains an early active endosperm promoter element operatively linked to an element which confers endosperm activity.
  • promoters which contain early active endosperm elements include 1) various zein promoters, such as the promoter for gamma, beta or alpha zein; 2) a 198 base pair portion of the low molecular weight glutenin (LMWG) promoter (as in Colot et al, 1987 EMBO J. 6(12):355-359, which is hereby incorporated by reference); and 3) a 227 base pair portion of the high molecular weight wheat glutenin (HMWG) promoter (as in Thomas and Flavell The Plant Cell 2:1171, which is hereby incorporated by reference).
  • LMWG low molecular weight glutenin
  • HMWG high molecular weight wheat glutenin
  • the aforementioned elements may be operatively linked to a normally constitutive promoter element such as the minimal 35S promoter (i.e. comprising the -46 base pair upstream region) or the minimal HSP82 promoter, such as described in co-pending application serial number 07/791,921 which is incorporated by reference.
  • a normally constitutive promoter element such as the minimal 35S promoter (i.e. comprising the -46 base pair upstream region) or the minimal HSP82 promoter, such as described in co-pending application serial number 07/791,921 which is incorporated by reference.
  • a normal sweet corn plant (su Sh-2) is transformed with one of the aforementioned constructs.
  • anti-sense Sh-2 mRNA is transcribed which hybridizes with the wild type Sh-2 mRNA, thereby blocking synthesis of the Sh-2 protein. Consequently, starch synthesis is impaired and sucrose accumulates.
  • this anti-sense approach can also be used with the Bt-2 gene.
  • the promoter constructs used for the expression and transcription constructs also contain other DNA sequences, such as enhancers and 3' termination sequences. Such sequences are well known in the art.
  • the cloned construct is then inserted into a plasmid or other vector suitable for transformation.
  • Maize type II friable callus suitable for transformation and regeneration is cultured using known techniques (Green, L.E. et al. 1975. "Plant Regeneration From Tissue Cultures of Maize.” Crop Science 15:417-421 and Vasil, V. et al. 1984. "Somatic Embryogenesis in Long-term Callus Cultures of Zea mays L. (Gramineae)" ⁇ mer. J. Bot. 71:158-61, both of which are incorporated herein by reference). Cells are transformed using the ballistic technique, detailed below in the Examples. Putative transformants are regenerated into intact plants.
  • the plants which have been transformed with a developmentally delayed promoter construct are grown to maturity and are seen to bear supersweet corn which later (approximately 25-30 dpp) becomes starchy.
  • the progeny of these plants (either inbred or the result of a cross with a non-related, transformed or non-transformed plant) also bear supersweet corn which ages into starchy corn. All of these progeny plants are included within the invention.
  • Polysomes are isolated according to the methods of Larkins et al. 1976 "Isolation and in Vitro Translation of Zein Messenger Ribonucleic Acid” Bioche 75 (25).: 5586, Larkins et al. 1976. "Storage Protein Synthesis in Maize” Plant Physiol 57:740-745, and Larkins et al. 1978. "Synthesis and Deposition of Zein in Protein Bodies of Maize Endosperm” Plant Physiol. 62:256-263, which are hereby incorporated by reference.
  • Isolated polysomes are resuspended in 1% w/v triisopropylnaphthalene-sulfonic acid, Na salt; 6% w/v p-amino-salicylic acid; 0.1 M TRIS-HC1, pH 7.6; 50 mM EGTA; 0.1 M NaCl; 1% w/v SDS; and 50 mM 2-mercaptoethanol (Rochester et al. 1986. EMBO J. 5:451-458). An equal volume of phenol/chloroform/isoamyl alcohol (25/24/1) is added. After centrifugation, the supernatant fluid is removed to a new tube and re-extracted.
  • cDNA clones for Sh-2 and Bt-2 are isolated as reported by Bhave et al., 1990, "Identification and Molecular Characterization of Shrunken-2 cDNA Clones of Maize” The Plant Cell 2:581-588, and Bae et al., 1990, Maydica 35:317-322, both of which are incorporated by reference.
  • a genomic library from public maize line W22 is made in Lambda EMBL3 according to standard procedures as described in Ausubel et al. 1987. Current Protocols in Biology, Wiley Interscience.
  • Approximately 5 X 10 6 clones are transferred to nitrocellulose and are screened using the radiolabelled cDNAs as probes, in 5X SSC, 0.1% SDS, IX Denhardt's solution, 50% formamide and 42°C for hybridization. Filters are washed four times for 30 minutes at 65°C in 0.1X SSC and 0.1% SDS.
  • Genomic clones isolated in this way are mapped first using Lambda Map (Promega). The rough map generated is then refined using conventional mapping techniques of probing Southern blots of various restriction digests of the clones. Maps thus generated are shown in Figures 5-6.
  • Subcloning of the genomic clones is done with pT7T3 vectors (Pharmacia). Nested deletions are performed with a commercial kit according to the directions of the manufacturer (Pharmacia). Sequencing is done by the dideoxy chain termination method using T7 polymerase (Pharmacia). Sequences of the Sh-2 genomic clone is given in TABLE 1 (SEQ.ID N0:1).
  • GST glutathione-S-transferase
  • Rabbits are immunized with 650-850 ⁇ g of either of the purified proteins in complete Freund's adjuvant and boosted every other week with 250 ⁇ g of protein in incomplete Freund's adjuvant. Anti-Bt-2 and Anti-Sh-2 sera is obtained.
  • Modified subtractive cDNA libraries are made using the procedure of Timblin et al.(1990 Nucl. Acids Res. 18(6).-1587-1593) which is hereby incorporated by reference.
  • mRNA populations are isolated 15, 25, and 35 dpp. Double stranded cDNAs from these populations are made blunt and linkers are added. The linkers are chosen so as to serve as a Polymerase Chain Reaction (PCR) oligomer binding site.
  • the resulting cDNAs are amplified according to the manufacturer's recommended instructions and are also used as probes.
  • Clones are identified which show differences (either quantitative or qualitative) in the mRNA populations. These represent potential developmentally delayed promoters. The copy numbers of these clones are determined using standard protocols, and those with a low copy number ( ⁇ 5 copies per genome) are selected. These are used for in situ hybridization to maize endosperm sections to determine which are active in appropriate cell layers.
  • genes are isolated from a maize genomic library. Promoter regions are isolated using conventional techniques. Putative promoters are tested by constructing b-glucuronidase (GUS) fusions, delivering the hybrid construct to longitudinal endosperm sections via the ballistic particle delivery process and visually determining their activity by GUS staining (Jefferson, R.A. 1988. in Genetic Engineering, Principles and Methods, Vol. 10:247-263 J.K. Setlow, Ed.) The promoters resulting from this isolation and evaluation are designated Promoter 20, which maximizes mRNA synthesis at approximately 20 dpp and Promoter 30 which maximizes mRNA synthesis at approximately 30 dpp. These are used to make transformation vectors, discussed in Example 7, below.
  • GUS b-glucuronidase
  • Example 4 The procedure of Example 4 is repeated, using polysomal RNA. The same developmentally delayed promoters are identified.
  • Promoter 20 from Example 5 is linked to the Sh-2 gene, along with the Sh-2 gene endogenous terminator. This construct is referred to as the agronomic cassette-20.
  • a similar cassette is constructed using Promoter 3C linked to the Bt-2 gene and terminator, referred to as agronomic cassette-30.
  • the vectors used for maize callus transformation are pUC19 derivatives a bacterial selectable marker ( ⁇ -lactamase, i.e. ampicillin/carbenicillin resistance) and a maize selectable marker.
  • the maize selectable marker has the cauliflower mosaic virus 35S promoter, a portion of intervening sequence II from maize alcohol dehydrogenase, and a selectable gene: neomycin phosphotransferase II (i.e. kanamycin resistance) or phosphinothricin acetyltransferase (i.e. resistance to the herbicides Basta and Bialophos) or acetolactate synthase (i.e. resistance to the herbicide chlorsulfuron).
  • the selectable gene sequence is followed by the nopalene synthase terminator.
  • agronomic cassette-20 is inserted using standard techniques into the above vector to create pSh-2.
  • agronomic cassette-30 is inserted into the vector to create pBt-2.
  • E. coli are transformed with either plasmid using standard techniques (Ausabel et al. , supra).
  • 1.5 mm embryos (10-13 dpp) from 29 sh-2 lines are explanted and placed on initiation medium (either Murashige-Skoog with 1.5 mg/1 Dicamba or N6 with 1 mg/1 2,4-D). Callus is transferred to and maintained on N6 containing 6 mM asparagine, 12 mM proline and 1 mg/12,4-D. Friable callus is selected and is used in the transformation procedure, below.
  • Plasmid pSh-2 is introduced into Type II maize callus of a sh-2 supersweet corn line using a ballistic process. Plants are regenerated following known procedures and are self-pollinated. ADP-GPP activity is determined prior to 20 dpp and after 30 dpp to ascertain the induction of Sh-2 transcription and its subsequent translation. Plants are then evaluated for agronomic performance, and those with the desired phenotype are identified.
  • Plasmid pBt-2 is introduced into Type II maize callus of a bt-2 line using a ballistic process. Plants are regenerated following known procedures and are self-pollinated. ADP-GPP activity is determined prior to 20 dpp and af er 30 dpp to ascertain the induction of Bt-2 transcription and translation. Plants are then evaluated for agronomic performance, and those with the desired phenotype are selected.
  • EXAMPLE 9 INDUCIBLE PROMOTER CONSTRUCTS
  • Vectors for callus transformation are constructed similarly to those described in Example 6, except for the agronomic cassettes.
  • the agronomic cassette contains the promoter which is inducible by the 20-hydroxyecdysone binding protein binary complex operably linked to either the Sh-2 gene or Bt-2 gene and their respective endogenous terminators.
  • the plasmid containing the inducible Sh-2 construct is designated pISh-2.
  • the plasmid containing the inducible Bt-2 construct is designated pIBt-2.
  • Maize calli are co-transformed with either pISh-2 and pRecept or pIBt-2 and pRecept.
  • pRecept is a compatible plasmid which codes for expression of the 20-hydroxyecdysone receptor. Transformants carrying both heterologous gene constructs are selected and regenerated into plants which inducibly express either Sh-2 or Bt-2.
  • Construct 1110 contains the 35S promoter, the maize IVS6 intron, EcRB gene construct and the Nos terminator.
  • Construct 1112 comprises the Steroid Response Element (SRE) linked to the -46 35S promoter fragment (base pairs -46 to +1), the maize IVS6 intron, GUS gene and the NOS terminator.
  • SRE Steroid Response Element
  • Construct 1113 comprises the SRE (as above) linked to a maize promoter, the IVS6 intron, the gene and the NOS terminator.
  • Black Mexican Sweet (BMS) suspension cells are transformed with the above constructs using the ballistic technique, incubated overnight, and then are overlaid with X-gluc staining solution which may contain 1 ⁇ M of the phytoecdysteroid Ponasterone A (PNA), as indicated below. Three plates are transformed for each construct. Results are presented below.
  • PNA phytoecdysteroid Ponasterone A
  • the ethanol phase is then concentrated in a vacuum and partitioned between methylene chloride: ethanol: water (2 ml each phase), and the methylene chloride phase is then concentrated to dryness.
  • This is dissolved in ethyl acetate: ethanol (2:1) as a 5% solution and filtered through neutral alumina (10% H 2 0, 2 g) and is eluted with further solvent (25 ml).
  • the total eluate is evaporated to dryness.
  • the crude ecdysteroids are then dissolved in methylene chloride: ethanol (2:1 to make a 5% solution w/v) and an aliquot is transferred to a TLC plate for purification.
  • the fractions are tested for ecdysone agonist activity by incubation with a genetically engineered Drosophila cell line which is ecdysone responsive. No ecdysone activity is observed in either the sweet corn endosperm or BMS cell extracts, with a limit of detection ⁇ 20 ng 20-0H-ecdysone equivalents/g. Hexane (lipophilic) and aqueous/ethanol (polar) fractions are also investigated for ecdysone agonist activity since transfection assays with the insect ecdysone receptor/hormone response element genes incorporated into BMS cells have indicated an endogenous supply of ecdysone agonist positive substance.
  • the lipophilic and polar fractions are assayed in the ecdysone responsive cell line. No activity is observed, but some toxicity is noted in the concentrated polar samples. However, it can be calculated that the level of 20-OH-ecdysone equivalents is ⁇ 200 ng/g endosperm or ⁇ 100 ng/g BMS cells, and may be much less.
  • Three dosages of 322-843 are prepared: 0, 20 mg and 200 mg active ingredient per plant.
  • 100 ml dimethylsulfoxide (DMSO) is added to 20 liters of water in a clean container and is mixed thoroughly.
  • Four liters are applied to each plant a 1, 7, or 14 days post pollination.
  • Each plant is in a 5 gallon container fitted with a base to catch leachate so that the leachate can be reabsorbed into the soil.
  • 20 mg dose the same procedures are followed except that 0.1 g 322-843 is dissolved in the 100 ml DMSO.
  • 1.0 g of 322-843 is dissolved in the DMSO. Regardless of the dose, all solutions are prepared just prior to use. Ears are harvested 16 days post-pollination and are frozen in liquid nitrogen.
  • Three dosages of 322-843 (0, 20, and 200 mg active ingredient per plant) are prepared as below just prior to application at 1, 7, or 14 days post pollination. Ears are harvested at 16 days post-pollination and are frozen in liquid nitrogen. For 0 mg, 2.5 g Valent X-77 is dissolved in 500 ml acetone to make a 0.5% solution. 100 ml of this solution is mixed with 100 ml water. 40 ml of the solution are sprayed per plant. For 20 mg, 0.1 g 322-843 is dissolved in 100 ml of the 0.5% Valent X-77/acetone solution and then mixed with 100ml water. 40 ml are sprayed per plant. For 200 mg, 1.0 g 322-843 is dissolved in 100 ml of the 0.5% Valent X-77/acetone solution and mixed with 100 ml water. 40 ml are sprayed per plant.
  • Endosperm from plants treated by the soil drench or foliar spray is analyzed as follows. For a standard, 10 ⁇ l of a CH 3 CN solution of [14C]322-843 containing approximately 25,000 dpm (approximately 30 ng) is dispensed into 25 ml methanol in a 50 ml beaker. 3 g of heat-treated celite and 5 g of thawed endosperm are added, and then chilled over ice. (The blank does not contain any corn tissue). The corn tissue is homogenized, and the homogenate is filtered under a vacuum. The filter cake is returned to the original flask and 25 ml dichloromethane is added, and the procedure is repeated.
  • the solvent is then removed under a vacuum and mild heat, leaving an oily residue.
  • the residue is dissolved in 1-2 ml dichloromethane and water.
  • the solvent layer is removed, and the contents are extracted with 2 x 100 ⁇ l of acetonitrile and then dried.
  • the acetonitrile extract is re-dissolved in 50 ⁇ l dichloromethane and vortexed.
  • a sample is added to a silica GF TLC plate (1000 ⁇ l thick layer) which is predeveloped with methanol and air dried for 24 hours.
  • the solvent is evaporated from the plate and plates are developed in 3% methanol/97% dichloromethane. Plates are air-dried and scanned with a radioactivity monitor.
  • the radioactive zone is transferred to a fritted funnel and the 322-843 is eluted with acetonitrile under a slight vacuum.
  • the sample is redissolved in acetonitrile for quantification of recovery and subsequent HPLC analysis. Alternatively, it is redissolved in 50 ⁇ l DMSO for screening in the Drosophila ecdysone agonist insect cell line assay or HPLC analysis.
  • HPLC analysis is performed using a 25 x 0.46 cm Spheri-5 RP-8 column and a linear gradient system: initially 40% acetonitrile in 0.01 TFA and, after 20 minutes 90% acetonitrile in 0.01% TFA, with a 5 min hold at 90% acetonitrile and a 5 min return to starting conditions.
  • the flow rate of solvent is 1.5 ml/min.
  • RT for 322-843 is approximately 7.6 min.
  • the 322-843 zone is collected for liquid scintillation counting and the UV peak area (255 nm) is integrated. After correcting for 100% recovery of the radiolabelled internal standard, total mass of 322-843 is calculated. Additional HPLC using a normal phase system was also utilised in further analysis of the samples.
  • a soil application at 200 mg is suffucient for uptake of 322-843 and transport to endosperm in quantities sufficient for inducible activity.
  • constructs are made using conventional cloning techniques and are tested for endosperm activity using a GUS reporter gene. The number of blue stains were counted and the results obtained are given below for the constructs tested.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • CGGCCGGGAC CAATGTTCTG GTCTCTTCTT AATATAATAC CGGG ⁇ CAGTC TTTCCCTCTC 540
  • TTTTTTCAAC AAATAATGTG GTGAAATACC TAAGAGGGGT GCACCTAGCA TAGATTTTTT 900
  • AAAGGTATAA ACCACGGCTG TGCATTTTGG AAGTATCATC TATAGATGTC TGTTGAGGGG 1320
  • CTATGGACTT CTACCATTTA TGTTATTACT TTGCCTTAAT GTTCCATTGA ATAGGGCAAA 3960
  • CAAGCATCAC CAAATCACAC AGAACAATAG CAACAAAGCC TTTTAGTTCC AAGCAATTTA 5880
  • ATATAATCCT ATTCTAATCG AGAAGTCATC TGTATCTTCG TCTCTTGTTC GAACACTAGT 7080 CACAAATTTT TTTGTACATG TTCTTAATGA GTCCAACGTA ATATTCCTTG ATATTTTGTC 714
  • ATAAGCCCTC ATCAAGTCAA TGAAAATCGC GTGTAGGTCC TTCATTTGTT CCTTATACTG 720
  • GGTAGTGCCC TAAATAAATT TTAGTTCAAA AATGAATAAA AAT ⁇ TGGCTG ATTCTAACTT 9780

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Abstract

Plantes de maïs très sucré améliorées possédant, en plus de leurs gènes sh-2 ou bt-2 récessifs et homozygotes se trouvant dans la nature, un gène de sous-unité ADP-GPP commandé par un promoteur hétérologue. Le promoteur hétérologue peut être retardé vis-à-vis de son développement de manière qu'il ne soit actif que 25 ou 30 jours environ après la pollinisation, sinon il peut être un promoteur inductible. Les grains sont très sucrés si on les récolte en vue de les utiliser pour l'alimentation, mais sont amylacés si on les récolte comme culture de semence. A titre d'alternative, on peut utiliser un gène de sous-unité ADP-GPP commandé par un promoteur inductible ou par un promoteur qui n'est actif que pendant la période initiale du développement de l'endosperme, pour transformer une plante de maïs sucré de manière que la transcription du gène anti-sens bloque la synthèse de ADP-GPP, ce qui permet d'obtenir des grains très sucrés.
PCT/EP1992/002531 1991-11-05 1992-11-04 Mais tres sucre et ameliore WO1993009237A1 (fr)

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HU9401302A HU219313B (en) 1991-11-05 1992-11-04 Improved supersweet corn; transformation method and tools; growing method
EP92923267A EP0611395A1 (fr) 1991-11-05 1992-11-04 Mais tres sucre et ameliore
JP5508166A JPH07500965A (ja) 1991-11-05 1992-11-04 改良超甘味トウモロコシ
AU29203/92A AU670417B2 (en) 1991-11-05 1992-11-04 Improved supersweet corn

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IL (1) IL103647A (fr)
WO (1) WO1993009237A1 (fr)
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WO1993021334A1 (fr) * 1992-04-13 1993-10-28 Zeneca Limited Produits de recombinaison d'adn et plantes les incorporant
WO1994009144A1 (fr) * 1992-10-14 1994-04-28 Zeneca Limited Nouvelles plantes et leurs procedes d'obtention
WO1994011520A3 (fr) * 1992-11-09 1994-08-04 Zeneca Ltd Nouvelles plantes et procedes de production
EP0634491A1 (fr) * 1993-07-12 1995-01-18 Monsanto Company Semences à contenu en huile modifiée
WO1994028146A3 (fr) * 1993-05-24 1995-01-26 Hoechst Schering Agrevo Gmbh Sequences d'adn et plasmides destines a la production d'une betterave a concentration de sucre modifiee
WO1995034660A1 (fr) * 1994-06-16 1995-12-21 Advanced Technologies (Cambridge) Limited Modification de la teneur en amidon de plantes
US5498831A (en) * 1993-07-23 1996-03-12 Dna Plant Technology Corporation Pea ADP-glucose pyrophosphorylase subunit genes and their uses
WO1996027673A1 (fr) * 1995-03-03 1996-09-12 Novartis Ag Regulation de l'expression genique dans les plantes par transactivation induite par recepteur en presence d'un ligand chimique
WO1997013864A1 (fr) * 1995-10-10 1997-04-17 Novartis Ag Hormone juvenile ou l'un de ses agonistes utilises en tant que ligand chimique aux fins de reguler l'expression genique dans des plantes, a l'aide d'une transactivation induite par des recepteurs
WO1997026365A3 (fr) * 1996-01-19 1997-09-12 Dekalb Genetic Corp Mais transgenique a teneur accrue en mannitol
WO1998005785A1 (fr) * 1996-08-01 1998-02-12 Biocem Phytases de plantes et applications biotechnologiques
US5866790A (en) * 1993-05-24 1999-02-02 Hoechst Schering Agrevo Gmbh DNA sequences and plasmids for the preparation of sugar beet with changed sucrose concentration
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WO2000012733A1 (fr) * 1998-08-28 2000-03-09 Pioneer Hi-Bred International, Inc. PROMOTEURS PREFERES DE SEMENCES PROVENANT DE GENES $i(END)
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WO2000029597A3 (fr) * 1998-11-19 2000-11-09 Univ Florida Genes mutants codant pour l'adp-glucose pyrophosphorylase chez des vegetaux
WO2000078984A3 (fr) * 1999-06-21 2001-02-15 Pioneer Hi Bred Int Amelioration de la resistance de puits florale et augmentation de la stabilite de grenaison des plantes
FR2799203A1 (fr) * 1999-10-01 2001-04-06 Biogemma Fr Promoteurs specifiques de l'albumen des graines de vegetaux
US6379945B1 (en) 1995-05-26 2002-04-30 Zeneca Limited Gene switch
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US6960709B1 (en) 1993-08-25 2005-11-01 Dekalb Genetics Corporation Method for altering the nutritional content of plant seed
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US7285703B2 (en) 1998-04-03 2007-10-23 Basf Plant Science Gmbh Plant like starches and the method of making them in hosts
US7288403B2 (en) 1993-08-25 2007-10-30 Anderson Paul C Anthranilate synthase gene and method for increasing tryptophan production
US7615685B2 (en) 1990-01-22 2009-11-10 Dekalb Genetics Corporation Methods of producing human or animal food from stably transformed, fertile maize plants
US8030540B2 (en) 2004-04-21 2011-10-04 Basf Plant Science Gmbh Transgenic corn having enhanced nutritional qualities

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US6013861A (en) * 1989-05-26 2000-01-11 Zeneca Limited Plants and processes for obtaining them
US7064248B2 (en) 1990-01-22 2006-06-20 Dekalb Genetics Corp. Method of preparing fertile transgenic corn plants by microprojectile bombardment
US7615685B2 (en) 1990-01-22 2009-11-10 Dekalb Genetics Corporation Methods of producing human or animal food from stably transformed, fertile maize plants
US6057493A (en) * 1990-04-20 2000-05-02 Hoechst Schering Agrevo Gmbh Plasmids, plants and plant cells expressing anti-sense patatin and anti-sense ADP-glucose pyrophosphorylase sequences
US6605754B1 (en) 1992-04-13 2003-08-12 Syngenta Limited DNA constructs and plants incorporating them
WO1993021334A1 (fr) * 1992-04-13 1993-10-28 Zeneca Limited Produits de recombinaison d'adn et plantes les incorporant
WO1994009144A1 (fr) * 1992-10-14 1994-04-28 Zeneca Limited Nouvelles plantes et leurs procedes d'obtention
WO1994011520A3 (fr) * 1992-11-09 1994-08-04 Zeneca Ltd Nouvelles plantes et procedes de production
WO1994028146A3 (fr) * 1993-05-24 1995-01-26 Hoechst Schering Agrevo Gmbh Sequences d'adn et plasmides destines a la production d'une betterave a concentration de sucre modifiee
US5866790A (en) * 1993-05-24 1999-02-02 Hoechst Schering Agrevo Gmbh DNA sequences and plasmids for the preparation of sugar beet with changed sucrose concentration
EP0634491A1 (fr) * 1993-07-12 1995-01-18 Monsanto Company Semences à contenu en huile modifiée
US5773693A (en) * 1993-07-23 1998-06-30 Dnap Plant Technology Corporation Pea ADP-glucose pyrophosphorylase subunit genes and their uses
US5498831A (en) * 1993-07-23 1996-03-12 Dna Plant Technology Corporation Pea ADP-glucose pyrophosphorylase subunit genes and their uses
US7288403B2 (en) 1993-08-25 2007-10-30 Anderson Paul C Anthranilate synthase gene and method for increasing tryptophan production
US7547820B2 (en) 1993-08-25 2009-06-16 Dekalb Genetics Corporation Method for altering the nutritional content of plant seed
US6960709B1 (en) 1993-08-25 2005-11-01 Dekalb Genetics Corporation Method for altering the nutritional content of plant seed
US6096945A (en) * 1994-06-16 2000-08-01 Advanced Technologies (Cambridge) Limited Modification of starch content in plants
US6486383B1 (en) * 1994-06-16 2002-11-26 Advanced Technologies (Cambridge) Limited Modification of starch content in plants
WO1995034660A1 (fr) * 1994-06-16 1995-12-21 Advanced Technologies (Cambridge) Limited Modification de la teneur en amidon de plantes
AU711602B2 (en) * 1994-06-16 1999-10-14 British American Tobacco (Investments) Limited Modification of starch content in plants
WO1996027673A1 (fr) * 1995-03-03 1996-09-12 Novartis Ag Regulation de l'expression genique dans les plantes par transactivation induite par recepteur en presence d'un ligand chimique
US6147282A (en) * 1995-03-03 2000-11-14 Novartis Finance Corporation Method of controlling the fertility of a plant
US6939711B2 (en) 1995-03-03 2005-09-06 Syngenta Investment Corporation Control of gene expression in plants by receptor mediated transactivation in the presence of a chemical ligand
US5880333A (en) * 1995-03-03 1999-03-09 Novartis Finance Corporation Control of gene expression in plants by receptor mediated transactivation in the presence of a chemical ligand
US6379945B1 (en) 1995-05-26 2002-04-30 Zeneca Limited Gene switch
WO1997013864A1 (fr) * 1995-10-10 1997-04-17 Novartis Ag Hormone juvenile ou l'un de ses agonistes utilises en tant que ligand chimique aux fins de reguler l'expression genique dans des plantes, a l'aide d'une transactivation induite par des recepteurs
US6362394B1 (en) 1995-10-10 2002-03-26 Syngenta Participations Ag Juvenile hormone or one of its agonists as a chemical ligand to control gene expression in plants by receptor mediated transactivation
EA003423B1 (ru) * 1995-10-10 2003-04-24 Новартис Аг Штамм трансгенных растительных клеток и трансгенное растение, содержащие кассету экспрессии полипептида-мишени usp рецептора, способы получения их потомства, способы регулирования экспрессии полипептида-мишени в указанном растении с помощью ювенильного гормона или его агонистов, способы выявления и получения лиганда для полипептида usp рецептора и соответствующий лиганд
WO1997026365A3 (fr) * 1996-01-19 1997-09-12 Dekalb Genetic Corp Mais transgenique a teneur accrue en mannitol
US6610828B1 (en) 1996-05-24 2003-08-26 Syngenta Limited Heliothis ecdysone receptor
US7183061B2 (en) 1996-05-24 2007-02-27 Syngenta Limited Method of expressing Heliothis ecdysone receptor fusion protein
WO1998005785A1 (fr) * 1996-08-01 1998-02-12 Biocem Phytases de plantes et applications biotechnologiques
US7285703B2 (en) 1998-04-03 2007-10-23 Basf Plant Science Gmbh Plant like starches and the method of making them in hosts
US6528704B1 (en) 1998-08-28 2003-03-04 Pioneer Hi-Bred International, Inc. Seed-preferred promoters from end genes
WO2000012733A1 (fr) * 1998-08-28 2000-03-09 Pioneer Hi-Bred International, Inc. PROMOTEURS PREFERES DE SEMENCES PROVENANT DE GENES $i(END)
US6184438B1 (en) 1998-11-19 2001-02-06 University Of Florida Mutant genes encoding plant ADP-glucose pyrophosphorylase and methods of use
WO2000029597A3 (fr) * 1998-11-19 2000-11-09 Univ Florida Genes mutants codant pour l'adp-glucose pyrophosphorylase chez des vegetaux
WO2000078984A3 (fr) * 1999-06-21 2001-02-15 Pioneer Hi Bred Int Amelioration de la resistance de puits florale et augmentation de la stabilite de grenaison des plantes
US7193130B2 (en) 1999-06-21 2007-03-20 Pioneer Hi-Bred International, Inc. Expressing acid invertase or ADP-glucose pyrophosphorylase in floral tissue for enhanced floral sink strength and increased stability of seed in plants
US8026411B2 (en) 1999-06-21 2011-09-27 Pioneer Hi-Bred International, Inc. Enhanced floral sink strength and increased stability of seed set in plants
US7071378B1 (en) 1999-10-01 2006-07-04 Biogemma Plant seed endosperm specific promoter
WO2001025439A1 (fr) * 1999-10-01 2001-04-12 Biogemma Promoteurs specifiques de l'albumen des graines de vegetaux
FR2799203A1 (fr) * 1999-10-01 2001-04-06 Biogemma Fr Promoteurs specifiques de l'albumen des graines de vegetaux
WO2002036788A3 (fr) * 2000-11-06 2003-09-25 Agronomique Inst Nat Rech Acides nucleiques et polypeptides exprimes specifiquement dans les cellules de la zone de transfert d'un grain de plante et leurs applications
FR2818286A1 (fr) * 2000-12-19 2002-06-21 Agronomique Inst Nat Rech Acides nucleiques et polypeptides exprimes specifiquement dans les cellules de la zone de transfert d'un grain de plante et leurs applications
US8030540B2 (en) 2004-04-21 2011-10-04 Basf Plant Science Gmbh Transgenic corn having enhanced nutritional qualities

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