WO1997041239A2 - Transgenic plants with enhanced sulfur amino acid content - Google Patents

Transgenic plants with enhanced sulfur amino acid content Download PDF

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WO1997041239A2
WO1997041239A2 PCT/US1997/006180 US9706180W WO9741239A2 WO 1997041239 A2 WO1997041239 A2 WO 1997041239A2 US 9706180 W US9706180 W US 9706180W WO 9741239 A2 WO9741239 A2 WO 9741239A2
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sulfur
rich
plant
gene
nucleotide sequence
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PCT/US1997/006180
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English (en)
French (fr)
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WO1997041239A3 (en
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Larry R. Beach
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Pioneer Hi-Bred International, Inc.
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Priority to BR9710839-1A priority Critical patent/BR9710839A/pt
Priority to HU0202676A priority patent/HUP0202676A2/hu
Priority to DE0929685T priority patent/DE929685T1/de
Priority to AU27295/97A priority patent/AU2729597A/en
Priority to CA002253292A priority patent/CA2253292A1/en
Priority to EP97921184A priority patent/EP0929685A2/de
Publication of WO1997041239A2 publication Critical patent/WO1997041239A2/en
Publication of WO1997041239A3 publication Critical patent/WO1997041239A3/en

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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
    • 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
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C12N15/8253—Methionine or cysteine
    • 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

Definitions

  • the present invention relates to transgenic plants that have elevated levels of sulfur-containing amino acids. More specifically, the invention relates to the production of transgenic plants that contain a DNA segment with a coding sequence corresponding to that of a gene for a sulfur-rich rice prolamin or sunflower 2S albumin protein.
  • Sulfur content of animal feed formulations based on soybeans has been increased by using soybeans transformed to express Brazil nut 2S albumin gene.
  • the Brazil nut 2S albumin protein is a seed storage protein that is high in sulfur-containing amino acids, containing 18 mol per cent methionine and eight mol per cent cysteine. Since the Brazil nut 2S albumin is largely responsible for Brazil nut allergenicity, however, the use of seed from soybean plants transformed to express the Brazil nut 2S albumin gene is an unacceptable method of raising the sulfur content of animal feeds based on soybeans.
  • Brazil nut 2S albumin is a member of a superfamily of homologous proteins hypothesized to have a common ancestral qene(s) .
  • Kreis et al . J. Mol . Biol . 183: 499 (1985) .
  • Many other seeds that have 2S albumin storage proteins are known to contain- potent allergens. Melo et al . , Food & Agricul tural Immunology 6: 185 (1994) . This is not surprising, given the degree of homology of 2S albumin storage proteins.
  • Monocot plants also contain seed storage proteins.
  • the predominant storage proteins in most of the agronomically important cereals are the prolamins .
  • Rice and oats are unusual cereal crops, in that the major storage protein is an US-type globulin, and only small amounts of prolamin proteins are found.
  • Fewer monocot genes have been introduced successfully into dicot plants, for several reasons. There are definite differences in seed composition, gene regulation and post transciptional processing of storage proteins in dicots and monocots. In particular, monocot promoters typically do not function well in dicots. Other concerns relate to the stability of the sulfur-rich gene in the seed, and possible affects of the introduced gene on normal development of the plant. Altenbach et al . , TIBTECH 8: 156 (1990) .
  • a transgenic plant that contains a DNA molecule comprised of (A) a nucleotide sequence corresponding to a sulfur-rich rice prolamin gene or sulfur-rich sunflower 2S albumin gene and (B) a promoter, operably linked to the nucleotide sequence, to effect expression thereof by the transgenic plant.
  • the transgenic plant preferably is a corn or soybean plant.
  • a preferred rice prolamin gene is a sulfur-rich 10 kDa rice prolamin gene, while a preferred sunflower 2S albumin gene is SFA-7 and SFA-8, preferably SFA-8.
  • the invention also provides seeds produced by the transgenic plants.
  • a method of altering a plant to produce seed that contains levels of sulfur-containing amino acids that are higher than those of an unaltered plant comprising steps of (i) providing cells or tissues of a plant transformed with an expression vectoi that contains a nucleotide sequence corresponding to a sulfur-rich rice prolamin gene or sulfur-rich sunflower 2S albumin gene;
  • a method for increasing the content of sulfur-containing amino acids in an animal feed preferably without supplementation, which comprises steps of (i) providing seeds from a plurality of plants, at least some of which plants contain a DNA molecule comprised of a nucleotide sequence corresponding to a sulfur-rich rice prolamin gene or sulfur-rich sunflower 2S albumin gene, operably linked to a promoter to effect expression of the nucleotide sequence by the plants; and (ii) processing the seeds into animal feed.
  • the method results in a feed product that has not been supplemented by addition of sulf r-containing amino acids and that contains soybean or corn meal obtained from seed of transgenic plants.
  • Figure 2 is the nucleotide and deduced amino acid sequence o f the 10 kDa rice prolamin clone.
  • the present invention allows for the production of transgenic plants that bear seeds characterized by a higher content of sulfur-containing amino acids, relative to plants that lack the heterologous DNA in question.
  • an enhanced sulfur content in soybean ( Glycine max) or corn (Zea mays) is achieved, without significant allergenicity problems, by transforming soybean oi corn material with a heterologous DNA segment that has a nucleotide sequence corresponding to one or more copies of a gene for a rice ⁇ Oryza sativa L) 10 kDa or 16 kDa prolamin or a sunflower (Helianthus annus L) 2S albumin protein, which are rich in sulfur.
  • Sulfur-rich sunflower 2S albumin proteins are described, by example, by Lilley et al . , "Isolation and Primary Structure for a Novel Methionine-rich Protein from Sunflowerseeds (Helianthus annus . L) , " in PROCEEDINGS OF THE WORLD CONGRESS ON VEGETABLE PROTEIN UTILIZATION IN HUMAN FOODS AND ANIMAL FEEDSTUFFS 497-502 (1989) , and by Kortt et al . , Phytoche istry 29: 2805 (1990) , the respective contents of which are incorporated herein by reference. Eight proteins, denoted sunflower albumins (SFA) 1 to 8, are identified in the 2S albumin fraction.
  • SFA sunflower albumins
  • SFA-7 and SFA-8 are sulfur-rich. They contain about eight mol per cent cysteine and 16 mol per cent methionine. SFA-8 is present in greater amounts than SFA-7, and for this reason is preferred.
  • the nucleotide sequence of SFA-8 is shown in Figure 1.
  • a DNA molecule comprising a transformation/expression vector is engineered to include a sequence from one of the sulfur- rich sunflower 2S albumin genes or from the 10 kDa or 16 kDa sulfur-rich rice prolamin gene. Isolation and cloning of these genes by standard methodology is described in Lilley et al . , Kortt et al . and Matsumura et al . , supra .
  • the rice prolamin genes first may be modified to reflect preferred codon usage in dicots, prior to introduction into soybean.
  • the sulfur-rich sunflower 2S albumin genes and the 10 kDa or 16 kDa sulfur-rich rice prolamin gene of the present invention can be isolated from a natural source or synthesized from a known sequence.
  • the sequence may be obtained by making a cDNA from mRNA.
  • the sequence may also be derived from a genomic DNA sequence.
  • the sequence may be subcloned in a vector of choice .
  • the methodologies used would include identification of the gene by hybridization with probes, PCR, probe/primer/synthetic gene synthesis, sequencing, molecular cloning and other techniques which are well-known to those skilled in molecular biology.
  • the vector also could contain additional sequences that are necessary to allow for the eventual integration of the vector into a chromosome.
  • promoters are the promotor for the small subunit of ribulose-1, 5-bis- phosphate carboxylase, promoters from tumor-inducing plasmids of Agrobacterium tu efaciens, such as the nopaline synthase and octopine synthase promoters, and viral promoters such as the cauliflower mosaic virus (CaMV) 19S and 35S promoters or the figwort mosaic virus 35S promoter.
  • the promoter can be constitutive or inducible. - 7 -
  • seed tissue-preferred or seed tissue-specific promoters that is, promoters that drive high expression of the heterologous DNA segment in seed tissue where control of genes that are involved in seed metabolism is desired, and little or no expression in other parts of the plant. Manufacture of sulfur-rich proteins in other parts of the plant needlessly expends the plant's energy.
  • seed tissue- preferred or seed tissue-specific promoters include the soybean promoter of ⁇ -conglycinin, also known as the 7S protein, which drives seed-directed transcription (Bray, Planta 172: 364 (1987)) and seed-directed promoters from the zein genes of maize endosperm (Pedersen et al . , Cell 29: 1015 (1982)) .
  • Dicot promoters are preferred for use in soybeans, and a particularly preferred promoter is the bean phaseolin promoter.
  • one or more enhancers are useful in the invention to increase transcrip ion of the introduced DNA segment.
  • the enhancer or enhancer-like element can be inserted into the promoter to provide higher levels of transcription.
  • enhancers include inter alia , viral enhancers like those within the 35S promoter, as shown by Odell et al . , Plant Mol . Biol . 10: 263-72 (1988) , and an enhancer from an opine gene as described by Fromm et al . , Plant Cell 1: 977 (1989) .
  • Selectable marker genes in physical proximity to the introduced DNA segment, are used to allow transformed cells to be recovered by either positive genetic selection or screening.
  • the selectable marker genes also allow for maintaining selection pressure on a transgenic plant population, to ensure that the introduced DNA segment, and its controlling promoters and enhancers, are retained by the transgenic plant.
  • Many of the commonly used positive selectable marker genes for plant transformation have been isolated from bacteria and code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or a herbicide.
  • Other positive selection marker genes encode an altered target which is insensitive to the inhibitor.
  • a preferred selection marker gene for plant transformation is the neomycin phosphotransferase II (nptll) gene, isolated from Tn5, which confers resistance to kanamycin when placed under the control of plant regulatory signals. Fraley et al . , Proc . Nat ' l Acad . Sci . USA 30: 4803 (1983) .
  • Another useful selectable marker is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen et al . , Plan Mol . Biol . 5: 299 (1985) .
  • Additional positive selectable markers genes of bacterial origin that confer resistance to antibiotics include gentamicin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3' -adenyl transferase and the bleomycin resistance determinant. Hayford et al . , Plant Physiol .
  • genes for plant transformation are not of bacterial origin. These genes include mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase. Eichholtz et al . , Soma ti c Cell Mol . Genet . 13: 67 (1987) ; Shah et al . , Science 233: 478 (1986) ; Charest et al . , Plant Cell Rep . 8: 643 (1990) .
  • Another class of useful marker genes for plant transformation with the DNA sequence requires screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantitate or visualize the spatial pattern of expression of the DNA sequence in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include ⁇ -glucuronidase (GUS) , jS-galactosidase, luciferase, and chloramphenicol acetyltransferase. Jefferson, Plant Mol . Biol . Rep . 5: 387 (1987) ; Teeri et al .
  • GUS ⁇ -glucuronidase
  • jS-galactosidase luciferase
  • chloramphenicol acetyltransferase Jefferson, Plant Mol . Biol . Rep . 5:
  • an expression cassette In order to create an expression vector containing the gene or genes of interest, an expression cassette first is made by inserting a cloned sunflower 2S albumin or rice prolamin gene into a plasmid under the control of a regulatory sequence. The resulting expression cassette can be ligated back to itself to produce an expression cassette with a tandem repeat of the cloned gene. A further ligation can be performed to generate a construct that contains four tandem copies of the gene. One or more copies of the expression cassette containing the introduced DNA segment corresponding to the sulfur-rich 2S albumin sunflower gene or sulfur-rich 10 kDa or 16 kDa rice prolamin gene then is transferred to an expression vector.
  • the vector also contains a gene encoding a selection marker which is functionally linked to promoters that control transcription initiation.
  • a disarmed Ti- plasmid is used as a vector for foreign DNA sequences.
  • an expression vector containing a sulfur-rich 2S albumin sunflower gene or sulfur-rich 10 kDa or 16 kDa rice prolamin gene can be introduced into protoplasts; into intact tissues, such as immature embryos and meristems; into callus cultures or into isolated cells.
  • expression vectors are inserted into intact tissues, particularly explants derived from hypocotyl or cotyledonary nodes of a germinated seed.
  • an explant is a piece of tissue that is taken from a donor plant and is capable of producing callus in culture.
  • Hypocotyl tissue is that portion of the stem of a plant embryo or seedling below the cotyledons and above the root .
  • a cotyledon is an embryonic leaf
  • a cotyledonary node is that part of the seedling between the embryonic axis and the cotyledons which botanically defines the division of the hypocotyl and the epicotyl , or embryonic shoot .
  • General methods of culturing plant tissues are provided, for example, by Miki et al . , "Procedures for Introducing Foreign DNA into Plants," in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY 67-88 (CRC Press 1993) .
  • a preferred method for introducing an expression vector into plant tissue is direct infection or co- cultivation of plant tissue with A . tumefaciens that contains an expression vector with the gene of interest and associated regulatory elements. Horsch et al . , Science 227: 1229 (1985) .
  • a preferred expression vector is the vector pARC12 (pl830) , a plasmid which is part of a binary Ti plasmid system of A . tumefaciens and which contains nopaline synthase/neomycin phosphotransferase II (NPTII) as a promoter and selectable marker for transformed plant cells.
  • NPTII nopaline synthase/neomycin phosphotransferase II
  • the expression vector preferably is transformed into A . tumefaciens using a freeze-thaw technique, as described in PLANT MOLECULAR BIOLOGY (Gelvin and Schilperoort, eds., Kluwer Academic Publishers, 1988) .
  • a suitable model for demonstrating transformation of a plant by the sulfur-rich sunflower 2S albumin and sulfur-rich rice prolamin genes is Agrobacterium-mediated transformation of the tobacco plant, Nicotiana tabacum .
  • an Agrobacteriu strain confirmed to contain the construct is used to inoculate wounded tobacco plants to generate the transgenic events.
  • Agrojbac ⁇ eriuiT? is used to transfer an expression vector to soybean, induction of the virulence (vir) genes in Agrobacterium leads to enhanced transformation.
  • Cultured soybean cells lack or have a limiting amount of the necessary signal molecules to initiate the transformation process, and induction of the vir genes is necessary to achieving successful transformation.
  • Various physical parameters can be used to induce the vir genes.
  • Various compounds can be used, individually or in combination, to induce the vir gene. Exemplary of such compounds are phenolic compounds such as acetosyringone, ⁇ -hydroxyacetosyringone, acetovanillone, syringaldehyde, syringic acid and sinapinic acid.
  • Transformation of soybean by Agrobacterium also is dependent upon concentration of bacteria in the inoculum. In general, higher numbers of bacteria result in more transformation events. Preferably an inoculation period of at least 30-minutes, and a concentration of bacteria of at least 3xl0 8 viable cells/ml are used.
  • An especially preferred methodology in this regard entails subjecting an exposed corn meristem to biolistic bombardment, in order to target non-differentiated meristem cells for transformation, as described in international application PCT 96/04392. According to this approach, meristem tissue is manipulated following bombardment in order to enlarge transgenic sectors, either through selection and/or through effecting a proliferation from the tissue of shoots or multiple meristems per se .
  • the shoot population thus obtained then is screened, by means of a nonlethal enrichment assay, to identify either chimeric sectors that will contribute to germline transmission, or non-sectored, periclinal chimeras that will by definition transmit to progeny. Increased time in culture, under selection, enhances the prospects for sectoral-to-periclinal conversions, and also selects for Ll-to-L2 conversions which, through a shift in position, ultimately contribute to the germline.
  • transgenic plants are established from transformed explants by conventional techniques known to the skilled artisan. A preferred technique is cultivation of transformed explants in liquid counterselection medium, followed by transfer to solidified selection medium.
  • transgenic markers such as 3-glucuronidase ( gus) . McCabe et al . , Bio/Technology 6: 922 (1988) .
  • Shoots are induced in transgenic explants by known methods. Wright et al . , Plan t Cell Rep . 5: 150 (1986) ; Barwale et al . , Planta 167: 473 1986) .
  • the shoots are excised and, by the addition of pyroglutamic acid to a hormone-enriched growth medium, roots readily are induced.
  • Whole mature, reproductive plants are produced after transfer to greenhouse culture in soil .
  • Seed from transgenic plants according to the invention contain significant levels of sulfur-containing amino acids, particularly methionine.
  • Expression of the sulfur-rich genes was accompanied by a concomitant decrease in trypsin and chymotrypsin inhibitor activities.
  • These endogenous inhibitors are relatively rich in sulfur-containing amino acids, and the overexpression of the introduced genes may be depleting the normal free sulfur pools in the seed, with the result that sulfur is being scavenged from endogenous proteins.
  • the tran genic plants appear to accumulate the heterologous proteins at the expense of certain endogenous proteins to maintain a homeostatic condition. The changes produce no apparent effect on the viability of the transgenic plant or on the total seed protein.
  • tumefaciens is a preferred vector
  • other types of vectors can be used for transformation by procedures such as direct gene transfer, as described, for example, in PCT application WO 85/01856 and in European application 0 275 069; in vi tro protoplast transformation, which is the subject of U.S. patent No. 4,684,611, for instance; plant virus- mediated transformation, illustrated in European application 0 67 553 and U.S. patent No. 4,407,956; and liposome-mediated transformation according to U.S. patent No. 4,536,475, among other disclosures.
  • Direct transfer methods also may be employed, such as microprojectile- mediated delivery, DNA injection, and electroporation. See, for example, Gruber et al . and Miki et al . , both cited above, and Klein et al . , Bio/Technology 10: 268 (1992) .
  • EXAMPLE ACROBACTERIUM-MEDIATED TRANSFORMATION OF TOBACCO OR SOYBEAN TO PRODUCE A TRANSGENIC PLANT THAT HAS A HIGH CONCENTRATION OF SULFUR-CONTAINING AMINO ACIDS
  • the resulting expression cassettes contained SFA-8 (p6445) and 10 kDa rice prolamin (p6465) under control of the phaseolin regulatory sequences. p6445 and p6465 were then ligated back to themselves to produce p6668 and p6670, respectively, which contained tandem repeats of the expression cassettes. A final set of ligations was performed with p6668 and p6670 to generate p7518 and p7519, respectively, constructs which contain four tandem copies of the expression cassettes.
  • Tobacco plants (Ni cotiana tabacum) var. Xanthii, were cultured from 1 cm apical or axillary explants on OMS, according to Murashige et al . , Physiologia Plantarum, 15:473 (1962) , in a Magenta Box (Magenta Corp., Chicago, 111.) at 27°C with a 16 hour light/8 hour dark photoperiod. The first four fully expanded leaves below the apex (leaves 3, 4, 5 or 6) were removed. Leaves were placed, one at a time, in a 100 mm petri dish with several Whatman #1 filter discs, 7cm, soaked with liquid medium. A sterile, sharp, #2 cork bore (0.5 cm i.d.) was used to punch leaf discs, avoiding the midrib and veins. Discs were held on filter paper at 100% humidity in a separate petri dish with Whatman filters and liquid medium.
  • the medium was supplemented with phytohor ones and included major salts and MS micronutrients (Murashiga, op . ci t . ) supplemented with sucrose, 3.0% w/v; naphthalene acetic acid (NAA) , 0.1 mg/L and 6-benzylaminopurine (BAP) , 1.0 mg/L.
  • the medium was buffered to pH 5.7.
  • the filter was dry enough to wick away excess inoculum.
  • Ten to twelve discs per 7 cm filter per 100 mm plate were plated.
  • the petri dishes were covered with parafilm and kept in low light at 28°C for two days.
  • the explants were moved to a liquid counterselection medium, a medium having the same basic composition as that used during cocultivation, but with the addition of cefotaxime, 500 ⁇ g/ml and vancomycin, 100 ⁇ g/ml.
  • the discs were washed with gentle continuous gyratory shaking for 3-6 hours with at least one change of liquid medium.
  • the discs then were placed on solid medium, a medium having the same basic composition as that used during cocultivation, but with the addition of vancomycin, 100 ⁇ g/ml and carbenicillin, 500 ⁇ /ml .
  • Excess liquid was evaporated before plating ten to twelve discs per 100 mm plate.
  • the explants were cultures for 3 days at 28°C in low light .
  • Discs were transferred to solid selection medium, a medium having the same basic composition as that used during cocultivation, but with the addition of vancomycin, 100 ⁇ g/ml; carbenicillin, ⁇ 00 ⁇ g/ml; and kanamycin, 100 ⁇ g/ml.
  • the discs were cultured at 26°C in high light with 16 hour light/8 hour dark photoperiod.
  • Seeds of soybean ( Glycine max) , var. PHI9341, were surface sterilized by exposure to chlorine gas evolved in a glass bell jar. Gas was produced by adding 3.5 ml hydrochloric acid (34-37% w/w) to 100 ml to sodium hypochlorite (5.25% w/w) . Exposure was for 16-20 hours in a container approximately one cubic foot in volume. Surface sterilized seed was stored in petri dishes at room temperature. Seed was germinated by plating of 1/10 strength agar solidified medium according to Gamborg (B5 basal medium with minimal organics, Sigma Chemical Co., cat. no.
  • Inoculations were conducted in batches such that each plate of seed was treated with a newly resuspended pellet of A . tumefaciens harboring the 4X construct. One at a time the pellets were resuspended in 20 ml inoculation medium. Inoculation medium consisted of B5 salts (B5893) , 3.2 gm/L; sucrose, 2.0% w/v; BAP, 44 ⁇ M; and indolebutyric acid (IBA) , 0.5 ⁇ M. Acetosyringone (AS) , 100 ⁇ M was added and the medium was buffered to pH 5.5 with MES, 10 .
  • B5 salts B5893
  • sucrose 2.0% w/v
  • BAP 44 ⁇ M
  • IBA indolebutyric acid
  • AS Acetosyringone
  • AS 100 ⁇ M was added and the medium was buffered to pH 5.5 with MES, 10 .
  • the mixture was resuspended by vortexing and the inoculum was poured into a petri dish containing prepared seed and the cotyledonary nodes were macerated with a surgical blade. This was accomplished by dividing seed in half by longitudinal section through the shoot apex, preserving the two whole cotyledons. The two halves of the shoot apex were broken off at their respective cotyledons by prying them away with a surgical blade. The cotyledonary node was then macerated with the - 18 -
  • Counterselection medium consisted of B4 salts (G5893) , 3.2 gm/L; sucrose, 2.0% w/v; BAP, 5.0 ⁇ M; IBA, 0.5 ⁇ M; vancomycin, 200 ⁇ /ml ; cefotaxime, 500 ⁇ g/ml and was buffered to pH 5.7 with MES, 3 mM.
  • Ten explants were washed in each petri dish with constant, slow gyratory agitation at room temperature for four days. Counterselection medium was replaced four times.
  • Selection medium consisted of B5 salts (G5893) , 3.2 gm/L; sucrose, 2.0% w/v; BAP, 5.0 ⁇ M; IBA, 0.5 ⁇ M; kanamycin sulfate, 50 ⁇ g/ml; vancomycin, 100 ⁇ g/ml; cefotaxime, 30 ⁇ g/ml; timentin, 30 ⁇ g/ml and was buffered to pH 5.7 with MES, 3.0 mM. Selection medium was solidified with SeaKem agarose, 0.3% w/v. The explants sere embedded in the medium, adaxial side down and cultured at 28°C with a 16 hour day length and cool white fluorescent light, of 60-80 ⁇ Em 2 S' .
  • explants were washed with liquid medium on the gyratory shaker. This time the wash was conducted overnight in counterselection medium containing kanamycin sulfate, 50 ⁇ g/ml. The following day explants were picked to agarose solidified selection medium. Again they were embedded in the medium, adaxial side down, and cultured as before for another two weeks. - 19 -
  • Elongation medium consisted of B5 salts (G5893) , 3.2 gm/L; sucrose, 2.0% w/v; IBA, 3.3 ⁇ M; gibberellic acid, 1.7 ⁇ M; vancomycin, 100 ⁇ g/ml; cefotaxime, 30 ⁇ g/ml; timentin, 30 ⁇ g/ml and was buffered to pH 5.7 with MES, 3.0 mM. Elongation medium was solidified with gelrite, 0.2% w/v.
  • Rooting medium consisted of B5 salts (G5893), 3.2 gm/L; sucrose, 15 gm/L; nicotinic acid, 20 ⁇ M; pyroglutamic acid (PGA) , 900 mg/L and IBA, 10 ⁇ M.
  • the medium was buffered to pH 5.7 with MES, 3.0 mM and solidified with GelRite, 0.2% w/v. After ten days the shoots were transferred to the same medium without IBA or PGA.
  • Total amino acid composition can be determined by acid hydrolysis of tobacco or soybean meal by standard protocols. Seed proteins also can be analyzed for trypsin inhibitory activity according to the previously described protocols of Kollipara et al . , J. Agri cul . Food Chem . 40:2356 (1992) . Similarly chymotrypsin inhibitory assays can be done according to Geiger, Chymotrypsin. In "Methods of Enzymatic Analysis," pp. 99-109 (1984) .
  • transgenic tobacco plants expressing the rice prolamin gene Similar results were obtain for transgenic tobacco plants expressing the rice prolamin gene. A band was detected that comigrated with the purified rice prolamin in all of the transgenic tobacco lines. No signal was detected in protein samples from non-transformed tobacco. Using a dilution series of purified rice prolamin as a standard, the transgenic rice prolamin protein accumulated as a percentage of total seed protein was calculated to be 1-5%. Surprisingly, no larger precursor proteins were detected, indicating that the transgenic tobacco plant, a dicot, processed the protein in the same way as rice, a monocot.

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PCT/US1997/006180 1996-04-30 1997-04-24 Transgenic plants with enhanced sulfur amino acid content WO1997041239A2 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
BR9710839-1A BR9710839A (pt) 1996-04-30 1997-04-24 Plantas transgênicas com tero melhorado de aminoácido com enxofre.
HU0202676A HUP0202676A2 (hu) 1996-04-30 1997-04-24 Kéntartalmú aminosavakat megnövelt mennyiségben tartalmazó transzgenikus növények
DE0929685T DE929685T1 (de) 1996-04-30 1997-04-24 Transgene pflanzen mit erhöhtem gehalt an schwefelhätigen aminosäuren.
AU27295/97A AU2729597A (en) 1996-04-30 1997-04-24 Transgenic plants with enhanced sulfur amino acid content
CA002253292A CA2253292A1 (en) 1996-04-30 1997-04-24 Transgenic plants with enhanced sulfur amino acid content
EP97921184A EP0929685A2 (de) 1996-04-30 1997-04-24 Transgene pflanzen mit erhöhtem gehalt an schwefelhätigen aminosäuren.

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US60/016,560 1996-04-30

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ES (1) ES2142780T1 (de)
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WO1999004024A2 (en) * 1997-07-15 1999-01-28 Dow Agrosciences Llc Nucleotide sequences of genes encoding sink proteins and uses thereof for improving the nutritional quality of feeds
WO1999015004A1 (en) * 1997-09-19 1999-04-01 Commonwealth Scientific And Industrial Research Organisation Method for altering storage organ composition
WO1999040209A1 (en) * 1998-02-09 1999-08-12 Pioneer Hi-Bred International, Inc. Alteration of amino acid compositions in seeds
WO2002036787A2 (fr) 2000-10-30 2002-05-10 Bayer Cropscience S.A. Plantes tolerantes aux herbicides par contournement de voie metabolique
US6576819B1 (en) 1999-02-18 2003-06-10 Pioneer Hi-Bred International, Inc. Methods for modulating the levels of organic sulfur compounds in plants by transforming with (P)APS reductase DNA
US7038109B1 (en) 2001-03-29 2006-05-02 Pioneer Hi-Bred International, Inc. Enzymatic methods for modulating the levels of organic sulfur compounds in plants
WO2010046422A2 (en) 2008-10-22 2010-04-29 Basf Se Use of auxin type herbicides on cultivated plants
WO2010046423A2 (en) 2008-10-22 2010-04-29 Basf Se Use of sulfonylurea herbicides on cultivated plants
WO2011095460A1 (en) 2010-02-02 2011-08-11 Bayer Cropscience Ag Soybean transformation using hppd inhibitors as selection agents
US8003799B2 (en) 2000-01-06 2011-08-23 Bayer Sas Picolinic acid derivatives and their use as fungicides
WO2014053395A1 (en) 2012-10-01 2014-04-10 Basf Se Use of n-thio-anthranilamide compounds on cultivated plants
WO2014079820A1 (en) 2012-11-22 2014-05-30 Basf Se Use of anthranilamide compounds for reducing insect-vectored viral infections
EP3028573A1 (de) 2014-12-05 2016-06-08 Basf Se Verwendung eines triazolfungizids auf transgenen pflanzen
WO2016091674A1 (en) 2014-12-12 2016-06-16 Basf Se Use of cyclaniliprole on cultivated plants
WO2016162371A1 (en) 2015-04-07 2016-10-13 Basf Agrochemical Products B.V. Use of an insecticidal carboxamide compound against pests on cultivated plants
EP3338552A1 (de) 2016-12-21 2018-06-27 Basf Se Verwendung eines tetrazolinon fungizids bei transgenen pflanzen
WO2020027214A1 (ja) 2018-07-31 2020-02-06 住友化学株式会社 Ｑｏ阻害剤に対して耐性を有するダイズさび病菌の防除方法

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999004024A3 (en) * 1997-07-15 1999-04-22 Dow Agrosciences Llc Nucleotide sequences of genes encoding sink proteins and uses thereof for improving the nutritional quality of feeds
WO1999004024A2 (en) * 1997-07-15 1999-01-28 Dow Agrosciences Llc Nucleotide sequences of genes encoding sink proteins and uses thereof for improving the nutritional quality of feeds
WO1999015004A1 (en) * 1997-09-19 1999-04-01 Commonwealth Scientific And Industrial Research Organisation Method for altering storage organ composition
US6930223B2 (en) 1997-09-19 2005-08-16 Commonwealth Scientific And Industrial Research Organisation Method for altering storage organ composition
US7053282B1 (en) 1998-02-09 2006-05-30 Pioneer Hi-Bred International, Inc. Alteration of amino acid compositions in seeds
WO1999040209A1 (en) * 1998-02-09 1999-08-12 Pioneer Hi-Bred International, Inc. Alteration of amino acid compositions in seeds
US6576819B1 (en) 1999-02-18 2003-06-10 Pioneer Hi-Bred International, Inc. Methods for modulating the levels of organic sulfur compounds in plants by transforming with (P)APS reductase DNA
US8003799B2 (en) 2000-01-06 2011-08-23 Bayer Sas Picolinic acid derivatives and their use as fungicides
EP2141240A2 (de) 2000-10-30 2010-01-06 Bayer CropScience SA Herbizidtolerante Pflanzen durch Umgehung des Stoffwechselwegs
WO2002036787A2 (fr) 2000-10-30 2002-05-10 Bayer Cropscience S.A. Plantes tolerantes aux herbicides par contournement de voie metabolique
US7560623B2 (en) 2001-03-29 2009-07-14 Pioneer Hi-Bred International, Inc. Enzymatic methods for modulating the levels of organic sulfur compounds in plants
US7038109B1 (en) 2001-03-29 2006-05-02 Pioneer Hi-Bred International, Inc. Enzymatic methods for modulating the levels of organic sulfur compounds in plants
WO2010046422A2 (en) 2008-10-22 2010-04-29 Basf Se Use of auxin type herbicides on cultivated plants
WO2010046423A2 (en) 2008-10-22 2010-04-29 Basf Se Use of sulfonylurea herbicides on cultivated plants
WO2011095460A1 (en) 2010-02-02 2011-08-11 Bayer Cropscience Ag Soybean transformation using hppd inhibitors as selection agents
WO2014053395A1 (en) 2012-10-01 2014-04-10 Basf Se Use of n-thio-anthranilamide compounds on cultivated plants
WO2014079820A1 (en) 2012-11-22 2014-05-30 Basf Se Use of anthranilamide compounds for reducing insect-vectored viral infections
EP3028573A1 (de) 2014-12-05 2016-06-08 Basf Se Verwendung eines triazolfungizids auf transgenen pflanzen
WO2016091674A1 (en) 2014-12-12 2016-06-16 Basf Se Use of cyclaniliprole on cultivated plants
WO2016162371A1 (en) 2015-04-07 2016-10-13 Basf Agrochemical Products B.V. Use of an insecticidal carboxamide compound against pests on cultivated plants
EP3338552A1 (de) 2016-12-21 2018-06-27 Basf Se Verwendung eines tetrazolinon fungizids bei transgenen pflanzen
WO2020027214A1 (ja) 2018-07-31 2020-02-06 住友化学株式会社 Ｑｏ阻害剤に対して耐性を有するダイズさび病菌の防除方法

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CA2253292A1 (en) 1997-11-06
AU2729597A (en) 1997-11-19
HUP0202676A2 (hu) 2002-12-28
EP0929685A2 (de) 1999-07-21
WO1997041239A3 (en) 1998-07-30
ES2142780T1 (es) 2000-05-01
DE929685T1 (de) 2000-08-17

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