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WO1998013503A1 - Une plante, et technique de modification - Google Patents

Une plante, et technique de modification Download PDF

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Publication number
WO1998013503A1
WO1998013503A1 PCT/AU1997/000625 AU9700625W WO9813503A1 WO 1998013503 A1 WO1998013503 A1 WO 1998013503A1 AU 9700625 W AU9700625 W AU 9700625W WO 9813503 A1 WO9813503 A1 WO 9813503A1
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WIPO (PCT)
Prior art keywords
gene
plasmid
promoter
expression
genes
Prior art date
Application number
PCT/AU1997/000625
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English (en)
Inventor
Robert Dixon Teasdale
Original Assignee
F.B. Investments Pty. Ltd.
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Publication date
Priority claimed from AUPO2756A external-priority patent/AUPO275696A0/en
Priority claimed from AUPO5092A external-priority patent/AUPO509297A0/en
Application filed by F.B. Investments Pty. Ltd. filed Critical F.B. Investments Pty. Ltd.
Priority to AU41929/97A priority Critical patent/AU4192997A/en
Publication of WO1998013503A1 publication Critical patent/WO1998013503A1/fr

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    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8231—Male-specific, e.g. anther, tapetum, pollen
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8233—Female-specific, e.g. pistil, ovule
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • This invention relates to new plants and methods of modification thereof.
  • This invention has particular but not exclusive application to forestry and increasing the productive capacity of trees, and for illustrative purposes reference will be made to such application. However, it is to be understood that this invention could be used in other applications, such as the modification of other plants such as certain leafy food crops to increase the production of useful parts thereof. BACKGROUND OF THE INVENTION
  • reproductive structures are in competition in the plant with other non-essential structures for growth resources such as water, nutrients and photosynthesis products.
  • the development of reproductive structures on forest trees represents a significant burden on the resources of the trees, the reproductive effort occurring at the expense of vegetative growth.
  • the extent of the effect of reproductive burden on vegetative growth may be estimated by measuring the proportion of photo-assimilate which is directed to flowers and fruits, or strobili and cone growth for gymnosperms .
  • reproductive effort is variously estimated to be up to 16% of annual photosynthate.
  • the formation of reproductive structures imposes a burden on the tree beyond that of carbon allocation.
  • these structures have very large requirements for important nutrients at the expense of vegetative tissues so that, at least under conditions of nutrient deficiency, prevention of reproductive structure formations as proposed can lead to a greater increment in vegetative growth than the corresponding mass of reproductive structure otherwise formed.
  • Nutrient losses from pollen dispersal are not inconsiderable in most forest plantations, with an estimated annual production of 370 kg/ha of nutrient-rich pollen by Pinus radiata plantations.
  • Tree crop productivity of P. radiata is as yet only about 50% of that theoretically obtainable on the basis of incident solar radiation. Therefore, in many environments, there are good prospects for significant increase in harvest yield from trees which have been inhibited from producing reproductive structures.
  • Koltunow et al. discloses methods of inducing male sterility in plants by expression of a lethal gene (Bamase) in plant stamen cells ( tapetal ) .
  • a chimeric gene comprising the barnase gene inserted into and under the expression control of the TA29 gene of tobacco, a gene established to be specifically expressed in tapetal tissue.
  • the disclosure relates to the induction of sterility only, but does not overcome the problem of plants developing reproductive structures at the expense of vegetative growth.
  • the present invention aims to alleviate the above disadvantages and to provide plants of enhanced productive capacity and methods for the production thereof which will be reliable and efficient in use. Other objects and advantages of this invention will hereinafter become apparent.
  • this invention in one aspect resides broadly in a method of enhancing vegetative growth in a plant including the steps of: - identifying a gene having a substantially tissue- specific promoter expressing during the development of both male and female plant reproductive structures; constructing an expression cassette comprising a heterologous coding region capable of expressing a product which aborts said development under the expression control of said promoter; transforming plant cells with said expression cassette, and selecting and vegetatively propagating the transformants.
  • the non essential structure is selected from the reproductive structures of plants which are economically capable of artificial vegetative propagation.
  • the plant selected is a tree for timber, pulp or fibre production wherein poor or absent expression of reproductive structures may result in increase in vegetative growth of the valuable material .
  • a large number of genes are differentially expressed between sexual and vegetative buds. Accordingly, it is preferred that the gene be selected from those specific to sexual budding, specific to the production of other sexual structures, or specifically coding for a product essential in a developmental pathway for a reproductive structure.
  • the gene is preferably selected for its early expression, specifically in the developing reproductive tissues.
  • PrMADSl cDNA library prepared from immature female and male cones.
  • PrMADSl, 2 and 3 belong to the family of MADS-box genes showing homology to Arabldopsls AGL-2, AGL-4 and AGL6 genes and dall gene from another non-angiosperm, Picea abies (Norway spruce), respectively.
  • the PrFLl gene is the pine ortholog of AraJb ⁇ dopsis Leafy ( Lfy ) and Floricaula (Flo ) gene from Anth ⁇ rrhinv ⁇ n.
  • the PrConl shows strong homology to Arabldopsls CONSTANS (CO) gene. A significantly lower level of expression was detected in vegetative tissues: vegetative buds, needles, stem and roots. In situ hybridisation showed that expression of these genes is substantially detectable only in reproductive tissue cells.
  • PrMADSl, 2 and 3 genes are cone-specific in that expression of both genes was substantially restricted to reproductive organ primordium tissues. No detectable expression of these genes was observed in vegetative tissues such as vegetative buds, needles, stems, roots. For PrFLl and PrConl low detectable expression was observed in vegetative buds.
  • MADS-box genes of Eucalyptus spp. have been identified as having the reproductive organ specificity required of the present invention and identified hereinafter as genes EGM1, 2 and 3 respectively, gene EGM3 being highly specific for reproductive primordia.
  • the gene is identified in cDNA libraries prepared from mRNA isolated from reproductive tissues and selected by differential screening against mRNA of vegetative structures. Since the biochemical pathways for vegetative buds and developing reproductive structures may include common expression products and consequently similar mRNAs, it is preferred to differentially screen cDNA libraries against vegetative bud mRNA, with or without preceding enrichment of the cDNA for genes specifically expressed in reproductive tissues.
  • the selected gene may be any which when its expression is blocked or otherwise made ineffectual, results in the failure to produce a non essential plant structure.
  • the cDNAs may be utilized as probes to select corresponding genomic clones from genomic libraries.
  • the genomic clones may be used to isolate and identify gene promoters that specifically express genes unique to reproductive structures. Such promoters can then be combined with lethal genes which when expressed will inhibit or terminate growth of the cells; within which the lethal gene is expressed.
  • the promoter-gene fusions when stably incorporated into a plant, by any suitable known means, will result in failure to develop reproductive tissues in whole or in part.
  • Candidate cDNA clones may be raised and are preferably selected for the presence of genes expressed in both male and female buds. The selection may be directed to those genes which are naturally produced or those which are induced by plant growth regulators which promote flowering, such as various gibberellins. Preferably, the specific expression in floral tissues is confirmed using an in situ RNA hybridization procedure with a wide range of different plant tissues. The preferred cDNA clones are those further selected for the characteristics of early appearance and highly specific expression.
  • the modification of the identified gene may be by fusing the tissue-specific promoter of said gene with a structural gene for a deleterious or lethal product such that regenerated plants transformed with said gene-fusion will not form said non- essential structure.
  • a critical function of said gene may be disrupted or modified by expression of the modified gene in transformed plants.
  • the modified gene is preferably introduced into a plant normally containing the identified gene such that a critical function of the identified gene is disrupted or modified.
  • introduction of the modified gene into plants not containing the specifically identified gene may result in useful reduction or elimination of a non-essential plant structure, particularly where the gene selected has analogues represented across several species, or in closely related species where the corresponding gene is essentially homologous with the gene in question.
  • Modification of the gene may be achieved by any suitable means, with the expression strategy desired being the primary arbiter of the modification process utilized.
  • it may be intended to constitutively express an antisense or perhaps ribozyme version of a gene which is critical to development of reproductive structures, so that the normal gene action is disrupted and vegetative development occurs instead.
  • the method may be to splice a promoter specific to a reproductive structure with a lethal gene which codes for an expression product which will cause abortion of the tissue in which it is produced, for example, buds that differentiate as reproductive structures.
  • Genes expressing antisense RNA against the mRNA coded by each of the selected target genes may be constructed. These and shorter RNA sequences that bind to the initiation regions of the target mRNA may be used for inhibition of translation. Of course, other critical regions apart from the translation initiation site may be targeted for binding of antisense RNA.
  • One option is to use an antisense or ribozyme version of a critical house-keeping gene, such as the actin gene or a gene coding for an enzyme of aromatic amino acid biosynthesis, for example enolpyruvyl shikimate phosphate synthase.
  • a deleterious enzyme such as a protease, ribonuclease, or deoxyribonuclease may be encoded for biosynthesis under control of a sexual promoter or any promoter specifically expressed in a non essential structure.
  • tissue ablation approach is the effect of promoter leakage or expression in other tissues.
  • any residual leakage is preferably overcome by modification of the expression cassette to include appropriate leakage control.
  • a second copy of the substantially tissue specific promoter, or a second tissue specific promoter may be used to promote a gene producing a control product capable of switching on production of an inhibitor to the lethal gene product in non target tissues.
  • One example of this approach is to construct an expression cassette including a control system (gene cascade) whereby expression of a lethal gene(s) is countered in non-target tissues, such as cassettes carrying both Barnase and Barstar where Barstar is under the constitutive control of a promoter responsive to repressor laclg, itself promoted by a second copy of the* selected tissue specific promoter.
  • a control system gene cascade
  • non-target tissues such as cassettes carrying both Barnase and Barstar where Barstar is under the constitutive control of a promoter responsive to repressor laclg, itself promoted by a second copy of the* selected tissue specific promoter.
  • this invention relates to a novel method for Agroiacterium-mediated transformation of Eucalyptus shoot and seedling explant tissue and regeneration of plants.
  • Eucalyptus species are known for their fast growth and high biomass productivity. They are grown in commercial forestry plantations around the world.
  • Reforestation the controlled regeneration of forests, has become an integral part of forest management in order to secure a renewable and sustainable source of raw material for production of paper and other wood-related products.
  • Forest trees can be regenerated by either sexual or asexual propagation.
  • Sexual reproduction of seedlings for reforestation has traditionally been the most important means of propagation. Regeneration by seed usually results in highly variable progeny.
  • vegetatively propagated clonal planting stock have a number of potential benefits including uniformity in quality and growth.
  • Eucalypts are primarily native trees to Australia with a small number of exceptions in the Asian region. However, they are now widely grown around the world. Several species of Eucalyptus are fast growing trees of importance for timber and paper pulp industries. Genetic improvement of eucalypts, as of other trees, is difficult with traditional breeding because of the constraint imposed by long developmental cycles.
  • the present invention is designed to produce elite Eucalyptus plants. These objectives are achieved by a multi-step method for the regeneration of Eucalyptus plants. Although other transformation and regeneration protocols have been published, none of these methods have proven totally effective with the Eucalyptus species in that none enabled the practitioner to reliably proceed from the beginning step of shoot explant transformation to completion of the regeneration process resulting in establishment of plants in field conditions.
  • the present protocols provide such a multi- step method for Eucalyptus plants. It incorporates two key steps. The first is keeping the shoot explant intact and upright during the co-cultivation step with Agrobacterium so that the leaves do not touch the tissue culture medium. The second key step is the incorporation of a liquid culture selection stage.
  • This liquid culture selection step is only effective once regenerated shoots have been produced on solid medium. Shoot material grew faster in liquid medium and formed more shoots when compared to shoots grown on solid medium.
  • This liquid selection step has the advantages of reducing false positives by increasing selection pressure, reducing residual Agrobacterium and increasing the amount of shoot tissue compared to solid grown cultures.
  • the process may proceed according to the following steps:
  • the Arohacterium-mediated transformation of Eucalyptus explant material with DNA may be from seedlings or micropropagated clonal plant tissue.
  • the Agroiacterium and explant are co-cultivated. In the case of shoot explants, the shoots are kept upright in the solidified medium so that the leaves are not in physical contact with the medium.
  • the explants are then transferred to medium containing an antibiotic which kills the Agrobacterium. This selection is maintained for the remainder of the plant regeneration procedure.
  • the explants are then transferred to callus induction medium containing a selective agent which only allows the growth of tissue containing the introduced DNA. This selection is also maintained for the remainder of the plant regeneration procedure.
  • the explants are then transferred to shoot induction medium.
  • the plantlets are transferred to soil or other growing medium in a container where root development and acclimatisation (gradually lowering relative humidity) continue.
  • Clones can be micropropagated by tissue culture propagation techniques and grown into trees of a size and form suitable for planting.
  • the method is well suited for large-scale production of clones of genetically modified and Improved Eucalyptus.
  • the present processes also provides a reliable multi-step regeneration method for the recalcitrant Eucalyptus species. It is the combined application of the progression of steps in this novel multi-step method that has enabled the first successful field planting of many different genotypes of Eucalyptus.
  • FIG 1 is expression of the PrMADSl, PrMADS2, PrMADS3, PrFLl and PrCONl genes in reproductive and vegetative tissues of P.radiata;
  • FIG 2 is expression of PrMADSl gene in female cones
  • FIG 3 is expression of PrMADS2 gene in female cones
  • FIG 4 is expression of PrMADS2 gene in male cones
  • FIG 5 is expression of PrMADS3 gene in male and female cones
  • FIG 6 is gel mobility shift assay of PrMADSl, PrMADS2 and PrMADS3;
  • FIG 7 is a promoter finder strategy;
  • FIG 8 is the nucleotide sequence of PrMADSl cDNA clone;
  • FIG 9 is the amino acid sequence of PrMADSl protein;
  • FIG 10 is the nucleotide sequence of PrMADS2 cDNA clone;
  • FIG 11 is the amino acid sequence of PrMADS2 protein;
  • FIG 12 is the nucleotide sequence of PrMADS2 promoter;
  • FIG 13 is the nucleotide sequence of PrMADS3 cDNA clone;
  • FIG 14 is the amino acid sequence of PrMADS3 prot €iin;
  • FIG 15 is the nucleotide sequence of PrMADS3 promoter;
  • FIG 16 is the nucleotide sequence of PrFLl cDNA clone;
  • FIG 17 is the amino acid sequence of PrFLl protein;
  • FIG 18 is the nucleotide sequence of PrFLl promoter;
  • FIG 19 is the nucleotide sequence of PrConl gene (partial ) ;
  • FIG 20 is a diagrammatic illustration of a control mechanism to protect non target tissues
  • FIG 21 is the plasmid pBR Bamase prom Barstar 1 containing VI-promoters
  • FIG 21A is a diagrammatic illustration of plasmid 7 prom Barstar comprising a promoter and barstar fragment cloned into pGem3zf ( Promega ) ;
  • FIG 22 is a diagrammatic illustration of plasmid containing V2-promoters;
  • FIG 24 is a Northern blot of total RNA extracted from Eucalypt tissue probed with the EGM2 cDNA (3 day exposure);
  • FIG 25 is a graphical representation of Northern blot of total RNA extracted from Eucalypt tissue probed with the EGM2 and EGM3 cDNA respectively (6 hour exposure);
  • FIG 26 is a Northern blot of total RNA extracted from Eucalypt tissue probed with the EGM3 cDNA (6 hour exposure);
  • FIG 28 is the nucleotide sequence of the EGM1 promoter;
  • FIG 28A is a diagrammatic illustration of the EGM1 promoter;
  • FIG 29 is the nucleotide sequence of the EGM3 promoter
  • FIG 29A is a diagrammatic representation of the EGM3 promoter
  • FIG 29B is the nucleotide sequence of the EGM3 promoter
  • FIG 30 is the nucleotide sequence of the EGM2 promoter
  • FIG 30A is a diagrammatic representation of the EGM2 promoter
  • FIG 31 is a diagrammatic representation of agarose separation of a Pstl digest of an EGM3 double bin. Given that the EcoRI cloning site is at base 6772 in EGM3 bin, the predicted fragment sizes from the map for this digestion are 4.9, 4.8, 4.4, 3.3, 1.9, 1.1, and 0.6 kb;
  • FIG 32 is a diagrammatic representation of two gel exposures of a Pstl digest of an EGM3 bin showing the other possible orientations of the cloned insert.
  • the sizes of fragments expected from this digest are 7.4, 4.9, 4.4, 1.1, 0.7, and 0.6 kb;
  • FIG 33 is a plasmid diagram of EGM3 double bin wherein the illustrated fragment is cloned in both orientations;
  • FIG 34 is a plasmid diagram of EGM3 VI sense
  • FIG 35 is a plasmid diagram of plasmid VI (Barnase- Barstar ) ;
  • FIG 36 is a plasmid diagram of pBR Bamase
  • FIG 37 is a plasmid diagram of plasmid V2 ( acIqNLS-35S pro oterOp-Barstar ) ;
  • FIG 38 is a plasmid diagram of p35Sop Barstar E-2;
  • FIG 39 is a plasmid diagram of p35Sop Barstar, and serves also to represent 35Sop Barstar E, which is the same plasmid with the EcoRI site at base508 removed;
  • FIG 40 is a plasmid diagram of pBRlac
  • FIG 41 is a plasmid diagram of pBRGUS 1;
  • FIG 42 is a plasmid diagram of pBRGUS 2
  • FIG 43 is a plasmid diagram of Bin 19+EGM3 VI sense (Orientation 2 as described in the following description of embodiments ) ;
  • FIG 44 is a plasmid diagram of EGM3 V2 sense;
  • FIG 45 is a plasmid diagram of plasmid pBRLacBH-;
  • FIG 46 is an illustration of Arabidopsis (FB.13L30) transformed with an antisense PrMADSl gene compared with a control plant; and
  • FIG 47 is a diagrammatic representation of the directed development strategy resulting in the transformed Arabidopsis of FIG 46.
  • Upstream sequences were isolated using a 'Promoter finder' strategy (Fig .7 ) .
  • a special adaptor was ligated to the ends of DNA fragments generated by digestion of genomic DNA from P. radiata with EcoRV, Seal, Dral, Pvull and Sspl separately. The enzymes used were selected because they have six-base recognition sites and generate blunt ends. Following adaptor ligation, these DNA fragments were used as a template for PCR using first adaptor primers API , AP2 and gene-specific primers GSP-1,2.
  • adaptor first sequence
  • adaptor-primers second sequence
  • polymerase blocking primer polymerase blocking primer
  • adaptor primer AP2 corresponds to bases 13 to 31
  • polymerase block corresponds to bases 41 to 48.
  • Adp ttnr 5 ' GTAATACGACTCACTA AGGGCACGCGTGGTCGACGGCCCGGGCTGGT-3 ' Pol wBrwM lni-.lfi 3 ' -NH 2 -CCCGACCA-P0,-5 ' Adaptor primer 1.
  • f API 5 * -GTAATACGACTCACTATAGGGC-3
  • the presence of the amine group on the 3 ' end of the lower strand blocks polymerase catalysed extension from free adaptor molecules that have not been ligated, thus preventing the generation of the primer binding site unless a defined, gene specific primer extends a DNA strand opposite the upper strand of the adaptor.
  • PrMADS2 5' CGGCGCTTCCGAACTCATAGAGTTTTCCTC 3'
  • PrMADS3 5 ' TAGCGCCACTTCGGCATCGCACAGC 3 '
  • PrFLl 5' CAAGGGACTTCAAATCCTTTCTCCCATTCATGG 3'
  • GSP-2 primers sequences The 1 ml of primary PCR was used in secondary PCR using the same cycling parameters and a second set of GSP primers with AP2 adaptor- primer.
  • PTMADS2 5' CGCCTTTTTCAGCAGACCATTCCGGC 3'
  • PRMADS3 5' CAGCAGTCCGTTTCCGGCGCTTCG 3'
  • PrFLl 5' CGTCCATGGTCCTTGTTAAAGACAGTTGTTGTTGG 3'
  • PCR fragments were cloned into the TA-type cloning vector and sequenced. Sequences of cDNAs, proteins and promoter regions PrMADS2, PrMADS3 and PrFLl are shown on Figs 12, 15 and 18.
  • Reverse primer 5 ' CCGGTCGACTTCTTTCCTTCTTTTCTTTCTGC 3 '
  • Reverse primer 5* ACGCGTCGACCAAGATCCCTCTGCTTCTTCACC 3'
  • Reverse primer 5' GCGAATTCCAAGATCCCTCTGCTTCTTCACC 3'
  • Reverse primer 5' GCG ECTTCATCTTACGTCACGCGAGG 3'
  • PCR fragments of Sail primers were digested with Sail restriction enzyme and introduced into the VI vector digested with Sail.
  • the orientation of promoters was checked using digestion with EcoRI enzyme.
  • PCR fragments of EcoRI primers were digested with EcoRI restriction enzyme and introduced into the VI vector digested with EcoRI. The orientation of promoters was checked using digestion with Sail enzyme. Maps of the resulting plasmids are shown in Figs. 21 and 22.
  • Binl9-Vl- promoter and Binl9 V2-promoter vectors were transformed into several Agrobacterium strains. Arabidopsis thaliana . Eucalyptus grandis and Plnus radiata embryos and explants were co-transformed with Agrobacterium strains.
  • the DNA encoding the laclq nuclear localisation signal peptide was amplified from the plasmid pGEM lacIqNLS by PCR using the 5' primer Lac I (this has an EcoRI site) and the 3' primer D23 which has a Kpnl site.
  • the amplified DNA fragment was restricted with EcoRI and Kpnl and cloned into pBRGUS 2 cut with the same enzymes.
  • the resulting plasmid was called pBRLac.
  • the pBRLac plasmid was then cut with Sphl and run on an agarose gel to allow the purification of the plasmid fragment containing the Laclq gene, Ampicillin resistance gene, and origin of replication away from the small Sphl fragment, which contained restriction sites that we wished to remove.
  • the resulting plasmid was called pBRLacBH- .
  • the second stage in the plasmid construction was the preparation for cloning of the Barstar gene under the regulation of the modified CaMV plus lac operator promoter. This was amplified from the plasmid p35S-op-Barstar EcoRI- which contained the promoter/gene sequence which had been modified to remove the EcoRI site between the promoter and coding sequence. This modification was accomplished by cutting the 35S-op-Barstar plasmid with EcoRI, carrying out a blunting reaction with T4 DNA polymerase, and then religating the blunted plasmid. The PCR was carried out using the 5 ' primer pQE-F and the 3 ' primer Bar-3 ' . The PCR fragment was cut with Xhol and Kpnl .
  • the plasmid was then ready for the cloning of, in this case, a flower specific promoter, into the unique EcoRI cloning site 5 prime of the laclq gene.
  • the EGM3 promoter from the Eucalyptus MADS gene EGM3 was used for this purpose. This was cut from plasmid pEGM3 with EcoRI. This promoter was cloned in both orientations, and the resulting plasmids were called V2 EGM3 sense and V2 EGM3 antisense.
  • the plasmid V2 EGM3 sense was used as a source the EGM3 regulated laclq and CaMVop Barstar genes for the plant transformation vector.
  • the orientation of the EGM3 promoter was determined by an Xbal Pstl digest. The presence of a 2.3 and 0.9 kb band was indicative of the correct orientation.
  • the second construct VI containing the promoterless Barnase gene, was constructed using pBrGUS 2 as the starting point.
  • a Barnase gene was amplified from genomic Bacillus amylollquefacien ⁇ using the primers Barnase 5 prime Sal, and Barnase 3 prime Kpn.
  • the resulting PCR product was restricted with Kpn I and Sal I as was pBRGUS 1 and a ligation reaction performed.
  • the plasmids from the resulting colonies were used as templates for sequencing using the amplification primers. Plasmid SB4 was found to contain a
  • the PCR fragment was restricted with Kpnl as was pGEM 3f and a ligation reaction performed. The resulting white colonies were screened for inserts.
  • the DNA from colony 7 was used for sequencing and found to contain the Barstar promoter plus Barstar sequence as in the genomic B. amyloliguefaciejjs DNA.
  • This plasmid was call 7 prom Barstar, as shown in Fig 21A.
  • the 7 prom Barstar plasmid was restricted with Kpn I as was pBRBarnase and the Promoter Barstar fragment was cloned into the Kpn I site of the pBRBarnase DNA. This resulted in a plasmid known as VI.
  • EGM3 VI sense The EGM3 promoter was cut out of pEGM3 using EcoRI, blunted using DNA polymerase I ( Klenow fragment ) and cloned into the unique Sail cloning site of the VI DNA which had also been restricted and blunted.
  • the resulting plasmid with the promoter inserted in the correct orientation was called EGM3 VI sense.
  • EGM3 VI sense DNA was linearised with Hindlll and cloned into the plant transformation vector Binl9 also linearised with Hindlll. This resulted in two plasmids, EGM3 VI Bin 1 and 2, corresponding to the two orientations of insertion. In orientation 2, the two EcoRI sites present in EGM3 VI Bin are approximately 80 bp apart.
  • the EGM3 VI Bin 2 was cut with EcoRI and blunted using DNA polymerase I (Klenow fragment ) .
  • the EGM3 promoter laclq gene, and the CaMV 35S op Barstar genes were cut out of EGM3 V2 sense using Aatll and Hindlll.
  • Northern blots were performed on a range of tissues in order to determine the tissue specificity of the expression of the three EGM MADS-box genes.
  • Tissue used for isolation of RNA from roots, seedlings, stems, shoots, leaves and mature flowers was obtained from Eu ⁇ aTyp-fcus grandis plants.
  • Tissue for isolation of RNA from floral tissues including receptacles, petals, stamens, carpels, and styles was collected from E. globulus flowers. This species was used because it produces very large flowers, making collection of sufficient amounts of RNA much easier.
  • the northern blots indicated that all three EGM genes are expressed at a high level in eucalypt flowers. When northern blots are exposed to film for long periods, weak expression of the EGM2 and EGM1 genes was detected in floral tissue. The EGM3 gene was specifically expressed in vegetative tissue. Within the flower, the EGM2 gene was observed to be expressed in stamens and petals. The EGM3 gene is expressed in receptacles, petals, stamens, carpels and styles.
  • This strategy involves the expression of an antisense; version of a gene critical to the development of reproductive structures so that vegetative development occurs instead.
  • the MADS-box region of PrMADSl gene was fused in the frame to the amino end of GUS reporter gene's coding region in sense and antisense orientations (Fig. 47). Constitutive expression of these genes will lead to high level of accumulation of the corresponding products in cytoplasm and nucleus. Accumulation of the fusion proteins containing sense orientation of the MADS-box region could lead to the inhibition of downstream transcription processes through competitively binding to specific trans-acting elements in the promoter regions.
  • Antisense orientation could block expression of MADS-box genes through RNA-RNA interactions between fusion antisense MADS- GUS mRNA and target MADS-box mRNAs in floral meristem.
  • Constructs were introduced into the binary vector Binl9 and transformed into AGL1 Agrobacterium strain. Arabidopsis thaliana plants were transformed using a root transformation procedure and transformants were selected on medium containing kanamycin antibiotic. Only antisense constructs were analysed for floral inhibition.
  • FB13L plants there was no detectable floral induction even after 25-35 leaves.
  • Leaves of FB13L line plants were stained with X-gluc (histological GUS assay). Most of the plants have shown the blue color of the leaves which indicates a high level of accumulation of GUS proteins.
  • BA benzyladenine also known as 6-benzylaminopurine.
  • IBA indole-3-butyric acid.
  • NAA 1-naphthaleneacetic acid
  • TDZ thidiazuron also known as l-phenyl-3-[l,2,3-thiadiazol- 5-yl] urea.
  • An Agrobacterium suspension with an optical density of 1.0 at 600nm diluted 1/20 is approximately 1X10 ⁇ cfu L "1 . Best results when wound near base of leaf. Alternatively, instead of leaving the wounded shoots in an Agrobacterium suspension for 1
  • the shoots can be vacuum-infiltrated with the Agrobacterium for 10-30 mins at 40-100 Kpa.
  • the shoots do not need to be wounded for the vacuum infiltration procedure.
  • callus formation is greater when wounding is effected with a needle rather than by vacuum-infiltration.
  • the cotyledons can be vacuum- infiltrated with the Agrobacterium for 20 mins at 95 KPa ( 28mm " 1 Hg ) .
  • the cotyledons do not need to be wounded for the vacuum infiltration procedure.
  • the hypocotyls require wounding even when using vacuum infiltration.
  • GBA 30 i.e. G22 containing 5mM BA and 0.5mM NAA
  • GBA 30 i.e. G22 containing 5mM BA and 0.5mM NAA
  • This liquid selection step has the advantages of reducing false positives by increasing selection pressure, reducing residual Agrobacterium and increasing the amount of shoot tissue compared to solid grown cultures.
  • Clones can be micropropagated by tissue culture propagation techniques and grown into trees of a size and form suitable for planting.
  • the media used in this study were G22, GBA and KG as described by Laine and David (1994). However, various basal media, including MS, B5 and P24, have been tested and found to support shoot regeneration.
  • the plant growth regulator regimes were generally different from those of Laine and David (1994), which were in the combination of 1-3 mM BA, 0.05-2 mM TDZ and 0.5-2.5 mM NAA for callus induction from leaves; 1-3 mM BA, 0.05-1 mM TDZ and 0.5-2.5 mM NAA for cotyledons; and 1-3 mM BA, 0.05-2 mM TDZ and 0.5-2.5 mM NAA for hypocotyls.
  • the differentiation medium was generally a GBA medium, which was a G22 but supplemented with 2.5 - 5 mM BA and 0.5 mM NAA (Laine and David, 1994).
  • a KG medium containing 0.2 mM BA is generally used as subculture medium for clone materials. All media were solidified with 0.25% 5 Gelrite or Phytagel. pH was adjusted to 5.7 - 5.8 using potassium hydroxide before autoclaving for 15 minutes at 121 C.

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Abstract

La présente invention concerne une technique de modification d'une plante visant à accroître la croissance végétale des structures végétales commercialement intéressantes au détriment des structures non essentielles et non commerciales. Des tissus reproductifs de Pinus radiata et de Eucalyptus grandis on a isolé des ADNc correspondant à des gènes qui s'expriment de façon spécifique pendant le développement précoce des structures végétales tant de sujets mâles que de sujets femelles. On a isolé de leurs ADN génomiques les régions promotrices destinées à ces gènes et on les a fusionnées pour former un gène structurel destiné à une ribonucléase bactérienne, la Barnase. Pour une variante de la technique, on a utilisé un système de contrôle de l'expression permettant de contrôler la déperdition de promoteur. On a également préparé des constructions géniques qui ont exprimé des formes antisenses de gènes essentiels pour le gemmage MADS destiné à ces plantes. On a utilisé des constructions de gènes candidates pour transformer les tissus de formation des pousses des espèces d'eucalyptus et de pins visés. On a testé des explants de transformants réussis, en vue de l'incorporation des constructions géniques et de la production de plants stériles.
PCT/AU1997/000625 1996-09-23 1997-09-23 Une plante, et technique de modification WO1998013503A1 (fr)

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000037663A3 (fr) * 1998-12-23 2000-11-09 Samuel Roberts Noble Found Inc Procede de transformation de vegetaux
WO2000023578A3 (fr) * 1998-10-16 2000-12-07 Univ California Procedes de suppression de la floraison de vegetaux transgeniques
WO2000053724A3 (fr) * 1999-03-11 2000-12-28 Genesis Res & Dev Corp Ltd Compositions et procedes convenant a la modification de la transcription de genes
WO2001012798A3 (fr) * 1999-08-18 2001-06-07 Suedwestdeutsche Saatzucht Plantes steriles males
WO2001012799A3 (fr) * 1999-08-18 2001-08-23 Horst Loerz Sequences regulatrices pour genes d'expression specifiques du pollen ou nombreux dans le pollen des plantes
WO2001031017A3 (fr) * 1999-10-25 2002-04-25 Thomas Dresselhaus Plantes a fleurs et a graines a croissance modifiee
EP1163340A4 (fr) * 1999-03-25 2002-08-07 Genesis Res & Dev Corp Ltd Compositions et methodes de modification de l'expression genique
US6563024B1 (en) 1999-05-07 2003-05-13 Oji Paper Co., Ltd. Process for transformation of mature trees of Eucalyptus plants
WO2003066823A3 (fr) * 2002-02-07 2003-12-04 Hybrigene Inc Prevention de la propagation des transgenes de plantes perennes genetiquement modifiees
JP2004511216A (ja) * 2000-06-20 2004-04-15 ジェネシス　リサーチ　アンド　デベロップメント　コーポレイション　リミテッド 植物遺伝子発現の改変のための核酸配列および方法
US6858776B1 (en) * 1999-03-17 2005-02-22 Carter Holt Harvey Limited Plants having modified reproductive capacity
US6987214B1 (en) 1998-10-16 2006-01-17 The Regents Of The University Of California Methods of suppressing flowering in transgenic plants
JP2008513029A (ja) * 2004-09-22 2008-05-01 アーバージェン リミテッド ライアビリティ カンパニー 生殖性除去構築物
US7667096B2 (en) * 2003-06-03 2010-02-23 University Of Georgia Research Foundation, Inc. Conditional sterility in plants
RU2410112C1 (ru) * 2009-07-07 2011-01-27 Государственное учреждение Всероссийский научно-исследовательский институт лекарственных и ароматических растений ("ВИЛАР") Лекарственное средство для профилактики и лечения нарушений нормальной микрофлоры (дисбактериозы)
US7910326B2 (en) 1996-09-11 2011-03-22 Arborgen, Inc. Materials and methods for the modification of plant lignin content

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU8622491A (en) * 1990-09-26 1992-04-15 Fb Investments Pty Ltd A plant and method of modification
WO1994000582A2 (fr) * 1992-06-30 1994-01-06 Bruinsma Seeds B.V. Procede d'obtention d'une plante a morphologie florale modifiee, et procede de protection des plantes contre les insectes nuisibles
WO1994025613A1 (fr) * 1993-05-03 1994-11-10 Cornell Research Foundation, Inc. Elements d'adn isoles dirigeant l'expression d'un gene specifique au pistil et a l'anthere, et leur procede d'utilisation
AU1713297A (en) * 1996-02-19 1997-09-02 Forbio Research Pty Ltd Regulation of gene expression in eukaryotes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU8622491A (en) * 1990-09-26 1992-04-15 Fb Investments Pty Ltd A plant and method of modification
WO1994000582A2 (fr) * 1992-06-30 1994-01-06 Bruinsma Seeds B.V. Procede d'obtention d'une plante a morphologie florale modifiee, et procede de protection des plantes contre les insectes nuisibles
WO1994025613A1 (fr) * 1993-05-03 1994-11-10 Cornell Research Foundation, Inc. Elements d'adn isoles dirigeant l'expression d'un gene specifique au pistil et a l'anthere, et leur procede d'utilisation
AU1713297A (en) * 1996-02-19 1997-09-02 Forbio Research Pty Ltd Regulation of gene expression in eukaryotes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
MOLECULAR BREEDING, Vol. 1, (1995), STRAUSS S.H. et al., "Genetic Engineering of Reproductive Sterility in Forest Trees", pages 5-26. *
THE PLANT CELL, Vol. 5, (1993), KANDASAMY M.K. et al., "Ablation of Papillar Cell Function in Brassica Flowers Results in the Loss of Stigma Receptivity to Pollination", pages 263-275. *
THE PLANT CELL, Vol. 5, (1993), THORSNESS M.K. et al., "Genetic Ablation of Floral Cells in Arabidopsis", pages 253-261. *
THE PLANT CELL, Vol. 9, (1997), COLUMBO L. et al., "Downregulation of Ovule-Specific MADS BOX Genes from Petunia Results in Maternally Controlled Defects in Seed Development", pages 703-715. *

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7910326B2 (en) 1996-09-11 2011-03-22 Arborgen, Inc. Materials and methods for the modification of plant lignin content
WO2000023578A3 (fr) * 1998-10-16 2000-12-07 Univ California Procedes de suppression de la floraison de vegetaux transgeniques
US7485772B2 (en) 1998-10-16 2009-02-03 The Regents Of The University Of California Methods of suppressing flowering in transgenic plants
US6987214B1 (en) 1998-10-16 2006-01-17 The Regents Of The University Of California Methods of suppressing flowering in transgenic plants
WO2000037663A3 (fr) * 1998-12-23 2000-11-09 Samuel Roberts Noble Found Inc Procede de transformation de vegetaux
JP2003525024A (ja) * 1999-03-11 2003-08-26 ジェネシス リサーチ アンド デベロップメント コーポレイション リミテッド 遺伝子転写改変のための組成物と方法
WO2000053724A3 (fr) * 1999-03-11 2000-12-28 Genesis Res & Dev Corp Ltd Compositions et procedes convenant a la modification de la transcription de genes
US6833446B1 (en) 1999-03-11 2004-12-21 Agrigenesis Biosciences Limited Compositions and methods for the modification of gene transcription
US6858776B1 (en) * 1999-03-17 2005-02-22 Carter Holt Harvey Limited Plants having modified reproductive capacity
EP1163340A4 (fr) * 1999-03-25 2002-08-07 Genesis Res & Dev Corp Ltd Compositions et methodes de modification de l'expression genique
JP2002539834A (ja) * 1999-03-25 2002-11-26 ジェネシス リサーチ アンド デベロップメント コーポレイション リミテッド 遺伝子発現の改変のための組成物と方法
US6563024B1 (en) 1999-05-07 2003-05-13 Oji Paper Co., Ltd. Process for transformation of mature trees of Eucalyptus plants
WO2001012798A3 (fr) * 1999-08-18 2001-06-07 Suedwestdeutsche Saatzucht Plantes steriles males
WO2001012799A3 (fr) * 1999-08-18 2001-08-23 Horst Loerz Sequences regulatrices pour genes d'expression specifiques du pollen ou nombreux dans le pollen des plantes
WO2001031017A3 (fr) * 1999-10-25 2002-04-25 Thomas Dresselhaus Plantes a fleurs et a graines a croissance modifiee
JP2004511216A (ja) * 2000-06-20 2004-04-15 ジェネシス　リサーチ　アンド　デベロップメント　コーポレイション　リミテッド 植物遺伝子発現の改変のための核酸配列および方法
EP1294870A4 (fr) * 2000-06-20 2005-09-07 Genesis Res & Dev Corp Ltd Sequences nucleotidiques et methodes pour la modification de l'expression de genes de plantes
WO2003066823A3 (fr) * 2002-02-07 2003-12-04 Hybrigene Inc Prevention de la propagation des transgenes de plantes perennes genetiquement modifiees
US7525015B2 (en) 2002-02-07 2009-04-28 Hybrigene, Inc. Prevention of transgene escape in genetically modified perennials
US7667096B2 (en) * 2003-06-03 2010-02-23 University Of Georgia Research Foundation, Inc. Conditional sterility in plants
JP2008513029A (ja) * 2004-09-22 2008-05-01 アーバージェン リミテッド ライアビリティ カンパニー 生殖性除去構築物
JP2011101659A (ja) * 2004-09-22 2011-05-26 Arborgen Inc 生殖性除去構築物
US8476493B2 (en) 2004-09-22 2013-07-02 Arborgen Inc. Reproductive ablation constructs
RU2410112C1 (ru) * 2009-07-07 2011-01-27 Государственное учреждение Всероссийский научно-исследовательский институт лекарственных и ароматических растений ("ВИЛАР") Лекарственное средство для профилактики и лечения нарушений нормальной микрофлоры (дисбактериозы)

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