WO1994000582A2 - Procede d'obtention d'une plante a morphologie florale modifiee, et procede de protection des plantes contre les insectes nuisibles - Google Patents

Procede d'obtention d'une plante a morphologie florale modifiee, et procede de protection des plantes contre les insectes nuisibles Download PDF

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Publication number
WO1994000582A2
WO1994000582A2 PCT/NL1993/000121 NL9300121W WO9400582A2 WO 1994000582 A2 WO1994000582 A2 WO 1994000582A2 NL 9300121 W NL9300121 W NL 9300121W WO 9400582 A2 WO9400582 A2 WO 9400582A2
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Prior art keywords
plant
gene
plants
fbpl
recombinant polynucleotide
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PCT/NL1993/000121
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English (en)
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WO1994000582A3 (fr
Inventor
Arjen J. Van Tunen
Chris Mollema
Gerco C. Angenent
Johannes J. M. Dons
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Bruinsma Seeds B.V.
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Priority to EP93916273A priority Critical patent/EP0672155A1/fr
Publication of WO1994000582A2 publication Critical patent/WO1994000582A2/fr
Publication of WO1994000582A3 publication Critical patent/WO1994000582A3/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • 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

  • a method for obtaining a plant having altered floral morphology and a method for protecting plants against pest insects is provided.
  • the invention is related to recombinant DNA, more in particular to recombinant DNA in relation to genetic manipulation of plants.
  • the invention is further related to plants which have modified floral organs and enhanced resistance to insect plagues and to secondary fungal infections, due to expression of the said recombinant DNA, as well as part of the said plant which are either sexually or asexually reproducible, or both.
  • the proteins encoded by these homeotic genes contain a region with a striking homology to the putative DNA binding domain of transcription factors from humans (SRF; Norman et al. , 1988) and Yeast (MCM1; Passmore et al. , 1988). This conserved motif was designated as the "MADS box” (Schwarz-Sommer et al. , 1990).
  • MADS box genes are only expressed during floral development and serve important functions with respect to the development of floral organs. For instance the agamous and deficiens genes play important roles in the development of petals and stamens. Natural mutants for these genes possess flowers without petals and stamens and instead
  • Antisense gene a gene, or a nucleotide sequence derived thereof, having 40 a homology of more than 50%, preferably more than 80% with a target gene as defined herein and which is linked to a promoter in the inverse 5' to 3' orientation with respect to the target gene.
  • Apomixis a form of asexual reproduction in which seed is produced but the embryo develops from an unreduced cell of the ovule without fusion of male and female gametes.
  • Gene a nucleotide sequence that can be expressed in the form of an RNA molecule and/or a polypeptide.
  • Inhibitory gene a gene or antisense gene, expression of which ultimately leads to inhibition of expression of a target gene as defined herein.
  • MADS box gene a gene having a region of about 55 amino acids with more than 30% ,• preferably more than 50%, amino acid homology with the yeast MCMl gene, with the Arabidopsis Agamous gene, the Antirrhinum Deficiens gene and/or the human SRF gene.
  • Parthenocarpy the phenomenon that (seedless) fruits are formed without a fertilization event.
  • Promoter a nucleotide sequence which is capable of promoting expression of a gene or antisense gene, or nucleotide sequences derived thereof, said expression being in the form of an RNA molecule and/or polypeptide.
  • Sense/co-suppression gene a gene, or a nucleotide sequence derived thereof, having a homology of more than 50%, preferably linked to a promoter in the normal 5' to 3' orientation with respect to the target gene.
  • Target gene a gene, expression of which is to be inhibited by proper expression of a suitable inhibitory gene as herein defined.
  • the present inventors have found that it is possible to disturb insect/flower relations in such a way that the pest insects are not attracted by the crop plants as a result of genetically modified target flowers. More specifically, the present inventors have found a method for protecting plants against pest insects by altering the flower morphology of the plant such that the petals and/or stamens are completely or partially removed.
  • the modification of the flower morphology of the plant is suitably and preferably carried out by introducing a specific recombinant polynucleotide in the genome of the plant. It has been found that the genetically modified plants according to the invention possess an increased resistance against pest insects attracted by flowers or insects feeding on the pollen. Moreover, it has been found that said genetically modified plants are less sensitive to organisms such as fungi which cause secondary infections at lesion sites caused by insect predators.
  • the present invention provides recombinant polynucleotides which can be suitably used for obtaining a plant having the petals and/or stamens completely or partially removed, essentially comprising: (a) an inhibitory gene either being a gene capable of inhibiting expression of a target gene in the said plant encoding a MADS box protein involved in flower organogenesis developmental programmes or being a toxin gene causing cell death, and
  • a preferred target gene according to the invention encodes MADS box proteins specifying the determination of floral primordia into petal and/or stamen primordia.
  • Especially preferred target genes encode the fJbpl or fbp2 proteins as disclosed hereinafter or proteins which are homologous therewith.
  • a preferred toxin causing cell death encodes products toxic for plant cells.
  • RNA degrading enzyme RNA degrading enzyme
  • Such ribonucleases can be used for a depletion of specific cell types as was for instance shown in the case of tapetal cell from tobacco anthers by Mariani et al. (1990) .
  • the inhibitory gene is an antisense or sense/co-suppression gene directed against a MADS box arget gene.
  • the promoter that is active in the floral primordia that give rise to the formation of petal and/or stamen organs comprises a highly active CaMV 35S promoter.
  • the highly active promoter comprises a MADS box gene promoter.
  • Preferred MADS box gene promoters are of the B-type e.g. the deficiens and the globosa promoters of Antirrhinum, and the apeta2a 3 and the pistillata promoters of Arabidopsis.
  • the high level promoter comprises the fjbpl promoter from Petunia.
  • the invention also provides a method for obtaining a plant with modified floral organs, comprising the steps of
  • the invention provides in another aspect a recombinant plant genome, comprising incorporated therein a recombinant polynucleotide as defined above.
  • the present invention is especially useful for plant species for which in the course of crop production fully developed petals and/or stamens are not necessary.
  • Preferred plant species according to the invention are parthenocarpic and/or apomictic plants.
  • Highly preferred plant species according to the invention are Cucurbita pepo, Pyru ⁇ co munis, Viti ⁇ vinifera, Solanum melongena, Carum carvi and Lycopersicum esculentum.
  • the most preferred plant species is cucumber ⁇ Cucumi ⁇ ⁇ ativi ⁇ ) .
  • the invention further encompasses a cell, organ, fruit, seed or progeny derived from a plant having altered flower morphology as defined above.
  • the present invention provides plants without or with reduced petals and/or stamen organs and as a result of that have a disturbed relation with pest insects which are attracted by and feeding on the flowers.
  • a thrips population developed slower on cucumber plants with modified flowers and the amount of leaf damage was reduced.
  • the genetic modification of floral organogenesis in which petals and/or anthers were completely or partly removed resulted in the formation of flowers which are more accessable for insecticides.
  • said plants have an increased resistance against those pest insects and the primary but also the secondary damage is significantly reduced.
  • the present invention thus provides an environmentally attractive and alternative method for obtaining plants with enhanced resistance against pest insects thereby also reducing the need for extensive and highly polluting chemical spraying.
  • the present invention can also be used to produce plants without or reduced petals thereby decreasing the size of the often massive flower screens. This has the advantage that the amount of photosynthesis increases dramatically as was reported recently by Rao et al. (1991) for a natural apetalous oilseed rape variety. Furthermore the present invention can also be used to produce plants without or with reduced stamens. The resulting absence of pollen will enhance resistance of strawberries against fungi as was reported by Simpson (1992) for natural strawberry mutants and the fungus Botrytis . The present invention also provides plants with stamens transformed into carpels.
  • Preferred plant species according to the invention are belonging to the group of oilseed crops like safflower, sunflower, flax, lineseed, sesame and groundnut, the group of grain legume crops like amaranth, chenopod, oat, millet, barley, rice, rye, sorghum, triticale, wheat, maize, pigeon pea, chickpea, soybean, lentil, alfalfa, bean, pea, broad bean, cowpea and buckwheat and the group of stimulating beverages like coffee, cola and cacao.
  • a highly preferred plant species according to the invention is rape seed ⁇ Bra ⁇ ica napens) . DESCRIPTION OF THE FIGURES.
  • Figure 1 Nucleotide and deduced amino acid sequence of the fjbpl (A) and fp2 cDNA (B) used for reverse genetic techniques.
  • the conserved amino acids used for the degenerated PCR primers are underlined.
  • the intron/exon junctions are denoted by arrowheads.
  • the fJpl cDNA sequences present in primers 1 and 2 used for amplification of the coding region by PCR are indicated by lines.
  • Figure 2 Diagrammatic representation of the fbpl gene (A) , the chimeric GUS construct (B) , the chimeric fbpl sense/co-suppression construct (C) and the fbp2 sense/co-suppression construct (D) .
  • Figure 3 Fbpl driven GUS expression in eight transgenic Petunia plants (T5001, T5002, T5003, T5005, T5006, T5008, T5010, and T5011) containing pFBP12E. GUS activity was only detected in petals and stamens and notni (for instance) leaves.
  • FIG. 4 Photograph showing different developmental stages of cucumber seedlings.
  • the optimal stage for transformation is stage B (B) .
  • Figure 5 Number of thrips larvae on leaves of flowerless plants (compartments A and C) or control plants (compartments B and D) one month after inoculation (ten weeks after sowing) .
  • Figure 6 Damage to cucumber leaves of flowerless plants (compartments A and C) or control plants (compartments B and D) one month after inoculation (ten weeks after sowing) .
  • Figure 7 Damage to cucumber leaves of flowerless plants (compartments A and C) or control plants (compartments B and D) three months after inoculation (eighteen weeks after sowing) .
  • Figure 8 Mean fruit weight of cucumbers grown on flowerless plants from compartments A and C compared to control fruit from compartments B and D.
  • Figure 9 Phenotype of transgenic petunia plants with inhibited fbpl gene expression.
  • the flowers have the 5 stamens transformed into 5 carpels which are fused to form an extra whorl 3 gynoecium.
  • Petunia hybrida a flower-specific gene entitled fbpl was isolated. This gene has features of a transcription factor and belongs to the group of MADS box genes. Fbpl RNA was shown to be present only in whorl 2 and whorl 3 floral organs. It was also found that expression of a bacterial ⁇ -glucuronidase (GUS) reporter gene in transgenic Petunia plants driven by the fbpl promoter was only detectable in whorl 2 and 3 organs. From the analysis of the same transgenic plants it could also be concluded that the fbpl promoter is activated during the differentiation stage of floral organ primordia.
  • GUS bacterial ⁇ -glucuronidase
  • the first class represented untransformed petunia W115 plants without the gene construct and with a normal W115 petunia phenotype.
  • the second class represented a class of plants containing the transgenes in a heterozygote situation and showed a phenotype closely resembling the primary transge ⁇ nic plants.
  • the third class represented plants with the transgenes in a homozygote situation and a considerable more severe phenotype than the primary transformant.
  • the transformation of petals into sepals was almost complete and the stamens were completely replaced by carpels. These carpels fused to each other and to the whorl 4 carpels. Surprisingly extra seedset in whorl 3 was observed when the modified flowers were pollinated.
  • the whorl 1 and 4 organs were not effected in both the heterozygote or in the homozygote transgenic plants. These homeotic conversions observed reveal a close resemblance with the conversions observed in natural Arabidop ⁇ is, Antirrhinum and Petunia flower mutants in which petals are replaced by sepals and stamens are replaced by carpels. Thus it was concluded that by inhibition of fbpl mRNA synthesis the phenotype of Petunia whorl 2 and 3 floral organs could be altered without changing the whorl 4 pistel organs.
  • a second MADS box cDNA, designated fbp2 was isolated from a petal- specific cDNA library with the use of the MADS box of fbpl as a probe.
  • RNA blot analysis revealed that the fbp2 gene is expressed only in flowers and only in whorls 2, 3 and 4.
  • Sense/co-suppression experiments in which a 35S CaMV-fbp2 construct was transformed to Petunia yielded transgenic plants with green corollas and petaloid anthers. From this it was concluded that using such a procedure it was again possible to generate transgenic plants with modified whorl 2 and 3 organs. It will also be possible to introduce similar geneconstructs into the cucumber genome.
  • An experiment in this direction revealed that the cucumber accession IBPGR can be readily transformed provided that seedlings of the optimal developmental stage are used in the transformation procedure.
  • DNA isolation, subcloning, restriction analysis and sequencing were performed using standard procedures well known to persons skilled in the art (e.g. Maniatis et al. , 1982) . Isolation of DNA from individual Petunia transformants and DNA gel blot analysis were performed as described by Koes et al., 1987. Polvmerase Chain Reaction (PCR)
  • Single stranded cDNA was synthesized by priming with the oligonucleotide 5' -CCGGATCCTCTAGAGCGGCCGC(T) 17 -3' (prat 7) starting from 10 ⁇ g of total RNA isolated from young corolla tissue (according to Koe ⁇ et al. , 1989) .
  • PCR analysis was performed in 100 ⁇ l of PCR buffer (10 mM Tris, pH 8.3, 50 mM KC1, 2.5 mM MgCl 2 ) containing 80 and 100 pmol of 5' and 3' primer, respectively, and 200 ⁇ M of each deoxynucleotide triphosphate.
  • Amplification involved 30 cycles with a denaturing time of 20s at 94°C, cooling down in 90 sec to 37°C, an anealing time of 30s, and an extension time of 6 min at 60°C.
  • Amplified cDNA was fractionated on 1% agarose gel, revealing one clear fragment of about 0.8 kb.
  • This band was isolated and subcloned into M13mpl9 vector using BamHl and Kpnl restriction site present in the 5' and 3' primers, respectively.
  • This cDNA insert (designated as fbpl) was used to screen the cDNA library and a complete cDNA clone of fbpl was isolated. This clone was used to rescreen the cDNA library under low stringency conditions (2xSSC, 60°C) and five additional MADS box cDNA clones were isolated including fbp2.
  • Prat 11, 5' -CCGGATCCCTCTCCCCATGGTTTCCCTTTCTC-3' and prat 14 5'- CGGGTCGACGTAAAACGACGGCCAGTGAATTG-3' were used to isolate an fbpl promoter fragment by a PCR approach.
  • the PCR was performed in 100 ⁇ l PCR buffer (see above) containing 100 pmol of prat 11 and 14.
  • Amplification involved 30 cycles with a denaturing time of 20s at 94°C, an annealing at 55°C for 30s and an extension time for 2 min at 72°C.
  • the resulting fragment of 240 bp was isolated from a normal 1% agarose gel and used for further subcloning.
  • the genomic library was made by insertion of partial Sau3A fragments of R27 nuclear DNA into the vector lambda GEM12 (Pro ega) . Approximately 150,000 plaques were sreened with 32 P-labelled fbpl cDNA, and positive clones were isolated and purified. Subsequently, the inserts or part of the inserts were subcloned into pEMBL vectors (Boehringer) and further analyzed by restriction enzyme analyses, hybridization and sequencing.
  • GUS extractions were performed as described by Jefferson et al . , (1987), by grinding the tissue with liquid N2. Fluorimetric GUS activity measurements were performed according to Jefferson et al. , (1987) . Protein concentrations were determined using the Biorad protein assay with Bovine serum albumin as a standard. Histochemical localization of GUS activity was performed as described by Koes et al. , (1990) . Before staining the floral buds were cut into small slices with a razor blade. The X-Gluc staining was performed according to Jefferson et al.
  • Mature flowerbuds were collected from cucumber plants and layered on a MS/agar plate. Bombardment was performed with a DuPont PDS1000 apparatus at 28.5 inch immediately after the floral tissues were collected.
  • pFBP12E DNA (2.5 ⁇ g) was precipitated on 1.6 ⁇ m thungsten particles according to the method of van der Leede-P puts et al. (1992) .
  • the floral tissues were incubated for 2 days. Subsequently the floral tissue was stained for GUS enzyme activity according Jefferson et al. (1987) .
  • Petunia hybrida was transformed according to the method of Horsch et al. , (1985) . After shoot and root induction on kanamycin-containing media, plants were put in the soil and kept in a green house. Plants regenerated on kanamycin-less media) from leaf discs treated with the strain LBA4404 without a binary vector served as a control.
  • Regeneration of the cucumber variety IBPGR was done according to the method developed by Colijn-Hooymans et al . (1992) .
  • Cucumber seeds were sterilized for 15' in 2% (w/v) NaOCl and washed three times with sterile water.
  • the seeds were germinated in honey jars on sterilized (20' 120°C) MS medium supplemented with trypton L42 (Oxoid 500 mg/1) , sucrose (30 g/1) and Imperial agar (6 g/1) , pH5.6, at 25°C under cool white light (Philips FTD 36W/TL84, 16, 2500 lux) .
  • Cotyledons were excised from the seedlings at the developmental stage B and transversely cut into two parts.
  • stage B the cotyledons are green and in a vertical position ( Figure 4) which is in contrast to stage A were the cotyledons' are still folded and are light green.
  • the basal explant was inserted in induction medium with the basal wounded edge in contact with the medium.
  • This induction medium consisted of MS salts and vitamins supplemented with sucrose (40 g/1) , trypton L42 (500 g/1) , 50 ⁇ M kinetin, 0.1 ⁇ M indoleacetic acid (IAA) and Imperial agar (6 g/1), pH 5.6, sterilized for 20' at 120°C.
  • Petri dishes containing the explants were placed in a growth chamber at 25°C and 16 hrs of cool white light
  • Agrobacterium tumefaciens C58C1 containing a GUS-intron reporter gene (pMP90 + GUS intron; Van Canneyt et al. , 1990) was used.
  • the bacteria were grown for 16 hrs in 10 ml LB supplemented with kanamycin and rifampicin (50 mg/1) at 28°C in a shaking wather bath. After growth the bacteria were pelleted by centrifugation at 5000 rpm for 10' . The resulting pellet was resuspended in 10 ml liquid induction medium poured into petri dishes to be used for inoculation. Cotyledonary explants were placed for 2 minutes into the bacterial suspension.
  • the inoculated discs were co-cultivated with Agrobacterium for 3 days at 25°C and 16hrs of 750 lux cool white light. Subsequently they were removed to the same medium supplemented with 250 mg/1 cefotaxime, 200 mg/1 vancomycin and 75 mg/1 kanamycin. The cultures were placed at 25°C and 16 hrs of cool white light (Philips 36W/TL 84) at 750 lux. After 14 days the developed shoots were cut from the explants. Rooting of the developed shoots occured spontaneously on the same medium in honey jars. Young plants with two leaves were transferred to rockwool plugs and placed in the greenhouse. After this the plants were checked for GUS activity. Growth of cucumber plants and removal of floral parts.
  • parthenocarpic cucumberplants (variety "Corona", De Ruiter Seeds) were used for insect resistance experiments.
  • the plants were grown in pots containing peatsoil with osmocoat.
  • the experiment was performed in four compartments of a greenhouse (designated A, B, C and D) .
  • Each compartment was 16 square meters in size and contained 16 plants.
  • the compartments were strictly isolated from each other.
  • the plants were grown at 25°-C during day time (from 06.00 - 22.00) and 20°C during the night (from 22.00 - 06.00) .
  • Shoots were removed twice a week. Approximately two meters above the soil a wire was constructed which carried the top of the plant. From this top downwards two stems were maintained.
  • oligonucleotides and an oligo(dT) primer 5'- CCGGATCCTCTAGAGCGGCCGC(T) 17 -3' , were used for an amplification of MADS box cDNA clones synthesized from floral mRNA by polymerase chain reaction (PCR) . Subsequently, the resulting PCR products were used to screen a lambda gtll-based petal-specific library (van Tunen et al. , 1988) . Initially, one cDNA clone, designated as fbpl , was isolated and analyzed in more detail.
  • PCR polymerase chain reaction
  • the complete fbpl cDNA insert was used to isolate the fbpl gene from a genomic library of the Petunia line R27. Hybridization under stringent conditions revealed four positive clones, which were further purified. The nucleotide sequence of the fbpl gene in one of those clones as well as +/- 1 kb of upstream sequence were determined resulting in a genomic structure depicted in Figure 2A.
  • the plasmid pBI121 (Jefferson et al. , 1987) was digested with EcoRI and Sstl and the resulting nos terminator fragment was cloned into pBIN19 (Bevan et al. , 1984) rendering pBINT. Subsequently a pDIP22 (a Bluescript plasmid containing the full size fbpl cDNA) Xbal/Kpnl fragment was cloned into pBINT cut . ith Xbal and Kpnl. Finally in this subclone a Xbal/BamHI 35S CaMV fragment isolated from the plasmid pCALlGc (P tenants et al.
  • plasmid pFBP20 Figure 2C
  • pDIP63 a Bluescript plasmid containing the full size fbp2 cDNA
  • EcoRV/Xbal fragment was cloned into pBINT cut with Smal and Xbal. This yielded a subclone in which a Xbal/BamHI 35S CaMV fragment isolated from the plasmid pCALl was cloned yielding plasmid pFBP21 ( Figure 2D) .
  • the binary vector containing the fbpl-GUS construct (pFBP12E) or the 35S CaMV-fbpl construct (pFBP20) were transferred from E. Coli JM83 (Messing, -1978) to Agrobacterium tumefacien ⁇ strain 4404 (Hoekema et al. , 1983) by triparental mating (Rogers et al., 1986), using a strain containing plasmid pRK2013 (Ditta et al. , 1980) . Exconjugants were used to transform Petunia hybrida leaf discs, as described by Horsch et al (1985) .
  • Leaf disks were prepared from top leaves of young, non-flowering Petunia hybrida variety W115 plants. After shoot and root induction on kanamycin containing media, plants were put on soil and transferred to the greenhouse. Plants regenerated from leaf discs treated with the LBA 4404 strain lacking a binary vector served as a control.
  • transgenic plants expressing the GUS construct containing the 35S CaMV-fbpl construct or containing the 35S CaMV-fbp2 construct.
  • the transgenic petunia plants carrying the fbpl-GUS construct were analyzed for fbpl driven GUS expression using the methods described in the Experimental part.
  • Fourteen independent transgenic Petunia plants were generated and analyzed. From the transgenic plants eight showed a clear GUS expression in fluorigenic assays using petal or stamen tissue extracts ( Figure 3) . No GUS enzyme activity above the background was measured in other tissue (roots, stem, leaves, pedicel, sepals, carpels) than petals and/or stamens suggesting that the fbpl promoter is only active in those two types of tissues.
  • Class 1 6 plants
  • Class II plants (10 plants) had a phenotype resembling the primary transfor ant.
  • a backcross of this plant with W115 resulted in a 1:1 segregation of normal W115 plants and transgenic plants.
  • Class III (5 plants) had severely affected flowers with petals almost completely transformed into sepals and stamens converted into carpels. These carpels were partially or often also completely fused to each other but also to the two whorl 4 carpels.
  • transgenic plants were raised containing the 35S CaMV-fbp2 construct (pFBP21) .
  • Two of the transgenic plants had modified flower organs as the result of co-suppression inhibition.
  • One plant has flowers with a greenish corolla and petal tissue has been formed on top of the anther.
  • the other plant also exhibited a normal phenotype with the exception of a completely green, short corolla with unfused limbs and a short carpel.
  • the fbp2 gene has an important role in the formation of petals and stamens. Obviously this cDNA represents a molecular handle to modify flower morphology by reverse genetic methods.
  • pFBP12E containing the fbpl-GUS construct was introduced into cucumber petals.
  • Petals bombarded with pFBP12E showed a high number of blue staining cell groups as a result of transient activity of the fbpl promoter driving the GUS reporter gene. This indicates that fbpl promoter is active in cucumber petals.
  • the same fbpl-GUS construct was also transformed into the genome of cucumber variety Isfahan using Agrobacterium mediated transformation procedure starting from hypocotyls (see also under VII) .
  • Transgenic cucumber plants were generated and tested for GUS activity.
  • the transgenic plants showed a clear 35S CaMV driven GUS activity in the first leaves.
  • Southern blot and PCR analysis revealed that those plants contained one gene copy which was integrated in the proper way in different chromosomal positions. From these transformation experiments it was concluded that the developmental stage of the cucumber seedlings is essential for an efficient transformation and regeneration of cucumber transgenic plants.
  • Figure 4 shows the optimal developmental stage of the seedlings. Using our transformation procedure a transformation efficiency of 3 % was obtained.
  • Floricaula A homeotic gene required for flower development in Antirrhinum maju ⁇ . Cell , 63:1311-1322.
  • Glucuronidase as a sensitive and verisatile gene fusion marker in higher plants. EMBO J. , 6:3901-3907.

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Abstract

Plantes à morphologie florale modifiée, dans le génome desquelles est incorporé un polynucléotide recombiné essentiellement constitué d'un gène inhibiteur pouvant, lorsqu'il est exprimé correctement dans les cellules du méristème floral, inhiber le développement et la détermination des ébauches florales, susceptibles de transformer celles-ci en ébauches de pétal et/ou d'étamine. Lesdites plantes ont un rapport perturbé avec les insectes nuisibles attirés par les fleurs. De ce fait, ces plantes présentent une résistance accrue à ces insectes nuisibles, et non seulement les dégâts primaires mais aussi les dégâts secondaires provoqués par ces insectes sont sensiblement diminués.
PCT/NL1993/000121 1992-06-30 1993-06-07 Procede d'obtention d'une plante a morphologie florale modifiee, et procede de protection des plantes contre les insectes nuisibles WO1994000582A2 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0798958A1 (fr) * 1994-12-21 1997-10-08 The Salk Institute For Biological Studies Plantes modifiees genetiquement avec un developpement floral module
WO1998013503A1 (fr) * 1996-09-23 1998-04-02 F.B. Investments Pty. Ltd. Une plante, et technique de modification
FR2768746A1 (fr) * 1997-09-23 1999-03-26 Agronomique Inst Nat Rech Promoteur specifique des petales et procede d'obtention de plantes a fleurs sans petale
WO2000032780A1 (fr) * 1998-12-03 2000-06-08 Commonwealth Scientific And Industrial Research Organisation Regulation de la floraison
WO2000037488A2 (fr) * 1998-12-21 2000-06-29 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Nouveaux genes a boite mads et utilisation de ces genes
WO2002029028A2 (fr) * 2000-10-03 2002-04-11 Bayer Bioscience N.V. Brassicaceae a developpement floral modifie
WO2004035797A2 (fr) * 2002-09-27 2004-04-29 Dlf - Trifolium A/S Promoteurs specifiques aux tissus issus de plantes
AU779114B2 (en) * 1998-12-03 2005-01-06 Commonwealth Scientific And Industrial Research Organisation Control of flowering
US8022274B2 (en) 1998-09-22 2011-09-20 Mendel Biotechnology, Inc. Plant tolerance to low water, low nitrogen and cold
US8669413B2 (en) 2009-11-25 2014-03-11 Hm.Clause, Inc. Breeding and selection for resistance to melon severe mosaic virus (MeSMV)
CN104450735A (zh) * 2014-11-19 2015-03-25 江西农业大学 黄瓜CsMADS1基因过表达载体及其应用
US11124801B2 (en) 2018-04-18 2021-09-21 Pioneer Hi-Bred International, Inc. Genes, constructs and maize event DP-202216-6

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1993021322A1 (fr) * 1992-04-13 1993-10-28 The Rockefeller University Procede de regulation et de determination de la morphogenese d'organes vegetaux, un gene homeotique, un element promoteur utilise a cet effet, et utilisations connexes

Patent Citations (1)

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WO1993021322A1 (fr) * 1992-04-13 1993-10-28 The Rockefeller University Procede de regulation et de determination de la morphogenese d'organes vegetaux, un gene homeotique, un element promoteur utilise a cet effet, et utilisations connexes

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CELL vol. 63 , 21 December 1990 pages 1311 - 1322 COEN, E.S., ET AL. 'floricaula: A homeotic gene required for flower development in Antirrhinum majus' *
GENES AND DEVELOPMENT vol. 5 , 1991 pages 484 - 495 MA, H., ET AL. 'AGL1-AGL6, an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes' *
J. CELL. BIOCHEM. SUPPL., CROP IMPROVEMENT THROUGH BIOTECHNOLOGY, SYMPOSIUM HELD APRIL 10-16, 1992. vol. 16F , April 1992 page 231 DONS, J.J.M., ET AL. 'Differential expression of fbp1: a gene involved in flower mophogenesis of petunia' *
PLANT MOLECULAR BIOLOGY. vol. 19, no. 5 , August 1992 , DORDRECHT, THE NETHERLANDS. pages III - VI LIFSCHITZ, E., ET AL. 'News and views' & WORKSHOP ON MOLECULAR CONTROL OF FLOWER DEVELOPMENT AND PLANT REPRODUCTION, AMSTERDAM, NETHERLANDS, APRIL 12-16, 1992. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA vol. 86 , March 1989 , WASHINGTON US pages 1934 - 1938 GOULD, S.J., ET AL. 'Use of the DNA polymerase chain reaction for homology probing: Isolation of partial cDNA or genomic clones encoding the iron-sulfur protein of succinate dehydrogenase from several species' *
SCIENCE vol. 250 , 16 November 1990 , LANCASTER, PA US pages 931 - 936 SCHWARZ-SOMMER, Z., ET AL. 'Genetic control of flower development by homeotic genes in Antirrhinum majus' *
See also references of EP0672155A1 *
THE PLANT CELL. vol. 4, no. 5 , May 1992 , ROCKVILLE, MD, USA. pages 507 - 512 CHASAN, R. 'Meeting report: A feast of maize genetics' & 34TH ANNUAL MAIZE GENETICS MEETING, HELD MARCH 19-22, 1992. *
THE PLANT CELL. vol. 4, no. 8 , August 1992 , ROCKVILLE, MD, USA. pages 867 - 870 CLARKE, A.E., ET AL. 'Forefronts of flowering: meeting report' & WORKSHOP ON MOLECULAR CONTROL OF FLOWER DEVELOPMENT AND PLANT REPRODUCTION , AMSTERDAM, NETHERLANDS, 12-16, APRIL, 1992. *
THE PLANT CELL. vol. 4, no. 8 , August 1992 , ROCKVILLE, MD, USA. pages 983 - 993 ANGENENT, G.C., ET AL. 'Differential expression of two MADS box genes in wild-type and mutant petunia flowers' *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0798958A4 (fr) * 1994-12-21 1998-05-27 Salk Inst For Biological Studi Plantes modifiees genetiquement avec un developpement floral module
EP0798958A1 (fr) * 1994-12-21 1997-10-08 The Salk Institute For Biological Studies Plantes modifiees genetiquement avec un developpement floral module
WO1998013503A1 (fr) * 1996-09-23 1998-04-02 F.B. Investments Pty. Ltd. Une plante, et technique de modification
FR2768746A1 (fr) * 1997-09-23 1999-03-26 Agronomique Inst Nat Rech Promoteur specifique des petales et procede d'obtention de plantes a fleurs sans petale
WO1999015679A1 (fr) * 1997-09-23 1999-04-01 Institut National De La Recherche Agronomique Promoteur specifique des petales et procede d'obtention de plantes a fleurs sans petale
US8022274B2 (en) 1998-09-22 2011-09-20 Mendel Biotechnology, Inc. Plant tolerance to low water, low nitrogen and cold
AU779114B2 (en) * 1998-12-03 2005-01-06 Commonwealth Scientific And Industrial Research Organisation Control of flowering
WO2000032780A1 (fr) * 1998-12-03 2000-06-08 Commonwealth Scientific And Industrial Research Organisation Regulation de la floraison
WO2000037488A2 (fr) * 1998-12-21 2000-06-29 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Nouveaux genes a boite mads et utilisation de ces genes
WO2000037488A3 (fr) * 1998-12-21 2000-09-14 Max Planck Gesellschaft Nouveaux genes a boite mads et utilisation de ces genes
WO2002029028A2 (fr) * 2000-10-03 2002-04-11 Bayer Bioscience N.V. Brassicaceae a developpement floral modifie
WO2002029028A3 (fr) * 2000-10-03 2002-07-25 Aventis Cropscience Nv Brassicaceae a developpement floral modifie
WO2004035797A2 (fr) * 2002-09-27 2004-04-29 Dlf - Trifolium A/S Promoteurs specifiques aux tissus issus de plantes
WO2004035797A3 (fr) * 2002-09-27 2005-11-10 Dlf Trifolium As Promoteurs specifiques aux tissus issus de plantes
US8669413B2 (en) 2009-11-25 2014-03-11 Hm.Clause, Inc. Breeding and selection for resistance to melon severe mosaic virus (MeSMV)
CN104450735A (zh) * 2014-11-19 2015-03-25 江西农业大学 黄瓜CsMADS1基因过表达载体及其应用
US11124801B2 (en) 2018-04-18 2021-09-21 Pioneer Hi-Bred International, Inc. Genes, constructs and maize event DP-202216-6
US11421242B2 (en) 2018-04-18 2022-08-23 Pioneer Hi-Bred International, Inc. Genes, constructs and maize event DP-202216-6

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