WO1997025433A1 - Controle de la floraison de plantes - Google Patents

Controle de la floraison de plantes Download PDF

Info

Publication number
WO1997025433A1
WO1997025433A1 PCT/EP1997/000161 EP9700161W WO9725433A1 WO 1997025433 A1 WO1997025433 A1 WO 1997025433A1 EP 9700161 W EP9700161 W EP 9700161W WO 9725433 A1 WO9725433 A1 WO 9725433A1
Authority
WO
WIPO (PCT)
Prior art keywords
plants
plant
flowering
safpfl
chimeric gene
Prior art date
Application number
PCT/EP1997/000161
Other languages
English (en)
Inventor
Siegbert Melzer
Thomas Kania
Klaus Apel
Original Assignee
Eidg. Technische Hochschule Zürich Ethz
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eidg. Technische Hochschule Zürich Ethz filed Critical Eidg. Technische Hochschule Zürich Ethz
Publication of WO1997025433A1 publication Critical patent/WO1997025433A1/fr

Links

Classifications

    • 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]

Definitions

  • This invention relates to DNA constructs comprising genes coding for flowering promoting factors (FPFs) in plants, i. e. factors causing earlier than normal flowering in plants, transgenic plants showing early flowering comprising such constructs constitutively expressing FPFs, and methods of using such early flowering plants.
  • FPFs flowering promoting factors
  • FPF1 is the earliest gene that is expressed in the apical meristem after the induction of flowering and in particular it is expressed before the floral meristem identity genes LEAFY and APETALA1 .
  • FPFs flowering promoting factors
  • this invention is mainly based on the finding that the gene SaFPFl is able to reduce the time to flowering, and of homologous genes thereof with similar activities, and provides DNA constructs which are stably introduced into plants, and are able to induce flowering before the naturally occurring timepoint of flowering.
  • the invention provides transgenic plants of significant economic value which are transformed with expression vectors that constitutively express a homologous or heterologous FPF. Such transgenic plants exert precocious flowering with a shortening of the life cycle of the plants.
  • a further advantage of the invention is seen in overcomming detrimental influences of environmental factors, such as the daylength and vernalization dependent timing and the extent of the time to flowering in certain species. Furthermore, the controlled induction of early flowering by constitutively expressing a FPF overcomes the limitations of geographic distribution of crops and in commercial flower and fruit producing species yields higher productivity rates by growing more generations of plants per year.
  • the present invention relates in a first aspect to a chimeric gene construct comprising a recombinant DNA containing a DNA sequence encoding a polypeptide which has the activity of a flowering promoting factor (FPF) and an expression cassette and which can be expressed within a flowering plant.
  • FPF flowering promoting factor
  • a chimeric gene construct according to the invention is in particular a recombinant DNA construct comprising heterolo ⁇ gous DNA and a gene coding for a FPF, e.g. coding for SaFPFl, SaFPF2, AtFPFl or AtFPF2, or for a FPF which is homologous to any of said FPFs and which has FPF activity, and preferably has from about 60 to 99% homology to SaFPFl.
  • the Sa stand for Sinapis alba (white mustard) , and the At for Arabidopsis thaliana .
  • Such chimeric gene constructs wherein the DNA sequence encoding the FPF may be under the control of the CaMV promoter, or genes or homologous sequences having the properties of expression signals, including bacterial expression signals, such as the expression signals of the CaMV promoter (Odell et al., 1985), nopaline synthase gene
  • octopine synthase gene from the Ti- plasmid of Agrobacterium tumefaciens (Croy, 1993) , or of the meristem specific histone H4 gene (Atanassova et al., 1992), or of developmental and tissue specific promoters like the Shootmeristemless (Long et al., 1996) and Knotted promoters (Lincoln et al., 1994), the heat shock inducible promoters (Schoffl et al. 1989), or of promoters that are inducible by chemicals like the steroid inducibele gene expression system (Schena et al. 1991) .
  • a particular chimeric gene construct according to the inven ⁇ tion is for example the plasmid pBIN19-SaFPFl, pBIN19-SaFPF2, pBIN19-AtFPFl, or pBIN19-AtFPF2.
  • the gene SaFPFl was identified in a screening for genes that are induced during the transition from vegetative to generative growth in mustard. Therefore, a subtractive hybridization technique was used as described by Melzer et al. (1990).
  • phage From an enriched cDNA library of 5-day-induced plants 32 phage were selected as putative flowering specific cDNA clones. The identity of the putative early flowering-specific cDNAs was tested by mRNA dot blot hybridization. Among the recombinant phage selected only three represented transcripts that were specifically expressed in the apex but not in the leaf, and only one cDNA was identified that shows a dramatic upregulation of the expression of the gene pSFD5.04 after the induction of flowering.
  • the gene has the potential to shorten the time to flowering even in the heterologous Arabidopsis thaliana the gene was renamed to SaFPFl ⁇ Sinapsis alba Flowering Promoting Factor 1) .
  • a chimeric gene construct according to the invention is in particular comprising a DNA sequence selected from the group consisting of DNA sequences coding for SaFPFl, SaFPF2, AtFPFl and AtFPF2, from Sinapis alba and Arabidopsis thaliana, respectively.
  • the gene SaFPFl is expressed during the transition from vegetative to reproductive growth in the apical meristem of mustard. It is after the transformation constitutively expressed under the control of inducible and constitutive promoters in Arabidopsis thaliana and also in other heterologous plants comprising this DNA sequence.
  • the invention is directed also to gene constructs, wherein the DNA sequence encodes a FPF which is homologous to SaFPFl, SaFPF2, AtFPFl, or AtFPF2, that is to say which hybridizes under normal, in particular stringent hybridi ⁇ zation conditions to the named genes, has the flowering promoting activity, and preferably has from about 60 to 99% homology to the SaFPFl or any other FPF gene shown in the Examples.
  • Such genes may originate from mustard or from other plants, or are synthesised by methods known in the art, e. g. with an automated DNA synthesizer, or are modified from its original form, e. g. by synthetic methods known in the art, and exhibit the early flowering promoting biological activity.
  • cDNAs 95G12T7 and 98K16t7 have been identified as cDNAs by T. Newman et al. (1994) in a random sequencing project of cDNAs from Arapidopsis that are called Expressed Sequenc Tags (ESTs) .
  • the cDNAs 95G12T7 and 98K16T7 have 100% identity and are two cDNAs derived from the same gene.
  • the cDNAs are stored in and are available from the Arabidopsis Biological Resoure Center (ABRC) at the Ohio State University, Ohio, USA, and their sequence is disclosed in common data bases like GenBank (National Institute of Health, USA, Computer Database) or EMBL (Heidelberg, Germany) .
  • the three Arabidopsis thaliana cDNA clones with stock number EST 40B10T7, EST 95G12T7 and98K16T7 were inserted into a vector that allows expression only in a bacterium, however, they cannot be expressed in a plant. They have 72,527% and 89.655% identity with SaFPFl, respectively, and are now named AtFPFl and AtFPF2, respectively. Their FPF activity was not disclosed up to now.
  • a chimeric gene construct is a DNA construct comprising a structural FPF gene operably linked to an expression cassette which permits controlled induction of gene expression and termination. It comprises promoter and terminator sequences. Constitutively or developmentally regulated or inducible promoters are included. Particularly useful are expression signals originating from genes of plants or plant viruses, e.g. of the Cauliflower Mosaic Virus (CaMV) genes or homologous sequences having the aforementioned properties of the expression signals. Also bacterial expression signals usable in plants are suitable, especially the expression signals of the nopaline synthase gene (nos) or the octopine synthase genes (ocs) from the Ti-plasmid of Agrobacteri um tumefaciens .
  • CaMV Cauliflower Mosaic Virus
  • tissue- preferential or tissue-specific promoters like those from histone or cyclin genes, which may direct higher expression in particular tissues of the plant, e.g. the apical meristem, as well as promoters from tissue specific and developmentally regulated genes like the Shootmeristemless gene and Knotted like genes from Arabidopsis and their ho ologs from other plants, and promoters from genes that are inducible by chemicals like the steroid inducibele gene expression system.
  • DNA sequences which are potentially useful in the chimeric constructs are known sequences which themselves may carry regulation signals such as ribosome binding sites, and translational enhancer sequences, e.g. an ⁇ -element (Holtorf et al. 1995) .
  • the invention relates in particular to a chimeric gene construct which is the plant transformation vector pBIN19 comprising the coding region of SaFPFl, SaFPF2, AtFPFl, or AtFPF2 flanked by a CaMV 35S promoter and a termination signal (e. g. pBinl9-SaFPFl; see Fig. 4).
  • a chimeric gene construct which is the plant transformation vector pBIN19 comprising the coding region of SaFPFl, SaFPF2, AtFPFl, or AtFPF2 flanked by a CaMV 35S promoter and a termination signal (e. g. pBinl9-SaFPFl; see Fig. 4).
  • the construction of binary vector systems is achieved in a way, that the tumor genes are deleted from the former TI- plasmid, which carries only the virulence functions and serves as a helper plasmid and that a second plasmid serves as the plant transformation vector like pBIN19. It has T-DNA borders on a compatible replicon that will function in both E. coli and Agrobacterium. DNA that is inserted between the T-DNA borders, like SaFPFl and a selectable marker gene, will be efficiently transfered to and stably maintained within the plant genome (Hoekeema et al. 1983, Bevan 1984, and Klee et al. 1985) .
  • Another aspect of the invention concerns a host cell transformed with a chimeric DNA construct according to the invention.
  • Such host cell is of bacterial or plant origin, e.g. Agrobacterium tumefaciens, and in particular is Agrobacterium tumefaciens transformed with the plasmids according to the Examples, e. g. with pBIN19-SaFPFl.
  • the invention concerns a method for the production of a transgenic plant or a part thereof, comprising transformation of a plant or a part therof with a chimeric gene construct according to the invention, and when required growing the transformed plant or transformed part thereof under conventional growing conditions.
  • Agrobacteria are capable of transfering a defined piece of DNA (T-DNA) containing tumorigenic genes into the genome of a large number of gymnosperms and angiosperms (Chilton et al. 1977, Willmitzer et al. 1980).
  • Transformation of plants are possible by other techniques, like icroinjection (Neuhaus et al. 1994), direct DNA trans ⁇ fer to protoplasts and subsequent regeneration of whole plants (Paszkowski et al. 1984), and direct gene transfer mediated by electroporation (Fromm et al. 1986) or micro- projectile bombardment (Klein et al. 1987) .
  • the invention concerns a transgenic plant cell or plant comprising stably integrated into its genome a DNA sequence encoding a recombinant FPF under control of a promoter causing constitutive expression of the FPF.
  • Such transgenic plant cell or plant including its seed has been transformed for example with a chimeric DNA construct described above, in particular such cell or plant including its seed, which has stably integrated into its genome a DNA sequence encoding a FPF and regulatory sequences causing constitutive expression of the FPF, and which may have been transformed with a host cell, in particular Agrobacteri um tumefaciens transformed with the plasmid pBIN19-FPFl, or any other plasmid comprising an FPF gene according to the Examples.
  • transgenic plant cell or plant may also be selected from the group of ornamental plants and vegetable and fruit producing plants of the various plant groups. Special mention has to be made of trees, that flower normally after 10-20 years of vegetative growth, having the limitation of a fast outbreeding of usefull traits.
  • Such a transgenic plant cell or plant is preferably selected from the group of higher plants, preferably a crop plant.
  • Some suitable plants include species of the genera e.g. Tri ticum, Oryza , Zea, Hordeum, Sorghum, Avena , Secale, Loli um, Festuca , Glycine, Brassica , Solanum, Lotus, Medicago, Trifolium, Petunia , Gerbera , elianthus and Beta, and in particular the Arabidopsis thaliana ecotypes.
  • the invention concerns a progeny or propa- gule, including seed, of a transgenic plant described above.
  • the invention concerns a method for promo ⁇ ting early flowering of a flowering plant, comprising trans- forming said plant or cells thereof with a chimeric gene con ⁇ struct according to the invention, and growing the trans ⁇ formed plant or cells under normal conditions.
  • the invention concerns a method for redu- cing the time for the selection of .agronomically and horti ⁇ culturally improved traits of a plant, comprising trans- forming said plant or cells thereof with a chimeric gene construct according to the invention, and growing the transformed plant or cells under normal conditions.
  • the invention concerns a method for growing plants in higher or cooler latitudes where they normaly do not flower, comprising transforming said plants or cells thereof with a chimeric gene construct according to the invention, and growing the transformed plants or cells under normal conditions.
  • the invention concerns a method for obtai ⁇ ning higher productivity rates of plants by growing more generations per year, comprising transforming said plants or cells thereof with a chimeric gene construct according to the invention, and growing the transformed plants or cells under normal conditions.
  • the invention concerns a method for increa- sing the productivity rate of ornamental flower plants, or food producing plants, comprising transforming said plants or cells thereof with a chimeric gene construct according to the invention, and growing the transformed plants or cells under normal conditions.
  • the invention concerns a method for retarding or abolishing flowering in crop plants, comprising downregulation of a gene by an antisense approach achieved by the constitutive expression of a gene in a reverse orientation.
  • This method is useful when flowering is normally not a desired trait, e.g. in sugar beet or different cabbage varieties, were flowering starts often at the expense of the vegetative structures that are harvested.
  • Downregulation of the FPF genes in those plants by an antisense approach can be used to prevent or retard flowering.
  • Downregulation of a gene by an antisense approach is achieved by the constitutive expression of a gene in a reverse orientation (Ecker and Davis, 1986) . Although such etnod is well known to those skilled in the art it is surprising that it can be achieved by using the FPFl genes in reverse orientation, were the expression is controlled by promoters also mentioned for the constitutive expression of the FPF genes.
  • SEQU ID NO 1 shows the DNA sequence of SaFPFl
  • SEQU ID NO 2 shows the DNA sequence of SaFPF2
  • SEQU ID NO 3 shows the DNA sequence of AtFPFl
  • SEQU ID NO 4 shows the DNA sequence of AtFPF2
  • SEQU ID NO 5 shows the deduced protein sequence of the 110 amino acids of SaFPFl
  • SEQU ID NO 6 shows the deduced protein sequence of the 109 amino acids of SaFPF2
  • SEQU ID NO 7 shows the deduced protein sequence of the 110 amino acids of AtFPFl
  • SEQU ID NO 8 shows the deduced protein sequence of the 112 amino acids of AtFPF2
  • Fig. 1 shows the vector pSH9 with the CaMV 35S promoter and terminator and a multiple cloning site MCS.
  • Fig. 2 shows the recombinant vector pSH9-SaFPFl with the coding region of SaFPFl ligated in the Ncol and BamHI site of pSH9.
  • Fig. 3 shows the pBIN19 plant transformation vector with the multiple cloning site MCS inserted in the lac Z gene of the T-DNA.
  • the T-DNA contains the selectable kanamycin marker under control of the nos promoter.
  • Fig. 4 shows the recombinant vector pBIN19-SaFPFl which contains the CaMV 35S promoter, the coding region of the cDNA FPFl and the terminator subcloned as a Hindlll fragment from the recombinant vector pSH9-SaFPFl.
  • Fig. 5 shows a photo of an Arabidopsis thaliana ecotype Columbia wildtype plant (left side) and a transgenic plant (right side) of the same age, transformed with pBIN19-SaFPFl and grown under short day conditions. The transformed plant has already build up an inflorescence with the first opened flowers, wheras the wildtype plant has remained in the vegetative rosette stage.
  • Example 1 Establishing subtracted phage cDNA libraries Changes in gene expression during flower formation were studied in the long-day plant Sinapis alba . The day length dependence was exploited to synchronize flower formation in a large population of mustard plants. From vegetative mustard plants grown for 56 days under short-day conditions and from plants grown for additional 5 days under an inductive photoperiod of 16 hours per day, apical buds were harvested.
  • Ten ⁇ g of mRNA was reverse transcribed into the first strand of cDNA in a 100 ⁇ l volume containing 50 mM Tris-HCl, pK 8.3, 50 mM KCl, 8 mM MgCl 2 / 500 ⁇ M deoxynucleotide triphosphates, 5 ⁇ g oligo-p(dT)i 2 - l ⁇ , 4 mM sodium pyropnospate, 10 mM DTT, 100 units RNasin, 10 ⁇ Ci of 3 P-dATP, and 100 units of reverse transcriptase (Boehringer) .
  • RNA template was removed by alkaline hydrolysis with 0.4 M NaOH for 2 hr at 56°C, and the cDNA was then desalted on a Sephadex G50 column with TES (10 mM Tris- HCl, pH 7.5, 1 mM EDTA and 0.1% SDS (Sodium Dodecyl Sulfate)) and precipitated with ethanol after addition of mRNA from young leaves.
  • TES Tris- HCl, pH 7.5, 1 mM EDTA and 0.1% SDS (Sodium Dodecyl Sulfate)
  • the single-stranded cDNA of both developmental stages was incubated with a 100 fold excess of mRNA from young leaves of noninduced control plants under hybridization conditions with 240 mM phosphate buffer pH7, 1 mM EDTA and 0.1% SDS at 68°C for 24 hours.
  • the remaining single stranded cDNA was separated from cDNA/mRNA hybrids by HPLC chromatography on a Bio-Gel high performance hydroxylapatite column (Bio-Rad) according to the instructions of the supplier with an increasing gradient of phosphate buffer.
  • the eluted single stranded cDNA was concentrated by several butanol extractions and was desalted on a Sephadex G50 column with TES and ethanol precipitated after addition of 10 ⁇ g of glycogen as a carrier.
  • the single stranded cDNA was tailed at the 3'end with a 50- fold molar excess of dCTP.
  • the reaction was performed in 20 ⁇ l tailing buffer (100 mM potassium cacodylate, pH 7.2, 2 mM CoCl 2 , and 0.2 mM DTT) with 12 units terminale transferase (Boehringer) at 30°C for 30 min.
  • the tailed cDNA was phenol and chloroform extracted and ethanol precipitated.
  • oligo-pd(G) ⁇ 2 - ⁇ 8 was annealed to the dC-tail in 100 mM Hepes-KOH, pH 6.9, 10 mM MgCl 2 , 70 mM KCl, 1 mM DTT, 250 ⁇ M each of all four nucleotides, and 10 ⁇ Ci of 32 P-dATP. After 30 min 10 units of Klenow polymerase (Boehringer) were added and the synthesis was carried out at 16°C. 2 units of T4 DNA-polymerase
  • ⁇ gtlO phage arms was ligated with 20 ng of cDNA in a 10 ⁇ l reaction with 1 unit of T4 ligase for 16 hr at 14°C.
  • the ligation mixture was packaged with Gigapack Gold extracts (Stratagene) into phage particles and plated with E. coli C600hfl.
  • cDNA clones were selected from the phage cDNA library that show stronger hybridization signals with the enriched radioactive probe from the induced stage compared to the enriched vegetative stage.
  • Up to 25,000 recombinant phage were plated on Nunc- plates.
  • Replica filters were prepared on nylon membranes from 8 plates and the differential screenings were performed with oligolabeled probes that were prepared from subtracted single stranded cDNAs from vegetative and induced developmental stages. With less than 50 ng of cDNA and 100 ⁇ Ci of 3 P-dATP as a label, a second strand with a total incorporation of 1 x 10 8 cpm was synthesized. Free nucleotides were separated from the cDNA on a Sephadex G50 column.
  • the filters were prehybridized for 6 hr in 6xSSC, 5xDenhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone and 0.1% BSA) , 0.1% SDS, and 100 ⁇ g/ml salmon sperm DNA at 65°C. the hybridization was performed in the same solution with lxlO 7 cpm/ml labeled cDNA for 36 hr at 65°C. Filters were washed twice in 2xSSC and 0.5xSSC with 0.1% SDS at 62°C and exposed to Kodak XAR films for 2 days to 4 days. Positive plaques were purified through a second screening at lower plaque density. Inserts of the purified phage were subcloned into Bluescribe plasmids (Stratagene) by standard methods.
  • phage From the enriched cDNA library of 5-day-induced plants 32 phage were selected as putative flowering specific cDNA clones. The identity of the putative early flowering-specific cDNAs was tested by mRNA dot blot hybridization. Among the recombinant phage selected only three represented transcripts that were specifically expressed in the apex but not in the leaf. And only one cDNA was identified that shows a dramatic upregulation of the expression of the gene SFD5. 04 (now SaFPFl) after the induction of flowering.
  • the nitrocellulose filters were washed first in lxSSC with 0.1% SDS at 68°C for 10 min and then two times in 0.1% SSC and 0.1% SDS at 68°C for 10 min.
  • This northern analysis revealed that SaFPFl is expressed in the apex of mustard after the induction of flowering and is not expressed in vegetative apices and in leaves.
  • a cDNA library from apices of plants that were induced to flowering by 7 long days was prepared.
  • the mRNA was isolated and reverse transcribed in 40 ⁇ l of 50 mM of Tris-HCl, pH 8.3, 10 mM of MgCl 2 , 10 mM of DTT, 100 mM of NaCl, 0.625 mM of dNTPs, 10 ⁇ Ci of 32 P-dATP, 100 ⁇ g of oligo d(T) 12 -i8, and 100 units of reverse transcriptase at 43°C for 1 hr.
  • AtFPFl is expressed in the periheral zone of the apical meristem after the induction of flowering.
  • AtFPFl SEQU ID NO 3
  • ESTs Expressed Sequence Tags
  • the cDNAs are stored in the ABRC Stock Center at Ohio State University and the sequences are stored in common DNA databases like GenBank (USA) or EMBL (Heidelberg, Germany) (Newman et al. 1994).
  • GenBank USA
  • EMBL Heidelberg, Germany
  • a search through the GenBank database led to the identification of three ESTs that show 87.7% and 79.7% homology on the DNA-level to SaFPFl .
  • One of those sequences (EST 40B10T7) is identical to AtFPFl isolated in our laboratory as described above.
  • the other two cDNAs (95G12T7 and 98K16T7) are identical and were not detected in our library screening.
  • AtFPF2 SEQU ID NO 4
  • the SaFPFl gene was inserted into the expression vector pBIN19 and constitutive expression studies were performed.
  • the protein coding region- of SaFPFl was amplified via the Polymerase Chain Reaction (PCR) by using primers which introduced restriction sites at the ends of the amplification products.
  • the primer CRD (5'-T GCA GGA TCC ACC ATG GCA GGC GTG TGG GTG TTC-3') of the 5' end of the coding region contains a Ncol site at the ATG start codon and a BamHI site before the Ncol site.
  • the 3'end primer CRU (5'-A TGC GGA TCC TTA ATT GGA GTC TCG GAC-3') introduced a BamHI site after the stop codon.
  • the PCR products were analyzed for the correct size on an agarose gel, the BamHI sites were cleaved and the PCR products first ligated via the BamHI sites into the plasmid vector pBluescript II SK * . By sequencing it was confirmed that the PCR reaction has not changed the D ⁇ A sequence.
  • the amplified coding region was then cut out with Ncol and
  • the vector pSH9 contains a CaMV 35S promoter with an ⁇ -element as a transla ⁇ tional enhancer adjacent to the Ncol site and a translational terminator with a polyadenylation site adjacent to the BamHI site (Holtorf et al. (1995) . From the recombinant vector pSH9-SaFPFl (FIG.
  • Example 5 The same procedure as described in Example 5 was applied to the second cDNA isolated from mustard, SaFPF2, to create the recombinant transformation vector pBIN19-SaFPF2.
  • Example 8 Production of transgenic Arabidopsis thaliana ecotype C24 plants containing the plasmid pBIN19-SaFPFl
  • transgenic plants containing the SaFPFl coding sequence or the coding sequence of homologous genes from other plants under the control of an inducible, tissue specific or a constitutive promoter can be performed by using well known transformation techniques as described in the Plant Molecular Biology Manual (1988. Gelvin, S.B., Schilperoort, R.A. and Ver a, D.P.S. eds.)
  • the gene SaFPFl under the control of the CaMV promoter as present in the recombinant vector pBIN19-SaFPFl was introduced into the Agrobacteri um tumefaciens strain C58C1 by a standard transformation procedure (Hofgen and Willmitzer 1988) using cells competent for the uptake of plasmids through the cell membrane.
  • the obtained recombinant bacteria were used to transform Arabidopsis thaliana ecotype C24 by the root transformation method from Valvekens et al. (1988) .
  • Arabidopsis seeds are germinated on agar medium containing MS salts and vitamins for 7 days under long-day conditions (16 hrs light, 8 hrs darkness) .
  • Roots are excised and incubated for three days under the described conditions on an agar medium containing 0.5mg/l 2.4- Dichlorophenoxyacetic Acid and 0.05mg/l Kinetin for callus induction (CIM-medium) .
  • Roots are cut into 0.5 cm explants and transferred to liquid CIM-medium containing 10 6 - 10 8 cells/ml of an overnight culture of the recombinant agrobacteria. Roots are incubated for 2-3 minutes, blotted on a sterile filter paper and subsequently put on solidified CIM-medium.
  • Agrobacteria are cocultivated for 2 days to transform the roots before they are washed off using liquid CIM-medium. Root explants are blotted on sterile filter paper and transferred to agar medium containing 5mg/l 6- ( ⁇ , ⁇ -Dimethyl- allylamino)-purine and 0.15mg/l Indole-3-acetic Acid for shoot induction.
  • the medium furthermore includes Timenten
  • Kanamycin 50mg/l to select for tranformed tissues (SIM- edium) . After an incubation time of 3 weeks first green calli appear on the root explants which are now transferred to fresh medium of the same kind.
  • First transgenic shoots arise approximately 2 weeks later, are allowed to grow for another 1-2 weeks and are subsequently transferred to glass containers with solidified SIM-medium for further development.
  • Example 9 Production of transgenic Arab!d psis thaliana ecotype C24 plants containing the plasmid pBIN19-SaFPF2
  • transgenic plants containing the SaFPF2 coding sequence was performed in analogy to Example 8. Again five independent transgenic lines were analysed in more detail.
  • Example 10 Production of transgenic Arabidopsis thaliana ecotype Columbia plants containing the plasmid pBIN19-SaFPFl
  • Soil-grown Arabidopsis plants are induced to flowering in a green house under unsterile conditions. Transformation is by direct incubation of the whole inflorescence in a medium containing 5% sucrose, 0.44 ⁇ M 6-Benzylaminopurine and 10 6 - 10 8 cells/ml of an overnight agrobacteria-culture under vaccuum conditions for 20 minutes.
  • Plants are subsequently allowed to set seed in the greenhouse for another 3-4 weeks. Seeds are harvested, sterilized and plated on solidified medium containing MS-salts, vitamins and 500mg/l Timenten to suppress agrobacterial growth as well as 50mg/l Kanamycin for selection of transformants.
  • transformants After approximately 2 weeks transformants are clearly distin- 5 guishable from non transformed seedlings by healthy green leaves and extensive root growth. Transformants were trans ⁇ ferred to soil and seeds were harvested for further analysis.
  • Example 11 Production of the plasmids pBIN19-AtFPFl and pBIN19-AtFPF2 and transformation of the AraJbidqpsis thaliana 15 ecotype Columbia with said plasmids.
  • Example 10 The in planta-transformation procedure of Example 10 was also applied on the transformation of Arabidopsis thaliana ecotype Columbia with the coding region of the Arabidopsis cDNAs AtFPFl and AtFPF2. 0
  • the coding region of the cDNA was amplified via PCR as des ⁇ cribed in Example 6. Again the mustard primer CRD (5*-T GCA GGA TCC ACC ATG GCA GGC GTG TGG GTG TTC-3') of the 5' end of the coding region, that is homologous to the Arabidopsi s 5 sequence, was used. As a second primer the T7 primer with an additional BamHI site at the 5' end (5'-GCA GGA TCC ATA CGA CTC ACT ATA GGG-3) from the Bluescript plasmid vector was choosen.
  • Example 12 Determination of the flowering promoting effects of SaFPFl and SaFPF2
  • the first visible morphological change after the induction of flowering in Arabidopsis is the elongation of the central axis. A process which is called bolting and that results in the formation of an inflorescence.
  • Transgenic plants form less leaves and flower significantly earlier than wild type plants grown under identical conditions.
  • the shortening of the time to flowering is independent of the ecotype as well as of the photoperiodic conditions, and is obtained by using SaFPFl as well as SaFPF2 for the constitutive expression.
  • the results are compiled in Tables 1 and 2.
  • Example 5 The same procedure as described in Example 5 was applied to the ATFPF1 cDNA isolated from Arabidopsis, to create the recombinant vector pBIN19-AtFPFl.
  • Example 14 Production of transgenic Arabidopsis thaliana ecotype Columbia plants containing the plasmid pBINl9-AtFPFl.
  • Columbia plants were transformed with the in planta transformation as described in Example 10.
  • the homologous gene we see the same phenotype in transformed plants as described for the two mustard genes. Again the vegetative phase is shortened and therefore the time to flowering is shortened under long- and short-day conditions.
  • Table 1 Shortday Conditions (8hrs light/day)
  • Fig. 5 shows a photo of an Arabidopsis thaliana ecotype Columbia wild-type plant (left side) and transgenic plant (right side) grown under short day conditions.
  • a wild type, untransformed plant grown under short-day conditions (Fig. 5, left side) is still in a vegetative stage, where only a rosette of leaves is formed, whereas a plant of a transgenic line grown under the same conditions and of the same age has enlarged the length of the internodes, which results in the bolting process (Fig. 5, right side) , which is the first visible sign that the plant has started the flowering process.
  • the transgenic line has fewer rosette leaves and has started to to open the first flowers at the top.
  • CATCTTACTC CACGCTCGAG CAGATCCTCC GGAGTCTTGG ATGGGAGAGG TACTTCGGTG 240
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • ORGANISM Arabidopsis thaliana
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL YES
  • ORGANISM Arabidopsis thaliana

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Physiology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne l'assemblage de vecteurs chimères comprenant les gènes du facteur FDF (facteur de déclenchement de la floraison) de plants de moutarde et des gènes homologues d'autres plantes. Ce vecteur permet de diminuer, chez les plantes transformées, le délai avant la floraison.
PCT/EP1997/000161 1996-01-09 1997-01-02 Controle de la floraison de plantes WO1997025433A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP96810014 1996-01-09
EP96810014.9 1996-01-09

Publications (1)

Publication Number Publication Date
WO1997025433A1 true WO1997025433A1 (fr) 1997-07-17

Family

ID=8225530

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1997/000161 WO1997025433A1 (fr) 1996-01-09 1997-01-02 Controle de la floraison de plantes

Country Status (1)

Country Link
WO (1) WO1997025433A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047654A2 (fr) * 1998-03-19 1999-09-23 Eidgenössische Technische Hochschule Zürich Association de genes pour la regulation de l'induction de la floraison chez les plantes cultivees et les plantes d'ornement
WO1999051728A2 (fr) * 1998-04-07 1999-10-14 Cold Spring Harbor Laboratory Regulation de l'induction de la floraison chez les plantes et applications
WO2006067219A1 (fr) * 2004-12-22 2006-06-29 Vib Vzw Procede et moyen permettant d'augmenter les quantites de glucides dans des plantes
EP1659180A3 (fr) * 1999-10-12 2006-07-26 Mendel Biotechnology, Inc. Modification du temps de floraison
EP2410060A1 (fr) * 2000-08-22 2012-01-25 Mendel Biotechnology, Inc. Gènes servant à modifier des caractéristiques de plantes IV
CN103183732A (zh) * 2013-04-18 2013-07-03 中国农业科学院棉花研究所 一种棉花Gh FPF1蛋白及其编码基因和应用

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04258292A (ja) * 1991-02-14 1992-09-14 Japan Tobacco Inc タバコの花芽形成時に発現する遺伝子
WO1993012239A1 (fr) * 1991-12-18 1993-06-24 Zeneca Limited Modification des caracteristiques des plantes et de leur croissance cellulaire
WO1993016187A1 (fr) * 1992-02-14 1993-08-19 Verneuil Recherche Plante portant des genes codant pour des enzymes de la voie de biosynthese des phytosterols, et procede d'obtention
WO1993018154A2 (fr) * 1992-03-05 1993-09-16 Institut National De La Recherche Agronomique Procede pour accroitre la precocite d'une plante et/ou abaisser la teneur en nitrates stockes dans la plante
WO1994010831A1 (fr) * 1992-11-06 1994-05-26 Washington University Induction du nanisme et d'une floraison precoce a l'aide des proteines abondantes d'embryogenese tardive (aet) du groupe 3
WO1995024487A1 (fr) * 1994-03-09 1995-09-14 Hoechst Schering Agrevo Gmbh Procedes d'inhibition et de declenchement de la floraison des plantes
WO1996011566A1 (fr) * 1994-10-14 1996-04-25 Washington State University Research Foundation Regulation genique du developpement floral et de la dominance apicale dans des plantes
WO1996013595A1 (fr) * 1994-10-31 1996-05-09 Hoechst Schering Agrevo Gmbh Procede permettant de modifier la floraison de plantes
WO1996014414A1 (fr) * 1994-11-02 1996-05-17 John Innes Centre Innovations Limited Regulation genetique du fleurissement
WO1996019105A1 (fr) * 1994-12-21 1996-06-27 The Salk Institute For Biological Studies Plantes modifiees genetiquement avec un developpement floral module
WO1996034088A2 (fr) * 1995-03-16 1996-10-31 Cold Spring Harbor Laboratory Regulation de l'induction de la floraison chez les plantes et ses utilisations
WO1996038560A2 (fr) * 1995-06-02 1996-12-05 John Innes Centre Innovations Limited Regulation genetique de la floraison

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04258292A (ja) * 1991-02-14 1992-09-14 Japan Tobacco Inc タバコの花芽形成時に発現する遺伝子
WO1993012239A1 (fr) * 1991-12-18 1993-06-24 Zeneca Limited Modification des caracteristiques des plantes et de leur croissance cellulaire
WO1993016187A1 (fr) * 1992-02-14 1993-08-19 Verneuil Recherche Plante portant des genes codant pour des enzymes de la voie de biosynthese des phytosterols, et procede d'obtention
WO1993018154A2 (fr) * 1992-03-05 1993-09-16 Institut National De La Recherche Agronomique Procede pour accroitre la precocite d'une plante et/ou abaisser la teneur en nitrates stockes dans la plante
WO1994010831A1 (fr) * 1992-11-06 1994-05-26 Washington University Induction du nanisme et d'une floraison precoce a l'aide des proteines abondantes d'embryogenese tardive (aet) du groupe 3
WO1995024487A1 (fr) * 1994-03-09 1995-09-14 Hoechst Schering Agrevo Gmbh Procedes d'inhibition et de declenchement de la floraison des plantes
WO1996011566A1 (fr) * 1994-10-14 1996-04-25 Washington State University Research Foundation Regulation genique du developpement floral et de la dominance apicale dans des plantes
WO1996013595A1 (fr) * 1994-10-31 1996-05-09 Hoechst Schering Agrevo Gmbh Procede permettant de modifier la floraison de plantes
WO1996014414A1 (fr) * 1994-11-02 1996-05-17 John Innes Centre Innovations Limited Regulation genetique du fleurissement
WO1996019105A1 (fr) * 1994-12-21 1996-06-27 The Salk Institute For Biological Studies Plantes modifiees genetiquement avec un developpement floral module
WO1996034088A2 (fr) * 1995-03-16 1996-10-31 Cold Spring Harbor Laboratory Regulation de l'induction de la floraison chez les plantes et ses utilisations
WO1996038560A2 (fr) * 1995-06-02 1996-12-05 John Innes Centre Innovations Limited Regulation genetique de la floraison

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
CHUNG, Y.-Y., ET AL.: "Early flowering and reduced apical dominance result from ectopic expression of a rice MADS box gene", PLANT MOLECULAR BIOLOGY, vol. 26, October 1994 (1994-10-01), pages 657 - 665, XP002004927 *
COUPLAND, G.: "Leafy blooms in aspen", NATURE, vol. 377, 12 October 1995 (1995-10-12), pages 482 - 483, XP002004925 *
DATABASE WPI Section Ch Week 9243, Derwent World Patents Index; Class C06, AN 92-354683, XP002004923 *
MANDEL, M.A, ET AL.: "A gene triggering flower formation in Arabidopsis", NATURE, vol. 377, 12 October 1995 (1995-10-12), pages 522 - 524, XP002030346 *
MELZER S., ET AL: "EARLY CHANGES IN GENE EXPRESSION DURING THE TRANSITION FROM VEGETATIVE TO GENERATIVE GROWTH IN THE LONG-DAY PLANT SINAPIS-ALBA.", PLANT CELL 2 (10). 1990. 953-962., XP002004922 *
MENZEL, G., ET AL.: "Isolation and analysis of SaMADS C, the APATELA 1 cDNA homology from mustard", PLANT PHYSIOLOGY, vol. 108, 1995, pages 853 - 854, XP002030345 *
NEWMAN T., ET AL.: "3313 Arabidopsis thaliana cDNA clone 95G12T7.", EMBL SEQEUNCE DATABASE ACCESSION NO. T21305;27-JUN-1994 (REL. 40, CREATED),, XP002004919 *
NEWMAN T., ET AL.: "4197 Arabidopsis thaliana cDNA clone 98K16T7.", EMBL SEQUNCE DATABASE ACESSION NO. T22189; 27-JUN-1994 (REL. 40, CREATED), XP002004920 *
NEWMAN T., ET AL.: "449 Arabidopsis thaliana cDNA clone 40B10T7.", EMBL SEQUENCE DATABASE ACCESSION NO. T04402; 30-AUG-1993 (REL. 36, CREATED), XP002004921 *
PUTTERILL, J., ET AL.: "The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors", CELL, vol. 80, 24 March 1995 (1995-03-24), pages 847 - 857, XP002004926 *
WEIGEL, D., ET AL.: "A developmental switch sufficient for flower initiation in diverse plants", NATURE, vol. 377, 12 October 1995 (1995-10-12), pages 495 - 500, XP002004924 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6177614B1 (en) 1995-03-16 2001-01-23 Cold Spring Harbor Laboratory Control of floral induction in plants and uses therefor
WO1999047654A2 (fr) * 1998-03-19 1999-09-23 Eidgenössische Technische Hochschule Zürich Association de genes pour la regulation de l'induction de la floraison chez les plantes cultivees et les plantes d'ornement
WO1999047654A3 (fr) * 1998-03-19 1999-10-21 Eidgenoess Tech Hochschule Association de genes pour la regulation de l'induction de la floraison chez les plantes cultivees et les plantes d'ornement
WO1999051728A2 (fr) * 1998-04-07 1999-10-14 Cold Spring Harbor Laboratory Regulation de l'induction de la floraison chez les plantes et applications
WO1999051728A3 (fr) * 1998-04-07 1999-11-18 Cold Spring Harbor Lab Regulation de l'induction de la floraison chez les plantes et applications
EP1659180A3 (fr) * 1999-10-12 2006-07-26 Mendel Biotechnology, Inc. Modification du temps de floraison
EP2410060A1 (fr) * 2000-08-22 2012-01-25 Mendel Biotechnology, Inc. Gènes servant à modifier des caractéristiques de plantes IV
WO2006067219A1 (fr) * 2004-12-22 2006-06-29 Vib Vzw Procede et moyen permettant d'augmenter les quantites de glucides dans des plantes
CN103183732A (zh) * 2013-04-18 2013-07-03 中国农业科学院棉花研究所 一种棉花Gh FPF1蛋白及其编码基因和应用
CN103183732B (zh) * 2013-04-18 2014-08-20 中国农业科学院棉花研究所 一种棉花Gh FPF1蛋白及其编码基因和应用

Similar Documents

Publication Publication Date Title
AU705130B2 (en) Genetic control of flowering
EP0832234B1 (fr) Regulation genetique de la floraison
Prigge et al. The Arabidopsis SERRATE gene encodes a zinc-finger protein required for normal shoot development
EP0935658B1 (fr) Modulation genique d'une floraison
Tadege et al. Control of flowering time by FLC orthologues in Brassica napus
CA2230511C (fr) Genes de floraison
US6670526B1 (en) Arabidopsis ESD4 gene and method to control flowering time
AU708766B2 (en) Gene controlling floral development and apical dominance in plants
US20110010792A1 (en) Plant polynucleotides for improved yield and quality
JP2002535993A (ja) 組換え体細胞における代謝物の生合成を変調する方法
MX2010013722A (es) Composiciones y metodos de uso de proteina quinasa quinasa quinasa activada por mitogeno.
US7057087B2 (en) Application of aspen MADS-box genes to alter reproduction and development in trees
WO1997025433A1 (fr) Controle de la floraison de plantes
CA2258571A1 (fr) Reductases de sterol de plantes et leurs utilisations
AU701718B2 (en) Processes for modifying plant flowering behaviour
CN1423520B (zh) 调节植物细胞分裂的组合物和方法
US7560612B2 (en) Early-inflorescence-preferred regulatory elements and uses thereof
WO1999004003A1 (fr) Procede d'obtention de plantes transgeniques dont la floraison est inhibee, et sequences d'adn utilisees dans ledit procede
US5861542A (en) Gene controlling floral development and apical dominance in plants
US7220845B2 (en) Nucleic acid molecules associated with plant cell proliferation and growth and uses thereof
ZA200601498B (en) Plant cell cycle genes and methods of use
US20030018995A1 (en) Plants with a modified flower and seed development
KR100443488B1 (ko) 에틸렌 합성을 조절하는 신규한 유전자
Bradley Flowering genes
NZ502374A (en) Production of transgenic plants in which flowering is inhibited by using the FBP10 gene

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97524873

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase