WO2015150412A1 - Plantes transgéniques présentant un nombre accru de fruits et de graines et son procédé d'obtention - Google Patents

Plantes transgéniques présentant un nombre accru de fruits et de graines et son procédé d'obtention Download PDF

Info

Publication number
WO2015150412A1
WO2015150412A1 PCT/EP2015/057086 EP2015057086W WO2015150412A1 WO 2015150412 A1 WO2015150412 A1 WO 2015150412A1 EP 2015057086 W EP2015057086 W EP 2015057086W WO 2015150412 A1 WO2015150412 A1 WO 2015150412A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
nucleic acid
seq
acid construct
ful
Prior art date
Application number
PCT/EP2015/057086
Other languages
English (en)
Inventor
Irene MARTINEZ FERNANDEZ
Chloe Fourquin
Antonio SERRANO MISLATA
Vicente BALANZA PEREZ
Ana BERBEL TORNERO
M Cristina FERRÁNDIZ MAESTRE
Francisco MADUEÑO ALBI
Original Assignee
Consejo Superior De Investigaciones Cientificas
Universitat Politècnica De València
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 Consejo Superior De Investigaciones Cientificas, Universitat Politècnica De València filed Critical Consejo Superior De Investigaciones Cientificas
Publication of WO2015150412A1 publication Critical patent/WO2015150412A1/fr

Links

Classifications

    • 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
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • 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]
    • 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

  • the invention relates to transgenic plants with improved yield, related methods, nucleic acid constructs and uses thereof.
  • Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, for example, the number and size of the organs, plant architecture (for example, the number of branches), seed production, leaf senescence, abiotic and biotic stress. Root development, nutrient uptake, stress tolerance and early vigour may also be important factors in determining ultimate yield. Optimizing any of the abovementioned factors may therefore contribute to increasing crop yield and addressing efficiency of agriculture and providing food security.
  • Seed and fruit production in particular are global multi-billion dollar commercial industries.
  • Commercially valuable seeds include, for example, rapeseeds, cotton seeds and sunflower seeds, which are prized for the vegetable oil that can be pressed from the seed.
  • the seeds of leguminous plants such as peas, beans, soybeans and lentils and of many fruits, including, for example, corn, rice, wheat, barley and other cereals, nuts, legumes, tomatoes, and citrus fruits are also commercially valuable.
  • Plants like many other organisms, exhibit various life history patterns and possess a broad spectrum of longevity, ranging from a few weeks to several thousand years. Annuals (e.g. Arabidopsis [Arabidopsis thaliana]), biennials (e.g. wheat [Triticum aestivum]), and some perennials possess a monocarpic lifestyle, which is characterized by only a single reproductive event in the life cycle. After flowering (and setting seeds or fruits) the whole plant will senesce and die.
  • Annuals e.g. Arabidopsis [Arabidopsis thaliana]
  • biennials e.g. wheat [Triticum aestivum]
  • some perennials possess a monocarpic lifestyle, which is characterized by only a single reproductive event in the life cycle. After flowering (and setting seeds or fruits) the whole plant will senesce and die.
  • the monocarpic senescence includes three coordinated processes: (1 ) senescence of somatic organs and tissues such as leaves; (2) arrest of shoot apical meristems (SAM); and (3) suppression of axillary buds to prevent the formation of new shoots/branches. Plants have the capacity to develop new organs postembryonically. This potential to develop new organs is attributed to sets of cells, called meristems, which are found at the growing tips of the plants. Two meristematic cell populations are generated during embryogenesis. The SAM generates all of the aerial parts of the plant, whereas the root apical meristem generates the underground parts.
  • the SAM produces lateral organs from the cells on its flanks while simultaneously maintaining a central pool of pluripotent stem cells for future organogenesis.
  • maintenance of a functional SAM requires coordination between loss of cells from the meristem by differentiation and their replenishment by stem cell division.
  • SAM Different types of lateral organs are generated by the SAM during successive phases of development.
  • the SAM produces leaves and axillary meristems during the vegetative phase and floral meristems during the reproductive phase.
  • Floral meristems on the other hand produce flowers that usually consist of four whorls of organs. After producing these whorls, the activity of the floral meristem ceases, unlike the SAM, which continuously proliferates and produces organ primordia from its flanks.
  • SAM and floral meristems are therefore two different meristems.
  • both the SAM and the floral meristem are similar, because both contain a stem cell reservoir at the apex that contributes cells to organogenesis on the flanks.
  • both types of meristems share a number of regulatory genes and mechanisms for development and maintenance.
  • shoot and floral meristems also differ in several ways. One difference between them is the type and arrangement of the lateral organs that they produce.
  • the SAM generally forms leaves and their associated meristems in different phyllotactic patterns whereas floral meristems generate floral organs, such as sepals, petals, stamens, and carpels in concentric rings called whorls. Another critical difference is that the SAM is indeterminate and grows indefinitely, whereas the floral meristem is determinate and terminates once all the floral organs are made.
  • the stem cell reservoir in floral meristems is transient, and floral meristems must overcome the mechanisms that ensure stem cell maintenance at the correct stage of development to allow carpel formation in the center of the flower.
  • MADS box genes are involved in the control of reproductive development in plants.
  • MADS box genes encode DNA binding proteins that have a conserved N-terminal domain that shares similarity with transcription factors from yeast and mammals.
  • AP2 APETALA2
  • AGAMOUS ⁇ AG AGAMOUS ⁇ AG
  • AP2 APETALA2
  • AGAMOUS ⁇ AG AGAMOUS ⁇ AG
  • the two genes act antagonistically to restrict each other to their proper domains of action within the floral meristem.
  • miR172 a microRNA, serves as a negative regulator of AP2.
  • miR172 is initially present throughout the floral meristem but is concentrated in the inner two whorls after floral stage 7.
  • miR172-mediated repression of AP2 is crucial for maintaining floral meristem size and for the timely termination of floral stem cells. Elevated levels of mutant AP2 with disrupted miR172 base paring resulted in elevated levels of AP2 protein.
  • Plants expressing a miR172-resistant version of AP2 cDNA showed flowers with enlarged meristems surrounded by many whorls of stamens and petals. The flowers were infertile (Chen, Science; 2004, 303: 2022-2025; Zhao et al, The Plant Journal; 2007, 51 : 840-849).
  • FRUITFULL ⁇ FUL also known as AGL-8
  • AGL-8 is a MADS box gene and functions in controlling flowering time (Mandel MA, Yanofsky MF. Plant Cell. 1995 Nov;7(1 1 ):1763-71 ).
  • US 6,541 ,683 describes ectopic expression of AGL-8 to increase seed size and suppression of AGL-8 to achieve smaller seeds size
  • the inventors have surprisingly shown that when expression of miR172- resistant version of AP2 is specifically directed to the SAM, this leads to increased number of fruits and seeds and therefore increased yield. At the same time, the detrimental phenotypes that were observed when miR172- resistant version of AP2 cDNA were overexpressed in the plant were absent in the plant when expression is specifically directed to the SAM. The inventors have surprisingly shown that when targeted silencing of FUL RNA is specifically directed to the SAM, this leads to increased number of fruits and seeds and therefore increased yield. At the same time, the detrimental phenotypes that were observed in FUL knockout mutants were absent in the plant when expression of the silencing RNA is specifically directed to the SAM (Gu Q, et al. Development. 1998, 125: 1509-1517).
  • the invention is aimed at providing alternative transgenic plants and methods for increasing yield to provide benefits to agriculture.
  • the inventors have found that manipulating the expression of certain floral genes in a plant leads to increased number of fruits and seeds and therefore increased yield.
  • the invention relates to a transgenic plant comprising a nucleic acid construct said construct comprising a modified AP2 nucleic acid operably linked to a promoter that directs expression of the nucleic acid to the shoot apical meristem (SAM) wherein said modified AP2 nucleic acid encodes a modified polypeptide comprising a mutation in the miRNA172 binding site.
  • SAM shoot apical meristem
  • said modified AP2 nucleic acid encodes a modified polypeptide comprising a mutation in the miRNA172 binding site with reference to SEQ ID NO: 3, a functional variant, homologue or orthologue thereof.
  • said mutation is a deletion or substitution of one or more residues located from the positions 393 to 400 with reference to SEQ ID NO: 3, preferably, the one or more residues selected from the following AAASSGFS (SEQ ID NO: 12) with reference to SEQ ID NO: 3 or a deletion or substitution at a homologous position in a functional variant, homolog or ortholog of AP2 as defined in SEQ ID NO. 3.
  • said mutation is a substitution
  • said mutation is a substitution of G398 with reference to SEQ ID NO: 3 or a residue at a homologous position.
  • G398 with reference to SEQ ID NO: 3 or a residue at a homologous position is substituted with E (G398E).
  • said promoter is selected from TFL1 .
  • said plant is a crop plant or ornamental plant.
  • said plant is a dicot.
  • said plant is a crop plant is selected from maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • said functional variant, homolog or orthologue has at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO. 3.
  • said orthologue is AP2 from maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • the invention also relates to harvestable parts of a transgenic plant according to any of the preceding claims or a product derived therefrom.
  • the invention in another aspect, relates to a nucleic acid construct said construct comprising an AP2 nucleic acid comprising a mutation in the miRNA2 binding site operably linked to a promoter that directs expression of the nucleic acid to the shoot apical meristem (SAM).
  • SAM shoot apical meristem
  • said modified AP2 nucleic acid encodes a modified polypeptide comprising a mutation in the miRNA2 binding site with reference to SEQ ID NO: 3, a functional variant, homolog or ortholog thereof.
  • said mutation is a deletion or substitution of one or more residues located from the positions 393 to 400 with reference to SEQ ID NO: 3, preferably the one or more residues selected from the following AAASSGFS (SEQ ID NO: 12) with reference to SEQ ID NO: 3 or a deletion or substitution at a homologous position.
  • said mutation is a substitution. In one embodiment, said mutation is a substitution of G398 with reference to SEQ ID NO: 3 or a residue at a homologous position.
  • G398 with reference to SEQ ID NO: 3 or a residue at a homologous position is substituted with E (G398E).
  • said promoter is selected from TFL1 .
  • said functional variant, homolog or orthologue has at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 3.
  • said orthologue is a AP2 from maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • the invention also relates to a vector comprising a nucleic acid construct described above.
  • the invention also relates to a host cell transformed with a nucleic acid construct or a vector described above.
  • said host cell is a bacterial or a plant cell.
  • the invention also relates to a method for increasing yield of a plant said method comprising introducing and expressing a nucleic acid construct or a vector described above in a plant.
  • the invention also relates to a method for producing a plant with increased yield of a plant said method comprising introducing and expressing a nucleic acid construct or a vector as described above in a plant.
  • said plant is selected from maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • the invention further relates to a plant obtained or obtainable by a method described above.
  • the invention in another aspect, relates to a use of a nucleic acid construct or a vector as described above for increasing yield of a plant.
  • the invention also relates to a transgenic plant expressing a RNA molecule directed against a FUL nucleic acid wherein said RNA is operably linked to a promoter that directs expression in the SAM.
  • said RNA is RNAi, snRNA, dsRNA, siRNA, miRNA, ta- siRNA, amiRNA.
  • said FUL nucleic acid comprises SEQ ID NO: 5, a function variant, homologue or orthologue thereof.
  • said promoter is selected from TFL1 (SEQ ID NO: 4).
  • said plant is a crop plant or ornamental plant.
  • said plant is a dicot.
  • said plant is a crop plant is selected from maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • said functional variant, homolog or orthologue has at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 7.
  • said orthologue is FUL from maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • the invention related to harvestable parts of a transgenic plant described above or a product derived therefrom.
  • the invention also relates to a nucleic acid construct said construct comprising a siRNA directed against a FUL nucleic acid wherein said siRNA is operably linked to a promoter that directs expression in the SAM.
  • said FUL nucleic acid comprises SEQ ID NO: 5 or 6, a function variant, part, homologue or orthologue thereof.
  • said RNA is RNAi, snRNA, dsRNA, siRNA, miRNA, ta- siRNA, amiRNA.
  • said promoter is selected from TFL1 (SEQ ID NO: 4).
  • said functional variant, homolog or orthologue has at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 7.
  • said orthologue is FUL from maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • the invention further relates to a vector comprising a nucleic acid construct described above.
  • the invention also relates to a host cell transformed with a nucleic acid construct or a vector described above.
  • said host cell is a bacterial or a plant cell.
  • the invention also relates to a method for increasing yield of a plant said method comprising introducing and expressing a nucleic acid construct or a vector as described above in a plant.
  • the invention also relates to a method for producing a plant with increased yield of a plant said method comprising introducing and expressing a nucleic acid construct or a vector as described above in a plant.
  • said plant is selected from maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • the invention also relates to a plant obtained or obtainable by a method described herein.
  • the invention also relates to the use of a nucleic acid construct or a vector described above for increasing yield of a plant.
  • the invention also relates to the use of a nucleic acid construct comprising a siRNA directed against FUL or a vector comprising a siRNA directed against FUL wherein said siRNA for increasing yield of a plant wherein said plant is a legume and said FUL is a legume FUL.
  • Fig. 1 Schematic illustration of the AtAP2 protein. Checked boxes indicate the conserved AP2 DNA-binding domains. In grey, the position corresponding to the miR172 binding site in the mRNA is shown. White boxes indicate the three motifs characteristic of AP2-like genes.
  • Fig. 3 Number of fruits in the main inflorescence for transgenic plants expressing TFL1 ::AtAP2 170 and TFL1 ::RNAi-AtFUL respectively. Each bar represents the number of fruits in the main inflorescence of homozygous plants for independent transgenic insertions.
  • Fig. 4 Number of fruits in total inflorescence for transgenic plants expressing TFL1 ::AtAP2 170
  • Number of fruits produced by whole plants expressing TFL1 ::AtAP2 170 include main inflorescence, coflorescences and secondary inflorescences axilary to rosette leaves). Dark grey represents number of fruits in main inflorescence, light grey indicates fruits produced by the remaining inflorescences. Quantification was performed in Columbia wildtype and two independent transgenic lines (#1 and #3) previously identified for increased fruit production in the main inflorescence.
  • Seed number is an estimation calculated by quantification of average seed number per fruit (n>25) multiplied by average number of fruit in the main inflorescence.
  • FIG. 7 Schematic illustration of the AtFUL protein.
  • FIG. 8 FUL in pea Top panel, from left to right: Wildtype plant (cultivar Cameor), psfula mutant, psfulb mutant and psfula psfulb mutant after GPA has taken place. Bottom panels: quantification of total fruit number and total seed number produced per plant from the indicated genotypes.
  • nucleic acid As used herein, the words “nucleic acid”, “nucleic acid sequence”, “nucleotide”, “nucleic acid molecule” or “polynucleotide” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), naturally occurring, mutated, synthetic DNA or RNA molecules, and analogues of the DNA or RNA generated using nucleotide analogues. It can be single- stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products.
  • genes also encompass a gene.
  • the term “gene” or “gene sequence” is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences. In one embodiment, cDNA is preferred. Thus, in all of the aspects described herein that use nucleic acids, cDNA can be used unless otherwise specified.
  • transgenic means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either
  • genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or
  • the natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library.
  • the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part.
  • the environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp.
  • a naturally occurring expression cassette for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above - becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in US 5,565,350 or WO 00/15815 both incorporated by reference.
  • transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids used in the method of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously.
  • the plant expresses a transgene.
  • transgenic also means that, while the nucleic acids according to the different embodiments of the invention are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified, for example by mutagenesis or by modifying its methylation pattern.
  • Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place.
  • the transgene is stably integrated into the plant and the plant is preferably homozygous for the transgene.
  • the transgenic plant of the invention is non-naturally occurring.
  • non-naturally occurring when used in reference to a plant, means a seed plant that has been genetically modified by man.
  • a transgenic plant of the invention for example, is a non-naturally occurring plant that contains an exogenous nucleic acid molecule, such as a nucleic acid molecule encoding a modified AP2 or FUL gene product and, therefore, has been genetically modified by man.
  • the aspects of the invention involve recombination DNA technology and in preferred embodiments exclude embodiments that are solely based on generating plants by traditional breeding methods.
  • the inventors have surprisingly demonstrated that when expression of miR172- resistant version of AP2 is specifically directed to the SAM, this leads to increased number of fruits and seeds and therefore increased yield. At the same time, the detrimental phenotypes that were observed when a miR172- resistant version of AP2 cDNA was overexpressed in the plant were absent in the plant when expression is specifically directed to the SAM.
  • the invention relates to a transgenic plant comprising a nucleic acid construct said construct comprising a modified AP2 nucleic acid operably linked to a promoter that directs expression of the nucleic acid to the SAM wherein said modified AP2 nucleic acid encodes a modified AP2 polypeptide comprising a mutation in the miRNA172 binding site.
  • Said mutation confers at least partial miR172-resistance to AP2 and allows accumulation of the AP2 polypeptide in the SAM.
  • an exogenous modified AP2 nucleic acid is expressed in said plant.
  • Yield-related traits are traits or features which are related to plant yield, including yield- related traits. Yield-related traits may be quantitative or qualitative. For example, yield-related traits can comprise one or more of the following non-limitative list of features: biomass, fruit yield, size of fruits, seed yield, size of seeds, seed viability and germination efficiency, seed/fruit/grain size, starch content of grain, early vigour, greenness index, increased growth rate, delayed senescence of green tissue.
  • yield in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time.
  • yield of a plant may relate to vegetative biomass (root and/or shoot biomass), to reproductive organs, and/or to propagules (such as seeds) of that plant.
  • increased yield comprises one or more of and can be measured by assessing one or more of: increased seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, increased viability/germination efficiency, increased number or size of fruits/seeds/capsules/pods, increased growth or increased branching, increased biomass or grain fill.
  • increased yield comprises an increased number of grain/seed/capsules/pods, increased biomass, increased growth, increased number of floral organs and/or floral increased branching.
  • Yield is increased relative to a control plant.
  • a control plant is a plant that has not been transformed with a construct of the invention.
  • the control plant is a wild type plant.
  • yield for example are increased by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, for example at least 15% or 20%, or 25%, 30%, 35%, 40% or 50% in comparison to a control plant.
  • yield traits for example are increased by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.
  • said modified AP2 nucleic acid encodes a modified polypeptide comprising a mutation in the miRNA172 binding site with reference to protein SEQ ID NO: 3, a functional variant, homolog or ortholog thereof or an >AP2-like gene product.
  • Functionally characterized miR172 members and their targets in Arabidopsis, maize, rice, and barley are functionally characterized in Zhu & Heliwell. J. Exp. Bot. 201 1 , Vol. 62: 487-95.
  • a functional variant also comprises a variant of the gene of interest encoding a peptide which has sequence alterations that do not affect function of the resulting protein, for example in non-conserved residues.
  • Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, to the wild type sequences as shown herein and is biologically active.
  • An AP2- ⁇ ke gene product can have at least about 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to that particular non- variant nucleotide sequence or polypeptide as determined by sequence alignment programs described elsewhere herein.
  • An AP2-like gene product can also have, for example, substantially the amino acid sequence of a AtAP2 or an AtAP2 orthologue. The AP2-like gene product retains the biological function of the full non-variant sequence.
  • nucleic acid or peptide as described herein, but also functional variants or homologues thereof that do not affect the biological activity and function of the resulting protein.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also produce a functionally equivalent product.
  • Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.
  • variants of a particular nucleotide sequence or polypeptide of the invention will have at least about 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to that particular non-variant nucleotide sequence or polypeptide as determined by sequence alignment programs described elsewhere herein.
  • variants of a particular nucleotide sequence or polypeptide retain conserved domains.
  • fragments or parts
  • fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein.
  • homologue as used herein also designates an AP2/AP2 orthologue from other plant species.
  • a homologue of an AtAP2 polypeptide belongs to the euAP2 clade (Kim el al,. Molecular Biology and Evolution. 2006, vol. 23 (1 ): 107-200 and Tang et al., J Genet Genomics. 2007, vol. 34 (10): 930-8) and has the characteristic miRNA172 binding site.
  • an AP2INP2 orthologue has, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall
  • overall sequence identity is more than 70% or more than 73%.
  • overall sequence identity is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.
  • the homologue of a AtAP2 nucleic acid sequence has, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
  • overall sequence identity is more than 70% or more than 73%.
  • overall sequence identity is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.
  • the overall sequence identity is determined using a global alignment algorithm known in the art, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys).
  • Suitable homologues can be identified by sequence comparisons and identifications of conserved domains. There are predictors in the art that can be used to identify such sequences.
  • the function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function when expressed in a plant.
  • homologues could be selected from any of the following list: Brassica napus AP2 (ADU04499), Brassica rapa AP2 (AGD801 12), Zea mays SID1 (NP_001 139539), Zea mays IDS1 (NP_001 104904), Zea mays GLOSSY15 (NP_001 105890), Hordeum vulgare IDS1-like (AAL50205), Solanum lycopersicum AP2-like (NP_001234452), Phaseolus vulgaris (ESW27899), Cacao AP2 (EOX91494), Medicago truncatula AP2 (XP_00361 1692), Glycine max AP2-like (XP_003517312), Arabidopsis thaliana TOE1 (NP_001 189625), Arabidopsis thaliana TOE2 (gi/75264273), Arabidopsis thaliana SMZ (gi/75223382), Glycine
  • Variants of such homologues that have 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the polypeptide are also included.
  • SEQ ID NO: 1 and 2 are the cDNA and wild type genomic nucleic acid sequences encoding SEQ ID NO. 3.
  • the miRNA172 binding site is located at residues 393-400 of the protein sequence SEQ ID NO: 3, being the residues AAASSGFS (SEQ ID NO: 12), corresponding to nucleotides 1 177- 1200 in the cDNA sequence SEQ ID NO: 1 .
  • nucleotide sequences of the invention and described herein can be used to isolate corresponding sequences from other organisms, particularly other plants, for example crop plants.
  • methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences described herein.
  • Sequences may be isolated based on their sequence identity to the entire sequence or to fragments thereof.
  • hybridization techniques all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen plant.
  • the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labelled with a detectable group, or any other detectable marker.
  • probes for hybridization can be made by labelling synthetic oligonucleotides based on the ABA-associated sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Library Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
  • Hybridization of such sequences may be carried out under stringent conditions.
  • stringent conditions or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background).
  • Stringent conditions are sequence dependent and will be different in different circumstances.
  • target sequences that are 100% complementary to the probe can be identified (homologous probing).
  • stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
  • a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
  • stringent conditions will be those in which the salt concentration is less than about 1 .5 M Na ion, typically about 0.01 to 1 .0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Duration of hybridization is generally less than about 24 hours, usually about 4 to 12. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Preferred homologues of AtAP2 peptides are AP2 peptides from crop plants, for example cereal crops.
  • preferred homologues include AP2 in dicots.
  • preferred homologues include AP2 in maize, rice, wheat, sorghum, sugar cane, oilseed rape (canola), soybean, cotton, potato, tomato, tobacco, grape, barley, pea, bean, field bean or other legumes, lettuce, sunflower, cacao, lentils, alfalfa, sugar beet, broccoli or other vegetable brassicas or poplar.
  • Preferred homologues and their peptide sequences are selected from: Brassica napus AP2 (ADU04499), Brassica rapa AP2 (AGD801 12), Zea mays SID1 (NP_001 139539), Zea mays IDS1 (NP_001 104904), Zea mays GLOSSY15 (NP_001 105890), Hordeum vulgare IDS1-like (AAL50205), Solanum lycopersicum AP2-like (NP_001234452), Phaseolus vulgaris (ESW27899), Cacao AP2 (EOX91494), Medicago truncatula AP2 (XP_00361 1692), Glycine max AP2-like (XP_003517312), Arabidopsis thaliana TOE1 (NP_001 189625), Arabidopsis thaliana T0E2 (gi/75264273) and Arabidopsis thaliana SMZ (gi/75223382).
  • Glycine max (BI426798, BM892891 , BI893552 ,BU762655, BE659939), Medicago truncatula (BG447926, ABE87891 , CX541535), Persea americana (CV004845), Oryza sativa (05g03040, 03g60430), Saccharum officinarum (CA134360), Triticum aestivum (BE516333, BE430033, CAE53889, CJ706407), Helianthus tuberosus (EL444408 ,EL438914, EL469046), Citrus Clementina (DY276868, DY269701 ), Ipomoea nil (BAD36744), Populus trichocarpa (128415, 0019003502), Allium cepa (CF452606), Lactuca serriola (BQ996151 , BU000526), Solanum lycopersicum (
  • variants of AP2 orthologues for example orthologues listed above, which have overall sequence identity of at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% to these orthologues.
  • nucleic acid construct comprises or consists, as described above, an AP2 nucleic acid encoding an AP2 peptide which has a mutation in the miRNA binding site.
  • AP2 wild type sequence for example SEQ ID NO: 2, a homologue or orthologue thereof as described herein, comprises a mutation in the miRNA binding site.
  • the mutation may be a deletion or substitution of one or more of the following residues 393-400 (AAASSGFS) (SEQ ID NO: 12) of SEQ ID NO: 3, corresponding to nucleotides 1 177-1200 in SEQ ID NO: 1 .
  • the mutation is a substitution. In one embodiment, the mutation is a substitution of G398 with reference to SEQ ID NO: 3. In one embodiment, G398 is substituted with reference to SEQ ID NO: 3 substituted with E. Any other substitution is also within the scope of the invention. Introduction of a stop codon should be avoided.
  • the AP2 peptide (SEQ ID NO: 3) which has a mutation in the miRNA binding site, preferably a substitution of G398E with reference to SEQ ID NO: 3, is the SEQ ID NO: 13.
  • analogous amino acid substitutions listed above with reference to SEQ ID NO: 3 can be made in an AP2 homologue or orthologue from another plants by aligning the AP2 polypeptide sequence to be mutated with the AtAP2 polypeptide sequence as set forth in SEQ ID NO: 3 and identifying the miRNA172 binding site.
  • the mutation is at one or more of the residues listed above with reference to the position in SEQ ID NO:3, but in an AP2 homologue or orthologue the mutation will be at a homologous or equivalent position in the miRNA binding site.
  • the AP2 nucleic acid is operably linked to a promoter or regulatory sequence which directs expression of the nucleic acid to the SAM.
  • Said promoter is selected from TFL1 (SEQ ID NO: 4).
  • Other SAM-specific promoters can be selected from pATML.1 , which is active in the L1 layer of the SAM; pWUSCHEL (pWUS) which is active in the central part of the SAM, or ARR7.
  • operably or operatively linked means that the regulatory element confers regulated expression upon the operatively linked nucleic acid molecule.
  • operatively linked as used in reference to an exogenous regulatory element such as a constitutive regulatory element and a nucleic acid molecule encoding an AP2 related gene product, means that the constitutive regulatory element is linked to the nucleic acid molecule encoding an AP2 gene product such that the expression pattern of the constitutive regulatory element is conferred upon the nucleic acid molecule encoding the AP2 gene product. It is recognized that a regulatory element and a nucleic acid molecule that are operatively linked have, at a minimum, all elements essential for transcription, including, for example, a TATA box.
  • regulatory element may also include a terminator sequence.
  • the construct may comprise further regulatory elements, for example an inducible regulatory element, which is a regulatory element that confers conditional expression upon an operatively linked nucleic acid molecule, where expression of the operatively linked nucleic acid molecule is increased in the presence of a particular inducing agent or stimulus as compared to expression of the nucleic acid molecule in the absence of the inducing agent or stimulus.
  • an inducible regulatory element which is a regulatory element that confers conditional expression upon an operatively linked nucleic acid molecule, where expression of the operatively linked nucleic acid molecule is increased in the presence of a particular inducing agent or stimulus as compared to expression of the nucleic acid molecule in the absence of the inducing agent or stimulus.
  • the modified AP2 polynucleotide is a modified endogenous polynucleotide that is exogenously expressed in said plant.
  • said transgenic plant expressed a nucleic acid construct comprising a modified endogenous AP2 nucleic acid.
  • the plant may be oilseed rape expressing a nucleic acid construct comprising a modified endogenous oilseed rape AP2.
  • the modified AP2 polynucleotide is a modified exogenous polynucleotide that is exogenously expressed in said plant.
  • the plant may be oilseed rape expressing a nucleic acid construct comprising a modified AXAP2.
  • exogenous as used herein in reference to a nucleic acid molecule and a transgenic plant, means a nucleic acid molecule originating from outside the seed plant.
  • An exogenous nucleic acid molecule can have a naturally occurring or non-naturally occurring nucleotide sequence.
  • an exogenous nucleic acid molecule can be a heterologous nucleic acid molecule derived from a different plant species than the plant into which the nucleic acid molecule is introduced.
  • a plant according to the various aspects of the invention, including the transgenic plants described herein expressing a modified version of AP2 or which have suppressed expression of FUL in the SAM, methods and uses described herein may be a monocot or a dicot plant. In one embodiment, the plant is a dicot plant. In one embodiment, the plant is a seed plant.
  • a dicot plant may be selected from the families including, but not limited to Asteraceae, Brassicaceae (e.g. Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae.
  • the plant may be selected from lettuce, sunflower, Arabidopsis, broccoli, spinach, water melon, squash, cabbage, tomato, potato, yam, capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape vine, bell pepper, chilli or citrus species.
  • the plant is oilseed rape.
  • biofuel and bioenergy crops such as rape/canola, sugar cane, sweet sorghum, Panicum virgatum (switchgrass), linseed, lupin and willow, poplar, poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine.
  • high erucic acid oil seed rape, linseed and for amenity purposes (e.g. turf grasses for golf courses), ornamentals for public and private gardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the home (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber plant).
  • a monocot plant may, for example, be selected from the families Arecaceae, Amaryllidaceae or Poaceae.
  • the plant may be a cereal crop, such as wheat, rice, barley, maize, oat, sorghum, rye, millet, buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species, or a crop such as onion, leek, yam or banana.
  • the plant is a crop plant.
  • crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use.
  • the plant is a cereal or legume.
  • Most preferred plants are maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • plant as used herein in connection with the various aspects of the invention encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest.
  • plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
  • the invention in another aspect, relates to a nucleic acid construct said construct comprising an AP2 nucleic acid comprising a mutation in the miRNA2 binding site operably linked to a promoter that directs expression of the nucleic acid to the SAM.
  • the modified AP2 nucleic acid encodes a modified polypeptide comprising a mutation in the miRNA2 binding site with reference to SEQ ID NO: 3, a functional variant, homolog or orthologue thereof.
  • a functional variant, homolog or orthologue of AtAP2 is described above.
  • said mutation is a deletion or substitution of one or more residues selected from the following 393-400 (AAASSGFS; SEQ ID NO: 12) of SEQ ID NO: 3, corresponding to nucleotides 1 177-1200 in SEQ ID NO: 1 or a deletion or substitution at a homologous position.
  • said mutation is a substitution.
  • said mutation is a substitution of G398 with reference to SEQ ID NO: 3 or a residue at a homologous position.
  • G398 with reference to SEQ ID NO: 3 or a residue at a homologous position is substituted with E (G398E).
  • said promoter is selected from TFL1 (SEQ ID NO: 4).
  • the invention relates to a vector comprising a nucleic acid construct comprising a modified AP2 nucleic acid encodes a modified polypeptide comprising a mutation in the miRNA2 binding site with reference to SEQ ID NO. 3, a functional variant, homolog or ortholog thereof described above.
  • the invention relates to a host cell transformed with a nucleic acid construct or vector described above comprising a modified AP2 nucleic acid.
  • the host cell can be a bacterial, for example Agrobacterium, or a plant cell.
  • the invention relates to a culture medium or kit comprising a bacterial or a plant host cell as described above and a culture medium.
  • the invention relates to a plant transformed with a vector or nucleic acid construct comprising a modified AP2 nucleic acid which encodes a modified polypeptide comprising a mutation in the miRNA2 binding site with reference to SEQ ID NO: 2, a functional variant, homolog or ortholog thereof as described above.
  • the invention in another aspect, relates to a method for increasing yield, including yield-related traits, of a plant said method comprising introducing and expressing a nucleic acid construct comprising an AP2 nucleic acid comprising a mutation in the miRNA2 binding site operably linked to a promoter that directs expression of the nucleic acid to the SAM.
  • a nucleic acid construct comprising an AP2 nucleic acid comprising a mutation in the miRNA2 binding site operably linked to a promoter that directs expression of the nucleic acid to the SAM.
  • the invention in another aspect, relates to a method for producing a plant with increased yield of a plant said method comprising introducing and expressing a nucleic acid construct comprising an AP2 nucleic acid comprising a mutation in the miRNA2 binding site operably linked to a promoter that directs expression of the nucleic acid to the SAM.
  • a nucleic acid construct comprising an AP2 nucleic acid comprising a mutation in the miRNA2 binding site operably linked to a promoter that directs expression of the nucleic acid to the SAM.
  • the method is for increasing yield-related traits as described elsewhere herein.
  • these traits are selected from fruits/seed size and/or number.
  • the yield related trait is delay of global proliferation arrest (GPA).
  • the methods of the invention may also optionally comprise the steps of screening and selecting plants for those that comprise a polynucleotide construct as above and which have increased yield, including yield-related traits.
  • the progeny plant is stably transformed and comprises the exogenous polynucleotide which is heritable as a fragment of DNA maintained in the plant cell and the method may include steps to verify that the construct is stably integrated.
  • the method may also comprise the additional step of collecting seeds from the selected progeny plant.
  • the method includes the step of introducing an exogenous nucleic acid molecule encoding an AP2 gene product into the seed plant.
  • the invention relates to the use of a nucleic acid construct comprising an AP2 nucleic acid comprising a mutation in the miRNA2 binding site operably linked to a promoter that directs expression of the nucleic acid to the SAM in increasing yield of a plant.
  • a nucleic acid construct comprising an AP2 nucleic acid comprising a mutation in the miRNA2 binding site operably linked to a promoter that directs expression of the nucleic acid to the SAM in increasing yield of a plant.
  • the invention also relates to expression of FUL.
  • the inventors have shown that when FUL expression is specifically repressed in the SAM, an increase in yield, including yield-related traits can be achieved.
  • the invention therefore relates to a transgenic plant expressing a RNA sequence directed against FUL wherein said RNA is operably linked to a promoter that directs expression in the SAM.
  • expression of FUL is specifically suppressed, repressed or silenced in the SAM.
  • the terms "suppressed, repressed or silenced” as used herein in reference to expression of a FUL gene product mean that the amount of functional FUL gene product is reduced in a plant in comparison with the amount of functional FUL gene product in the corresponding control plant, for example a wild type plant. Thus, these terms encompasses the absence of FUL gene product in a plant, as well as expression that is present but reduced as compared to the level of this gene product in control plant.
  • the terms "suppressed, repressed or silenced” also encompass an amount of FUL gene product that is equivalent to the amount of FUL gene product in a corresponding control plant, but where the FUL gene product has a reduced level of activity.
  • an FUL gene product can contain a conserved MADS domain; thus, for example, point mutations or gross deletions within the MADS domain that reduce the DNA-binding activity of an FUL gene product can reduce or destroy its activity and, therefore, "suppress" FUL gene product expression as defined herein.
  • FUL gene product expression is essentially absent in the plant or the FUL gene product is essentially non-functional.
  • Gene silencing is a term generally used to refer to suppression of expression of a gene via sequence-specific interactions that are mediated by RNA molecules. The degree of reduction may be so as to totally abolish production of the encoded gene product, but more usually the abolition of expression is partial, with some degree of expression remaining. The term should not therefore be taken to require complete “silencing" of expression.
  • Transgenes may be used to suppress endogenous plant genes. This was discovered originally when chalcone synthase transgenes in petunia caused suppression of the endogenous chalcone synthase genes and indicated by easily visible pigmentation changes. Subsequently it has been described how many, if not all plant genes can be "silenced" by transgenes. Gene silencing requires sequence similarity between the transgene and the gene that becomes silenced. This sequence homology may involve promoter regions or coding regions of the silenced target gene. When coding regions are involved, the transgene able to cause gene silencing may have been constructed with a promoter that would transcribe either the sense or the antisense orientation of the coding sequence RNA. It is likely that the various examples of gene silencing involve different mechanisms that are not well understood. In different examples there may be transcriptional or post-transcriptional gene silencing and both may be used according to the methods of the invention.
  • RNA-mediated gene suppression or RNA silencing include antisense RNA to reduce transcript levels of the endogenous FUL gene in a plant.
  • RNA silencing does not affect the transcription of a gene locus, but only causes sequence-specific degradation of target mRNAs.
  • An "antisense" nucleic acid sequence comprises a nucleotide sequence that is complementary to a "sense" nucleic acid sequence encoding a FUL protein, or a part of a FUL protein, i.e. complementary to the coding strand of a double- stranded cDNA molecule or complementary to an mRNA transcript sequence.
  • the antisense nucleic acid sequence is preferably complementary to the endogenous FUL gene to be silenced.
  • the complementarity may be located in the "coding region” and/or in the "non-coding region” of a gene.
  • coding region refers to a region of the nucleotide sequence comprising codons that are translated into amino acid residues.
  • non-coding region refers to 5' and 3' sequences that flank the coding region that are transcribed but not translated into amino acids (also referred to as 5' and 3' untranslated regions).
  • Antisense nucleic acid sequences can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid sequence may be complementary to the entire FUL nucleic acid sequence, but may also be an oligonucleotide that is antisense to only a part of the nucleic acid sequence (including the mRNA 5' and 3' UTR).
  • the antisense oligonucleotide sequence may be complementary to the region surrounding the translation start site of an mRNA transcript encoding a polypeptide.
  • the length of a suitable antisense oligonucleotide sequence is known in the art and may start from about 50, 45, 40, 35, 30, 25, 20, 15 or 10 nucleotides in length or less.
  • an antisense nucleic acid sequence according to the invention may be constructed using chemical synthesis and enzymatic ligation reactions using methods known in the art.
  • an antisense nucleic acid sequence e.g., an antisense oligonucleotide sequence
  • an antisense nucleic acid sequence may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acid sequences, e.g., phosphorothioate derivatives and acridine-substituted nucleotides may be used.
  • the antisense nucleic acid sequence can be produced biologically using an expression vector into which a nucleic acid sequence has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • production of antisense nucleic acid sequences in plants occurs by means of a stably integrated nucleic acid construct comprising a promoter, an operably linked antisense oligonucleotide, and a terminator.
  • the nucleic acid molecules used for silencing in the methods of the invention hybridize with or bind to mRNA transcripts and/or insert into genomic DNA encoding a polypeptide to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid sequence which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • Antisense nucleic acid sequences may be introduced into a plant by transformation or direct injection at a specific tissue site. Alternatively, antisense nucleic acid sequences can be modified to target selected cells and then administered systemically.
  • antisense nucleic acid sequences can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid sequence to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid sequences can also be delivered to cells using vectors.
  • RNA interference is another post-transcriptional gene-silencing phenomenon which may be used according to the methods of the invention. This is induced by double-stranded RNA in which mRNA that is homologous to the dsRNA is specifically degraded. It refers to the process of sequence-specific post-transcriptional gene silencing mediated by short interfering RNAs (siRNA).
  • siRNA short interfering RNAs
  • the process of RNAi begins when the enzyme, DICER, encounters dsRNA and chops it into pieces called small-interfering RNAs (siRNA).
  • This enzyme belongs to the RNase III nuclease family. A complex of proteins gathers up these RNA remains and uses their code as a guide to search out and destroy any RNAs in the cell with a matching sequence, such as target mRNA.
  • MicroRNAs miRNAs
  • miRNAs are typically single stranded small RNAs typically 19-24 nucleotides long. Most plant miRNAs have perfect or near-perfect complementarity with their target sequences. However, there are natural targets with up to five mismatches. They are processed from longer non-coding RNAs with characteristic fold-back structures by double-strand specific RNases of the Dicer family. Upon processing, they are incorporated in the RNA-induced silencing complex (RISC) by binding to its main component, an Argonaute protein.
  • RISC RNA-induced silencing complex
  • miRNAs serve as the specificity components of RISC, since they base-pair to target nucleic acids, mostly mRNAs, in the cytoplasm. Subsequent regulatory events include target mRNA cleavage and destruction and/or translational inhibition. Effects of miRNA overexpression are thus often reflected in decreased mRNA levels of target genes. Artificial microRNA (amiRNA) technology has been applied in Arabidopsis thaliana and other plants to efficiently silence target genes of interest. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weigelworld.org).
  • a plant may be transformed to introduce a RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule that has been designed to target the expression of an FUL gene and selectively decreases or inhibits the expression of the gene or stability of its transcript.
  • the RNAi, snRNA, dsRNA, siRNA, miRNA, amiRNA, ta-siRNA or cosuppression molecule used according to the various aspects of the invention comprises a fragment of at least 17 nt, preferably 22 to 26 nt and can be designed on the basis of the information shown in SEQ ID NO: 5 and 6.
  • a short fragment of the target gene sequence (e.g., 19-40 nucleotides in length) is chosen as the target sequence of the siRNA of the invention.
  • the short fragment of target gene sequence is a fragment of the target gene mRNA.
  • the criteria for choosing a sequence fragment from the target gene mRNA to be a candidate siRNA molecule include 1 ) a sequence from the target gene mRNA that is at least 50-100 nucleotides from the 5' or 3' end of the native mRNA molecule, 2) a sequence from the target gene mRNA that has a G/C content of between 30% and 70%, most preferably around 50%, 3) a sequence from the target gene mRNA that does not contain repetitive sequences (e.g., AAA, CCC, GGG, TTT, AAAA, CCCC, GGGG, TTTT), 4) a sequence from the target gene mRNA that is accessible in the mRNA, 5) a sequence from the target gene mRNA that is unique to the target gene, 6) avoids regions within 75 bases of a start codon.
  • a sequence from the target gene mRNA that is at least 50-100 nucleotides from the 5' or 3' end of the native mRNA molecule 2) a sequence from the target gene
  • the sequence fragment from the target gene mRNA may meet one or more of the criteria identified above.
  • the selected gene is introduced as a nucleotide sequence in a prediction program that takes into account all the variables described above for the design of optimal oligonucleotides.
  • This program scans any mRNA nucleotide sequence for regions susceptible to be targeted by siRNAs.
  • the output of this analysis is a score of possible siRNA oligonucleotides. The highest scores are used to design double stranded RNA oligonucleotides that are typically made by chemical synthesis.
  • degenerate siRNA sequences may be used to target homologous regions.
  • siRNAs according to the invention can be synthesized by any method known in the art. RNAs are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA RNA synthesizer. Additionally, siRNAs can be obtained from commercial RNA oligonucleotide synthesis suppliers. siRNA molecules according to the aspects of the invention may be double stranded. In one embodiment, double stranded siRNA molecules comprise blunt ends. In another embodiment, double stranded siRNA molecules comprise overhanging nucleotides (e.g., 1 -5 nucleotide overhangs, preferably 2 nucleotide overhangs).
  • the siRNA is a short hairpin RNA (shRNA); and the two strands of the siRNA molecule may be connected by a linker region (e.g., a nucleotide linker or a non-nucleotide linker).
  • the siRNAs of the invention may contain one or more modified nucleotides and/or non- phosphodiester linkages. Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the siRNA. The skilled person will be aware of other types of chemical modification which may be incorporated into RNA molecules.
  • siRNA An example of a siRNA that can be used is shown in the examples herein.
  • the silencing molecule comprises or consists of SEQ ID NO: 8.
  • recombinant DNA constructs as described in US 6,635,805, incorporated herein by reference, can be used.
  • the silencing RNA molecule is introduced into the plant using conventional methods, for example a vector and Agrobacterium-mediated transformation or particle bombardment. Stably transformed plants are generated and expression of the FUL gene compared to a wild type control plant is analysed.
  • the transgenic plant expresses a nucleic acid construct comprising a RNAi, snRNA, dsRNA, siRNA, miRNA, ta- siRNA, amiRNA that targets the FUL gene as described herein and reduces expression of the endogenous FUL gene.
  • a gene is targeted when, for example, the RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule selectively decreases or inhibits the expression of the gene compared to a control plant.
  • RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule targets FUL when the RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRN, amiRNA or cosuppression molecule hybridises under stringent conditions to the gene transcript.
  • the RNA preferably, to specifically target FUL, the RNA must comprise at least the same seed sequence.
  • said FUL nucleic acid sequence which is targeted by the RNA comprises SEQ ID NO: 5 or 6, a function variant, homologue or orthologue thereof or a FUL-Wke gene.
  • variants of a FUL nucleic acid or peptide sequence or FUL-Wke genes described herein will have at least about 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to that particular non- variant nucleotide sequence or polypeptide as determined by sequence alignment programs described elsewhere herein.
  • the term homologue as used herein also designates an FUL/FDL orthologue from other plant species.
  • a homologue of a AtFUL polypeptide has, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall
  • overall sequence identity is more than 70% or more than 73%.
  • overall sequence identity is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.
  • the homologue of a FUL nucleic acid sequence has, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at
  • overall sequence identity is more than 70% or more than 73%.
  • overall sequence identity is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.
  • Suitable homologs are selected from any of the following list: Hordeum vulgare BM5A (AAW82994), Triticum (AAO72630), Zea mays (ABW84393), Sorghum (XP_003559398), Brachypodium (XP_003559398), Oryza sativa (AAP68361 ), Gossypium hirsutum (AGA01526), Solanum lycopersicum (NP_001234173), Pisum sativum PsFULb (AFI08227), Pisum sativum PsFULa (AAX69065), Glycine max (NP_001242674), Phaseolus vulgaris (ESW10371 ) and Brassica oleracea (CAD47849).
  • Suitable homologs are selected from any of the following list: Hordeum vulgare BM5A (AAW82994), Triticum (AAO72630), Zea mays (ABW84393), S
  • Variants thereof that have at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identify to the sequence mentioned above are also included.
  • Suitable homologues can be identified by sequence comparisons and identifications of conserved domains. There are predictors in the art that can be used to identify such sequences. The function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function when expressed in a plant.
  • the wild type AtFUL protein is schematically illustrated in Figure 7 and shown in SEQ ID NO: 7.
  • SEQ ID NO: 5 and 6 are the cDNA and the wild type genomic nucleic acids respectively encoding SEQ ID NO: 7.
  • FUL like AGAMOUS and other AGL genes, is characterized, in part, in that it is a plant MADS box gene.
  • the plant MADS box genes generally encode proteins of about 260 amino acids including a highly conserved MADS domain of about 56 amino acids.
  • the MADS domain is the most highly conserved region of the MADS domain proteins, the major determinant of sequence specific DNA- binding activity and can also perform dimerization and other accessory functions.
  • the MADS domain frequently resides at the N-terminus, although some proteins contain additional residues N-terminal to the MADS domain (Riechmann and Meyerowitz. Biol. Chem., 1997; 378:1079-1 101 ).
  • the "intervening domain” or "l-domain,” located immediately C-terminal to the MADS domain, is a weakly conserved domain having a variable length of approximately 30 amino acids. In some proteins, the l-domain plays a role in the formation of DNA-binding dimers.
  • a third domain present in plant MADS domain proteins is a moderately conserved 70 amino acid region denoted the "keratinlike domain” or "K-domain.” Named for its similarity to regions of the keratin molecule, the structure of the K-domain appears capable of forming amphipathic helices and may mediate protein-protein interactions.
  • the most variable domain, both in sequence and in length, is the carboxy-terminal or "C- domain" of the MADS domain proteins. Dispensable for DNA binding and protein dimerization in some MADS domain proteins, the function of this C- domain remains unknown.
  • Arabidopsis AtFUL is a 242 amino acid MADS box protein (SEQ ID NO: 7) (Mandel et al., The Plant Cell. 1995; Vol. 7: 1763-1771 ).
  • the FUL MADS domain resides at amino acids 2 to 56 of SEQ ID NO: 7 and is as follows: RGRVQLKRIENKINRQVTFSKRRSGLLKKAHEISVLCDAEVALIVFSSKGK LFEY (SEQ ID NO: 9, residues 3-60 in figure 7).
  • the K-domain of FUL resides at amino acids 75-174 of SEQ ID NO: 5 and is as follows: YSDKQLVGRDVSQSENWVLEHAKLKARVEVLEKNKRNFMGEDLDSLSLKELQ SLEHQLDAAIKSIRSRKNQAMFESISALQKKDKALQDHNNSLLKKIKER (SEQ ID NO: 10)
  • Homologues/orthologues of AtFUL are therefore characterised by the presence of conserved MADS domain and K-domain domains.
  • FUL is also characterized by other sequence features for example the FUL-like motif in figure 7 (L/MPPWML; SEQ ID NO. 1 1 ).
  • the homologues/orthologues of AtFUL comprise a sequence corresponding to SEQ ID NO: 7, 8, 9, 10 and/or 1 1 or a sequence having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 95%, 96%, 97%, 98%, or at least 99% to SEQ ID NO: 7, 8, 9, 10 and/or 1 1 .
  • Preferred homologues/orthologues of AtFUL peptides are FUL peptides from crop plants, for example cereal crops.
  • preferred homologues include FUL in dicots.
  • preferred homologues include FUL in maize, rice, wheat, sorghum, sugar cane, oilseed rape (canola), soybean, cotton, potato, tomato, tobacco, grape, barley, pea, bean, field bean or other legumes, lettuce, sunflower, cacao, lentils, alfalfa, sugar beet, broccoli or other vegetable brassicas or poplar.
  • Suitable homologs are selected from any of the following list: Hordeum vulgare BM5A (AAW82994), Triticum (AAO72630), Zea mays (ABW84393), Sorghum (XP_003559398), Brachypodium (XP_003559398), Oryza sativa (AAP68361 ), Gossypium hirsutum (AGA01526), Solanum lycopersicum (NP_001234173), Pisum sativum PsFULb (AFI08227), Pisum sativum PsFULa (AAX69065), Glycine max (NP_001242674), Phaseolus vulgaris (ESW10371 ) and Brassica oleracea (CAD47849).
  • FUL orthologues for example orthologues listed above, which have overall sequence identity is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% to these orthologues.
  • the said promoter is selected from TFL1 (SEQ ID NO: 4).
  • the plant is a crop plant or ornamental plant.
  • the plant is a dicot.
  • the plant is a crop plant is selected from maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • the orthologue is FUL from maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • the invention also relates to harvestable parts of a transgenic plant wherein FUL expression is suppressed in the SAM as described above or a product derived therefrom.
  • the invention relates to a nucleic acid construct said construct comprising a siRNA directed against FUL wherein said siRNA is operably linked to a promoter that directs expression in the SAM.
  • said RNA is RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or longer nucleic acid sequences that are then transcribed and processed.
  • a nucleic acid construct comprising a siRNA directed against a FUL nucleic acid wherein said siRNA is operably linked to a promoter that directs expression in the SAM, wherein said FUL comprises SEQ ID NO: 5 or 6, a function variant, homologue or orthologue thereof and wherein said promoter is selected from TFL1 .
  • the pHANNIBAL system may also be used to design the silencing molecule as described in the examples.
  • the FUL comprises SEQ ID NO: 5 or 6 a functional variant, homologue or orthologue or part thereof.
  • FUL homologues, orthologues or functional variants are described above.
  • said functional variant, homolog or orthologue has at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 5, 6 or 7.
  • said orthologue is FUL from maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • the invention relates to a vector comprising a siRNA directed against FUL wherein said siRNA is operably linked to a promoter that directs expression in the SAM.
  • the invention in another aspect, relates to a host cell transformed with a nucleic acid comprising a siRNA directed against FUL wherein said siRNA is operably linked to a promoter that directs expression in the SAM or a vector comprising a siRNA directed against FUL wherein said siRNA is operably linked to a promoter that directs expression in the SAM.
  • the host cell can be a bacterial, for example Agrobacterium, or a plant cell.
  • the invention relates to a culture medium or kit comprising a bacterial or a plant host cell as described above and a culture medium.
  • the invention in another aspect, relates to a method for increasing yield, including yield-related traits of a plant said method comprising introducing and expressing a nucleic acid construct comprising a siRNA directed against FUL wherein said siRNA is operably linked to a promoter that directs expression in the SAM or a vector comprising a siRNA directed against FUL wherein said siRNA is operably linked to a promoter that directs expression in the SAM in a plant.
  • the invention in another aspect, relates to a method for producing a plant with increased yield of a plant said method comprising introducing and expressing a nucleic acid construct comprising a siRNA directed against FUL wherein said siRNA is operably linked to a promoter that directs expression in the SAM or a vector comprising a siRNA directed against FUL wherein said siRNA is operably linked to a promoter that directs expression in the SAM in a plant.
  • the plant is selected from maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • the invention relates to a plant obtained or obtainable by the methods.
  • the invention relates to the use of a nucleic acid construct comprising a siRNA directed against FUL wherein said siRNA is operably linked to a promoter that directs expression in the SAM or a vector comprising a siRNA directed against FUL wherein said siRNA is operably linked to a promoter that directs expression in the SAM for increasing yield of a plant.
  • the invention relates to a knockout mutant in a legume, wherein expression of the expression of the FUL gene is downregulated.
  • Mutant plants have point mutations either in PsFULa (G to T in beginning of third intron which causes missplicing and therefore renders a truncated PsFULa protein) or PsFULb (W 91 to STOP).
  • Homozygous mutant plants for each of these mutations display no alterations in vegetative growth, flowering time or plant size before flowering, but when flowering transition takes place, the SAM remains active for longer (2 to 4 weeks), extending the phase of flower and pod production (see figure 8).
  • said legume is of the family Fabaceae (or Leguminosae) and may be selected from pea, alfalfa, peas, beans, lentils, soybeans, peanuts and tamarind, chickpea, fava beans, cowpea, mung beans.
  • the legume is pea.
  • the invention in another aspect, relates to a transgenic legume plant comprising a nucleic acid construct comprising a RNA molecule directed against FUL wherein said RNA is operably linked to a promoter that directs expression in the SAM or a vector comprising a RNA directed against FUL.
  • the invention in another aspect, relates to a method for increasing yield, including yield related traits, of a plant said method comprising introducing and expressing a nucleic acid construct comprising a siRNA directed against FUL wherein said siRNA or a vector comprising a siRNA directed against FUL.
  • said plant is a legume and said FUL is a legume FUL.
  • said legume may be selected from pea, alfalfa, peas, beans, lentils, soybeans, peanuts and tamarind, chickpea, fava beans, cowpea, mung beans.
  • Yield can be measured using different yield related traits, these are described herein. For example, the number of seeds/fruit or the size of fruits can be measured.
  • the invention relates to a method for producing a plant with increased yield, including yield-related traits, of a plant said method comprising introducing and expressing a nucleic acid construct comprising a siRNA directed against FUL wherein said siRNA or a vector comprising a siRNA directed against FUL wherein said plant is a legume and said FUL is a legume FUL.
  • said legume may be selected from pea, alfalfa, peas, beans, lentils, soybeans, peanuts and tamarind, chickpea, fava beans, cowpea, mung beans.
  • the invention relates to a plant obtained or obtainable by the methods above.
  • the invention relates to the use of a nucleic acid construct comprising a siRNA directed against FUL or a vector comprising a siRNA directed against FUL wherein said siRNA for increasing yield of a plant wherein said plant is a legume and said FUL is a legume FUL.
  • said legume may be selected from pea, alfalfa, peas, beans, lentils, soybeans, peanuts and tamarind, chickpea, fava beans, cowpea, mung beans.
  • the methods of the invention may also optionally comprise the steps of screening and selecting plants for those that comprise a polynucleotide construct as above, have increased stress resistance to one or more of salinity, osmotic stress and/or oxidative stress compared to a control plant.
  • the progeny plant is stably transformed and comprises the exogenous polynucleotide which is heritable as a fragment of DNA maintained in the plant cell and the method may include steps to verify that the construct is stably integrated.
  • the method may also comprise the additional step of collecting seeds from the selected progeny plant.
  • the method includes the step of introducing an exogenous nucleic acid molecule encoding an FUL gene product into the plant.
  • a further step can include measuring yield and optionally comparing yield to a control plant.
  • the invention in another aspect of the invention, relates to a transgenic plant that expresses a nucleic acid construct comprising a dominant negative mutant of FUL operably linked to a promoter directing expression to the SAM.
  • the dominant negative construct also can be used to suppress FUL gene product expression in a seed plant.
  • a dominant negative construct useful in the invention generally contains a portion of the complete FUL gene product coding sequence sufficient, for example, for DNA-binding or for a protein-protein interaction such as a homodimeric or heterodimeric protein-protein interaction but lacking the transcriptional activity of the wild type protein.
  • a useful dominant negative construct can be a deletion mutant encoding, for example, the MADS box domain alone (“M”), the MADS box domain and “intervening” region (“Ml”); the MADS box, “intervening” and “K” domains ("MIK”); or the “intervening,” “K” and carboxy-terminal domains (“IKC”).
  • the invention in another aspect, relates to a transgenic plant comprising at least two exogenous nucleic acid constructs.
  • the first is a nucleic acid construct said construct comprising a modified AP2 nucleic acid operably linked to a promoter that directs expression of the nucleic acid to the shoot apical meristem (SAM) wherein said modified AP2 nucleic acid encodes a modified polypeptide comprising a mutation in the miRNA172 binding site and wherein said plant also expresses a RNA molecule directed against a FUL nucleic acid wherein said RNA is operably linked to a promoter that directs expression in the SAM.
  • SAM shoot apical meristem
  • Methods for increasing yield in a plant comprising expressing a first nucleic acid construct said construct comprising a modified AP2 nucleic acid operably linked to a promoter that directs expression of the nucleic acid to the shoot apical meristem (SAM) wherein said modified AP2 nucleic acid encodes a modified polypeptide comprising a mutation in the miRNA172 binding site and a second nucleic acid construct which comprises a RNA molecule directed against a FUL nucleic acid wherein said RNA is operably linked to a promoter that directs expression in the SAM are also within the scope of the invention.
  • SAM shoot apical meristem
  • the invention in another aspect, relates to a transgenic legume plant comprising at least two exogenous nucleic acid constructs.
  • the first is a nucleic acid construct said construct comprising a modified AP2 nucleic acid operably linked to a promoter that directs expression of the nucleic acid to the shoot apical meristem (SAM) wherein said modified AP2 nucleic acid encodes a modified polypeptide comprising a mutation in the miRNA172 binding site and wherein said plant also expresses a RNA molecule directed against a FUL nucleic.
  • SAM shoot apical meristem
  • Methods for increasing yield in a plant comprising expressing a first nucleic acid construct said construct comprising a modified AP2 nucleic acid operably linked to a promoter that directs expression of the nucleic acid to the shoot apical meristem (SAM) wherein said modified AP2 nucleic acid encodes a modified polypeptide comprising a mutation in the miRNA172 binding site and a second nucleic acid construct which comprises a RNA molecule directed against a FUL nucleic acid are also within the scope of the invention.
  • SAM shoot apical meristem
  • the invention also extends to harvestable parts of a transgenic plant of the invention as described herein such as, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs.
  • the invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
  • the invention also relates to food products and food supplements comprising the plant of the invention or parts thereof.
  • a nucleic acid described herein is introduced into a plant and expressed as a transgene.
  • the nucleic acid sequence is introduced into said plant through a process called transformation.
  • transformation or transformation as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer.
  • Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • tissue targets include leaf discs, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • the polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome.
  • the resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
  • Transformation of plants is now a routine technique in many species.
  • any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell.
  • the methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non- integrative) viruses and the like.
  • Transgenic plants, including transgenic crop plants are preferably produced via Agrobacterium tumefaciens mediated transformation.
  • the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants.
  • the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying.
  • a further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants.
  • the transformed plants are screened for the presence of a selectable marker such as the ones described above.
  • putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation.
  • expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
  • the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or T1 ) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
  • the generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
  • the various aspects of the invention described herein clearly extend to any plant cell or any plant produced, obtained or obtainable by any of the methods described herein, and to all plant parts and propagules thereof unless otherwise specified.
  • the present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
  • AP2 we amplified the sequence of AP2-170 allele from cDNA derived from the original mutant, and cloned it under the promoter of TFL1 (SEQ ID NO: 14) in a binary vector to transform Arabidopsis.
  • TFL1 SEQ ID NO: 14
  • FUL we used pHANNIBAL to clone one fragment of FUL mRNA in both sense and antisense orientations flanking the intron sequences in pHANNIBAL. Then, the cassette FULs-intron- FULas was cloned under the control of TFL1 promoter (SEQ ID NO: 15) as above.
  • Arabidopsis plants (Columbia ecotype) were infiltrated with Agrobacterium cultures following the protocol by Clough and Bent, 1998. Seeds were collected from these infiltrated plants and T1 transgenic plants were identified by growing the seeds in MS-Kan plates (both T-DNAs conferred Kanamicyn resistance).
  • Seeds were collected from individual T1 plants and at least 100 from each T1 line were grown on MS-kan plates to analyze the segregation of the resistance in the progeny. Those that segregated 3 Kan-Resistant: 1 Kan sensitive were identified as likely possessing a single T-DNA insertion. Presence of the transgene was further confirmed in selected T2 plants by PCR. Seeds were collected from individual T2 plants of those lines with good segregation ratios to identify homozygous plants for the T-DNA insertion (again by growing seeds in MS-Kan plates and selecting those that did not segregate Kan-sensitive plants). T3 homozygous plants were used to perform the quantifications on flower/fruit number, seed number and weight.
  • Table 1 Number of seeds for transgenic plants expressing TFL1 ::AtAP2 170 and TFL1 ::RNAi-AtFUL respectively and seed weight for transgenic plants expressing TFL1 ::AtAP2 170 and TFL1 ::RNAi-AtFUL, respectively.

Landscapes

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

Abstract

La présente invention concerne l'utilisation de gènes floraux de plante dans la modification de rendements de plantes. La présente invention concerne des plantes transgéniques présentant un rendement accru, un nombre de graines et de fruits accrus ainsi que des utilisations et des procédés associés. Spécifiquement, la présente invention se rapporte à des plantes transgéniques exprimant une version résistante au mi-172 de AP2 ou d'une molécule d'ARN dirigés contre un acide nucléique FUL, la version résistante au mi-172 de l'AP2 ou la molécule d'ARN sont liées de manière fonctionnelle à un promoteur qui dirige leur expression dans les méristèmes apicaux des pousses, conduisant à l'augmentation du nombre de fruits et de graines et par conséquent à un rendement accru dans les plantes transgéniques.
PCT/EP2015/057086 2014-03-31 2015-03-31 Plantes transgéniques présentant un nombre accru de fruits et de graines et son procédé d'obtention WO2015150412A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP14382126 2014-03-31
EP14382126.2 2014-03-31

Publications (1)

Publication Number Publication Date
WO2015150412A1 true WO2015150412A1 (fr) 2015-10-08

Family

ID=50434148

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2015/057086 WO2015150412A1 (fr) 2014-03-31 2015-03-31 Plantes transgéniques présentant un nombre accru de fruits et de graines et son procédé d'obtention

Country Status (1)

Country Link
WO (1) WO2015150412A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT201700065504A1 (it) * 2017-06-13 2018-12-13 Fond Parco Tecnologico Padano Nuove piante con fenotipo ‘fiore doppio’
US20210269817A1 (en) * 2015-10-30 2021-09-02 KWS SAAT SE & Co. KGaA Inhibition of bolting and flowering of a beta vulgaris plant

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999000503A1 (fr) * 1997-06-27 1999-01-07 The Regents Of The University Of California Procede permettant d'augmenter la taille d'un fruit dans une plante
WO2007144190A2 (fr) * 2006-06-15 2007-12-21 Cropdesign N.V. Plantes ayant des caractères liés au rendement accrus et procédé servant à fabriquer celles-ci

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999000503A1 (fr) * 1997-06-27 1999-01-07 The Regents Of The University Of California Procede permettant d'augmenter la taille d'un fruit dans une plante
WO2007144190A2 (fr) * 2006-06-15 2007-12-21 Cropdesign N.V. Plantes ayant des caractères liés au rendement accrus et procédé servant à fabriquer celles-ci

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
C. SMACZNIAK ET AL: "Developmental and evolutionary diversity of plant MADS-domain factors: insights from recent studies", DEVELOPMENT, vol. 139, no. 17, 7 August 2012 (2012-08-07), pages 3081 - 3098, XP055142964, ISSN: 0950-1991, DOI: 10.1242/dev.074674 *
CHEN X: "A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 303, no. 5666, 26 March 2004 (2004-03-26), pages 2022 - 2025, XP002300174, ISSN: 0036-8075, DOI: 10.1126/SCIENCE.1088060 *
FERRANDIZ CRISTINA ET AL: "Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER", DEVELOPMENT, THE COMPANY OF BIOLOGISTS LTD, GB, vol. 127, no. 4, 1 February 2000 (2000-02-01), pages 725 - 734, XP002351770, ISSN: 0950-1991 *
KEMPIN ET AL: "The FRUITFULL MADS-box gene mediates cell differentiation during Arabidopsis fruit development", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 125, no. 8, 1 April 1998 (1998-04-01), pages 1509 - 1517, XP002082111, ISSN: 0006-4971 *
MASA-AKI OHTO ET AL: "Effects of APETALA2 on embryo, endosperm, and seed coat development determine seed size in Arabidopsis", SEXUAL PLANT REPRODUCTION, SPRINGER, BERLIN, DE, vol. 22, no. 4, 25 October 2009 (2009-10-25), pages 277 - 289, XP019804132, ISSN: 1432-2145 *
T. WURSCHUM: "APETALA2 Regulates the Stem Cell Niche in the Arabidopsis Shoot Meristem", THE PLANT CELL ONLINE, vol. 18, no. 2, 1 February 2006 (2006-02-01), pages 295 - 307, XP055197009, ISSN: 1040-4651, DOI: 10.1105/tpc.105.038398 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210269817A1 (en) * 2015-10-30 2021-09-02 KWS SAAT SE & Co. KGaA Inhibition of bolting and flowering of a beta vulgaris plant
IT201700065504A1 (it) * 2017-06-13 2018-12-13 Fond Parco Tecnologico Padano Nuove piante con fenotipo ‘fiore doppio’

Similar Documents

Publication Publication Date Title
AU2014203225B2 (en) Woody plants having improved growth characteristics and method for making the same using transcription factors
US20200354735A1 (en) Plants with increased seed size
WO2009029739A2 (fr) Plantes avec architecture racinaire modifiée, constructions connexes et méthodes faisant intervenir des gènes codant des polypeptides de nucléoside diphosphatase kinase (ndk) et leurs homologues
CA2903700A1 (fr) Adaptation amelioree du mais
WO2014127744A1 (fr) Compositions et procédés impliquant des facteurs de transcription wrky de codage de gènes
WO2016124920A1 (fr) Plants de riz présentant une modification du phénotype et de la qualité des semences
WO2016000239A1 (fr) Plantes et procédés pour améliorer les caractéristiques agronomiques dans des conditions de stress abiotique
US20170121730A1 (en) Plants having altered agronomic characteristics under abiotic conditions and related constructs and methods involving abiotic tolerance genes
US20170183671A1 (en) Plants with improved agronomic traits
US20110138501A1 (en) Plants with altered root architecture, related constructs and methods involving genes encoding protein phophatase 2c (pp2c) polypeptides and homologs thereof
US8394634B2 (en) Plants having altered agronomic characteristics under nitrogen limiting conditions and related constructs and methods involving genes encoding LNT2 polypeptides and homologs thereof
US20160017347A1 (en) Terminating flower (tmf) gene and methods of use
US8541650B2 (en) Plants having altered agronomic characteristics under nitrogen limiting conditions and related constructs and methods involving genes encoding LNT1 polypeptides and homologs thereof
WO2010099083A1 (fr) Nouvel acide nucléique isolé et molécules protéiniques provenant de soja, et procédés d'utilisation de telles molécules
US20180066026A1 (en) Modulation of yep6 gene expression to increase yield and other related traits in plants
WO2015150412A1 (fr) Plantes transgéniques présentant un nombre accru de fruits et de graines et son procédé d'obtention
US20140007298A1 (en) Plants with altered root architecture, related constructs and methods involving genes encoding protein phophatase 2c (pp2c) polypeptides and homologs thereof
EP3191588A1 (fr) Procédés et matériaux de production de fruits de taille modifiée
Martínez Fernández et al. Transgenic plants with increased number of fruits and seeds and method for obtaining thereof
KR20150003099A (ko) 식물의 생산성 증대 기능, 스트레스 내성 기능 및 노화 지연 기능을 갖는 atpg6 단백질과 그 유전자 및 이들의 용도
US20110035837A1 (en) Plants having altered agronomic characteristics under nitrogen limiting conditions and related constructs and methods involving genes encoding lnt3 polypeptides
CA2729099A1 (fr) Plantes a l'architecture de racines modifiee, constructions associees et procedes impliquant des genes codant pour des polypeptides rep2 et leurs homologues

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15714202

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase
122 Ep: pct application non-entry in european phase

Ref document number: 15714202

Country of ref document: EP

Kind code of ref document: A1