US20080052793A1 - Seeds - Google Patents

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US20080052793A1
US20080052793A1 US10/591,418 US59141805A US2008052793A1 US 20080052793 A1 US20080052793 A1 US 20080052793A1 US 59141805 A US59141805 A US 59141805A US 2008052793 A1 US2008052793 A1 US 2008052793A1
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plant
gene
promoter
mnt
seed
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Roderick Scott
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University of Bath
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Priority claimed from GB0406275A external-priority patent/GB0406275D0/en
Priority claimed from GB0406729A external-priority patent/GB0406729D0/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—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
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8234—Seed-specific, e.g. embryo, endosperm
    • 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 present invention relates particularly, though not exclusively, to methods for modifying characteristics such as seed size in plants, especially flowering plants, and to plants and reproducible plant material produced by the methods.
  • the invention also relates to nucleic acid constructs for use in such methods, as well as to modified plants and reproducible plant material per se.
  • the seeds industry can be split into two high-value, commercial sectors: seeds for field crops such as corn, oil seeds, sugar beet and cereals, and vegetable and flower seed.
  • field crops such as corn, oil seeds, sugar beet and cereals
  • vegetable and flower seed The scientific improvement of crop plants has gone through a succession of innovations leading to the development of hybrid varieties for many crops and, most recently, to the introduction of genetically enhanced crops.
  • the worldwide commercial seeds market is valued at around $30 billion (International Seed Federation).
  • Seed size is positively correlated with a number of components of ‘seed quality’ such as the percentage of germination (Schaal, 1980; Alexander and Wulff, 1985; Guberac et al, 1998); time to emergence (Winn, 1985; Wulff, 1986); durability (survival under adverse growing conditions) (Krannitz et al, 1991; Manga and Yadav, 1995); and growth rate (Marshall, 1986).
  • Seed quality is an important factor in the cost of production of commercial seed lots since these must be tested before sale. Consequently, increasing total seed weight, even without increases in total seed yield, may have economic benefits through improvements in seed quality. Conversely, decreasing seed size may also be desirable in some circumstances, for example by facilitating water uptake required for germination (Harper et al., 1970), or in plants grown for their fruit.
  • Modification of seed size is also likely to improve yield through increasing the ‘sink strength’ of the seed (i.e. its capacity to demand nutrients from the seed parent), or increasing the period in which the seed is acting as a strong sink. It is well established that the demands of sink organs such as seeds have significant control over the rate of photosynthesis and the movement of photoassimilates from source to sink tissues (Patrick and Offler, 1995; Paul and Foyer, 2001). In wheat, the seed parent can supply more nutrients than developing seeds are able to demand for the first 15-20 days after pollination (Austin, 1980). Therefore modifications that enable seeds to draw nutrients earlier in development, for example by speeding up seed growth, will allow seeds to capture resources that would otherwise be wasted. An ‘improved source-sink balance permitting higher sink demand during grainfilling’ has also been proposed as a method for increasing yield in wheat (Reynolds et al., 2001).
  • Mature seeds of flowering plants consist of three components: the seed coat, which is of exclusively maternal origin; and the two fertilization products, embryo and endosperm, which have maternal and paternal genetic contributions. Seeds develop from fertilized ovules. Ovule development has been described for many species (Bouman, 1984), including Arabidopsis thaliana (Robinson-Beers et al., 1992; Schneitz et al., 1995). The main structures of the mature ovule are: the embryo sac, which contains the female reproductive cells (egg and central cell); the nucellus, which surrounds the embryo sac at least partially; and the inner and outer integuments, which envelop the embryo sac and nucellus. After fertilization the embryo and nutritive endosperm develop inside the embryo sac while the integuments differentiate into the seed coat, which expands to accommodate the growing endosperm and embryo.
  • Most monocotyledonous plants e.g. cereals including maize, wheat, rice, and barley (see Esau, 1965), produce albuminous seeds—that is, at maturity they contain a small embryo and a relatively massive endosperm.
  • Most dicotyledonous plants e.g. Brassica napus , (oil seed rape, canola), soybean, peanut, Phaseolus vulgaris (e.g. kidney bean, white bean, black bean) Vicia faba (broad bean), Pisum sativum (green pea), Cicer aeietinum (chick pea), and Lens culinaris (lentil), produce exalbuminous seeds—that is, the mature seeds lack an endosperm.
  • the embryo In such seeds the embryo is large and generally fills most of the volume of the seed, and accounts for almost the entire weight of the seed.
  • exalbuminous seeds the endosperm is ephemeral in nature and reaches maturity when the embryo is small and highly immature (usually heart/torpedo stage). Commonly embryo development depends on the presence of the endosperm, which is generally accepted to act as a source of nutrition for the embryo.
  • Seed size control can be viewed from the perspective of (1) ‘development’—the extent of cell division and expansion in one or more seed components (e.g. Reddy and Daynard, 1983; Swank et al, 1987; Scott et al., 1998; Garcia et al., 2003) or (2) ‘metabolism’ 13 metabolic activity and transport of nutrients within the seed and between the seed and seed parent (e.g. Weber et al., 1996, 1997).
  • development the extent of cell division and expansion in one or more seed components
  • ‘metabolism’ 13 metabolic activity and transport of nutrients within the seed and between the seed and seed parent
  • invertase activity involved in hexose transport
  • endosperm proliferation in maize
  • high invertase activity is correlated with increased cell numbers in broad bean seed coat (Weber et al., 1996), legume embryos (Weber et al., 1997), and barley endosperm (Weschke et al., 2003).
  • Our present investigations focus on the developmental aspects of seed size control, although it can be assumed that changes to cell division/expansion in the seed will also be correlated with changes in metabolic activity and nutrient flow.
  • Alonso-Blanco et al. (1999) investigated seed size in wild-type plants of two Arabidopsis thaliana accessions, Cvi and Ler: seeds of the former weigh 80% more than seeds of the latter and are 20% longer. In both accessions, the authors found that ‘seed coat and endosperm growth preceded embryo growth, determining the overall final length of the embryo and the seed’. They did find that the outer layer of the mature seed coat has more cells in Cvi than Ler, but did not investigate or comment on whether these extra cells were formed before or after fertilization.
  • ap2 mutant seeds have seed coat abnormalities including large and irregular outer integument cells, lack of mucilage, and hypersensitivity to bleach; and the increase in seed size was found to be a mainly (Jofuku et al., 2005) or wholly (Ohto et al., 2005) maternal effect.
  • the enlarged 35S: ANT fruit included T2 seeds that were larger than normal (not shown in the application), because of enlarged embryos.’
  • the large seed size of 35S::ANT seeds was attributed only to size of the nucellus and embryo.
  • US patent application no. 20030159180 describes uses of a modified ANT polypeptide for altering the size of plant organs including seeds. It was reported that the transgenic plants had varying degrees of fertility that were not correlated with organ size. There was no investigation of the effect of expressing the modified ANT polypeptide on integument or seed coat growth.
  • APETALA2 AP2
  • the mutations have a maternal effect on seed size but the only phenotype described for the integument/seed coat in ap2 mutants is that the cells of the outer layer of the seed coat are enlarged with an irregular shape, along with some other morphological abnormalities (Jofuku et al., 1994).
  • U.S. Pat. No. 6,329,567 describes methods of modulating seed mass using AP2 transgenes, but this patent does not assess any effect of the transgenes on the integuments or seed coat.
  • the BANYULS (BAN) gene is expressed exclusively in the inner layer of the inner integument (this layer is also called the endothelium) in early seed development (pre-globular stage) (Devic et al., 1999).
  • International patent application WO 03/012106 A2 describes use of the BAN promoter to drive expression of various genes specifically in the testa (the seed coat layer derived from the inner integument). The authors propose uses such as modifying the tannin or fibre composition, or the hormonal equilibrium, but no relevant expression cassettes were reported or described. Modification of seed size is also proposed but only in the context of reducing or ablating seeds in fruit crops.
  • a BAN promoter::BARNASE construct was shown to ablate the endothelium.
  • This patent application relates to expression of genes such as ipt that ‘affect metabolically effective levels of cytokinins in plant seeds, as well as in the maternal tissue from which such seeds arise, including developing ears, female inflorescences, ovaries, female florets, aleurone, pedicel, and pedicel-forming regions’, and to transgenic plants with enhanced levels of cytokinin that exhibit ‘improved seed size, decreased tip kernel abortion, increased seed set during unfavorable environmental conditions, and stability of yield’.
  • nucellus promoter is the maternal tissue surrounding the embryo sac and enclosed within the integuments
  • integuments are not specifically mentioned in the patent application, nor were any maternal tissue-specific expression cassettes described.
  • the disclosure of this patent application is particularly concerned with maize.
  • This patent application relates to methods for controlling endosperm size and development through use of an antisense DNA METHYLTRANSFERASE 1 gene that reduces cytosine methylation.
  • modification to the cytosine methylation status of the seed or pollen parent alters seed size by altering the rate and extent of endosperm proliferation. Therefore the disclosure of this patent application relates exclusively to ‘endosperm-led’ seed growth.
  • Increased stem diameter is also desirable in agriculture, as it may lead to an increase in plant biomass, which may in turn increase yield (Reynolds et al., 2001). Increased stem diameter and biomass are also desirable in certain crops such as trees and vegetables. Thicker stems are also desirable because this trait increases resistance to lodging, a serious problem that reduces yields in crops including cereals (Zuber et al., 1999), soybean (Board, 2001), and oilseed rape (Miliuviene et al., 2004). A further aspect of the mnt-1 mutant phenotype is increased diameter of the stems.
  • “Function” when used in relation to a gene embraces both the operation of that gene at a molecular level as well as the downstream effects of expression of the gene which may result in phenotypic changes.
  • Nucleic acid sequence refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end, including chromosomal DNA, plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
  • orthologues refers to genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologues when their nucleotide sequences and/or their encoded protein sequences have a high percentage of sequence identity and/or similarity. Functions of orthologues are often highly conserved among species.
  • “Homologue” A gene (or protein) with a similar nucleotide (or amino acid) sequence to another gene (or protein) in the same or another species.
  • Promoter a region or sequence located upstream and/or downstream from the start of transcription involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • Plant promoter refers to a promoter capable of initiating transcription in plant cells.
  • “Operably linked” refers to a functional linkage between a promoter and a DNA sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence.
  • “operably linked” means that the nucleic acid sequences being linked are contiguous, and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • Plant includes whole plants, plant parts, and plant propagative material including: shoot vegetative organs and/or structures (e.g. leaves, stems and tubers), roots, flowers and floral organs (e.g. bracts, sepals, petals, stamens, carpels, anthers), ovules (including egg and central cells), seed (including zygote, embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), seedlings, plant tissue (e.g., vascular tissue, ground tissue), cells (e.g., guard cells, egg cells, trichomes and the like), and their progeny.
  • shoot vegetative organs and/or structures e.g. leaves, stems and tubers
  • roots e.g. bracts, sepals, petals, stamens, carpels, anthers
  • ovules including egg and central cells
  • seed including zygote, embryo, endosperm, and seed coat
  • fruit e.g., the
  • Plant cell includes cells obtained from or found in seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores as well as whole plants.
  • plant cells also includes modified cells, such as protoplasts, obtained from the aforementioned tissues.
  • Wild type in the context of a plant or plant material which has been modified in some way refers to a comparable plant which has not been modified in that way and grown or produced under similar conditions. For a given plant it may be the genotype or phenotype that is found in nature or in standard laboratory stock. References in this specification to relative changes in characteristics of plants or plant material are relative to wildtype.
  • the invention provides a method of modifying cell proliferation in a plant which comprises modulating the expression of a gene whose expression or transcription product is capable of directly or indirectly modulating cell proliferation in the plant or plant propagating material, whereby cell proliferation, within the integuments and/or seed coats of the plant, is modified.
  • Cell proliferation in other parts of the plant may also be modified. For example, in the stem of the plant.
  • the present invention provides a method of modifying cell proliferation in a plant which comprises the step of transforming a plant or plant propagating material with a nucleic acid molecule comprising at least one regulatory sequence, typically a promoter sequence, capable of directing expression within the integuments and/or seed coat of at least one nucleic acid sequence whose expression or transcription product is capable of directly or indirectly modulating cell proliferation, whereby, on expression of that sequence, cell proliferation is modified.
  • the overall size of the integuments/seed coat in the plant is modified. This may be useful where a product is produced in the integument/seed coat. In some embodiments, this will be achieved without affecting the growth or development of any part of the plant other than the seed.
  • the function of a gene or gene product that promotes cell division is enhanced or the function of a gene or gene product that represses cell division is inhibited.
  • Cell division in the integuments/seed coat may be increased resulting in a larger seed compared to wild type. This may be advantageous because increases in seed size can be achieved which are desirable as mentioned above.
  • the seed may be at least 15%, or 25%, larger than wild type. More specifically, the seed may be at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, or even 200% heavier than wild-type.
  • the number of cells in the integuments/seed coat of the plant may be increased compared to wild type.
  • the number of cells in the integuments/seed coat of the plant may be increased by at least 30%, or 50%, compared to wild type.
  • the diameter of the stem of the plant may be greater, for example at least 10% greater, than wild type. Preferably, the diameter of the stem of the plant is at least 20% greater than wild type. This may be advantageous as discussed above.
  • the sepal length of the plant may sufficiently greater than wild type to inhibit flower opening.
  • the sepal length may be at least 20%, or at least 50% greater than wild type.
  • the method allows the production of smaller seeds which can also be advantageous as mentioned above, and in another embodiment, the function of a gene or gene product that promotes cell division is inhibited or the function of a gene product that represses cell division is enhanced.
  • Cell division in the integuments/seed coat may decreased resulting in a smaller seed compared to wild type.
  • the seed may be at least 5% smaller than wild type, preferably 25% or more.
  • the number of cells in the integuments/seed cost may be decreased compared to wild type. In particular the number of cells in the integuments/seed coat may be reduced by at least 30%, or 50%, compared to wild type.
  • the function of a gene that modulates cell proliferation may be enhanced compared to wildtype. Transcription of the gene is activated. Activation of transcription results in increased levels of mRNA and/or protein encoded by the gene. Typically, levels of mRNA may be increased by at least 20%. For example, the levels of mRNA may be increased by 50% or 75% or more.
  • a plant promoter may be operably linked to a coding region of the gene in the sense orientation.
  • the function of the gene may be modulated by operably linking a plant promoter to a nucleic acid fragment from the gene to form a recombinant nucleic acid molecule such that an antisense strand of RNA will be transcribed.
  • the function of a gene may be modulated by introducing a nucleic acid fragment of the gene into an appropriate vector such that double-stranded RNA is transcribed where directed by an operably linked plant promoter. Decreased levels of mRNA and/or protein encoded by endogenous copies of the gene may be produced. Levels of mRNA and protein encoded by homologues of the gene may be reduced.
  • the function of the gene may be modulated by operably linking a plant promoter to a ‘dominant negative’ allele of the gene, which interferes with the function of the gene product.
  • the plant may be monocotyledonous, and is preferably a crop plant.
  • the plant may be Tritcum spp (wheat), Oryza sativa (rice), Zea mays (maize), Hordeum spp. (barley), Secale cereale (rye), Sorghum bicolor (sorghum), or Pennisetum glaucum (pearl millet).
  • the plant is dicotyledonous.
  • the plant is Brassica napus (oil seed rape, canola) or any other Brassica species used to produce oilseeds (e.g.
  • Brassica carinata Glycine max (soybean), Arachis hypogaea (peanut), Helianthus annuus (sunflower), Phaseolus vulgaris (e.g. kidney bean, white bean, black bean), Vicia faba (broad bean), Pisum sativum (green pea), Cicer arietinum (chick pea), Lens culinaris (lentil), or Linum usitatissimum (flax, linseed).
  • Integument and seed coat development is similar in all species examined in the family Brassicaceae (Bouman, 1975), to which Arabidopsis thaliana belongs.
  • Brassica napus a crop plant closely related to Arabidopsis thaliana
  • the seed coat is also very similar in structure (Wan et al., 2002). Therefore modifications that affect growth and development of integuments/seed coat in Arabidopsis thaliana should be directly applicable to members of the Brassicaceae, including Brassica napus.
  • the mature seeds of monocots such as cereals have a distinct structure.
  • cereal ovules have fundamental similarities with ovules of Arabidopsis thaliana and other dicots, also consisting of integuments enclosing a nucellus and embryo sac.)
  • the inner integument encloses the ovule before fertilization, and its growth precedes that of the endosperm and embryo, as in Arabidopsis thaliana (Lopez-Dee et al., 1999). Therefore modification to growth of the integuments/seed coat may also be effective in altering overall seed growth in cereal crops.
  • a modification to growth of the inner integuments may be useful in modifying seed size.
  • the INO gene which in Arabidopsis thaliana is expressed in the outer integument and required for its growth (Villanueva et al., 1999), has been identified in Nymphaea alba (water lily), where it is also expressed in the integuments (Yamada et al., 2003).
  • Nymphaeaceae basal eudicots, which are ancestral to both dicots and monocots, this suggests that the sequence and expression patterns of at least some integument genes will be conserved even among distantly related groups of flowering plants.
  • the present invention is complementary to the invention disclosed in WO01/09299. Modifications to endosperm-led and integument-led seed growth could be combined for an even larger effect. In some situations integument-led seed growth alone may be preferable, as it only requires modification to the seed parent, while endosperm growth is determined both by maternal and paternal contributions.
  • the promoter sequence may be constitutive, directing gene expression in most or all cells of the plant.
  • An example of a constitutive promoter that may be used in some embodiments of the invention is the 35S promoter, derived from the gene that encodes the 35S subunit of Cauliflower Mosaic Virus (CaMV) coat protein.
  • the promoter sequence may be specific, directing expression exclusively or primarily in one organ, tissue, or cell type of the plant.
  • a variety of plant promoters can be used in the invention to direct expression exclusively or primarily in the integuments or seed coat. Suitable plant promoters include those obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells, such as Agrobacterium or Rhizobium .
  • promoters expressed in the pre-SUBSTITUTE fertilization integuments include but are not restricted to the promoters of the following genes: INO (Villanueva et al., 1999; At1g23420, accession no. AF195047) and BEL1 (Reiser et al., 1995; At5g41410; accession no. NM — 123506).
  • Other embodiments use promoters expressed in the seed coat after fertilization. These include but are not restricted to the promoters of the following genes: BAN (Devic et al., 1999; At1g61720, accession no.
  • TT1 Sesser et al., 2002
  • TT2 Nesi et al., 2001; At5g35550; accession no. NM — 122946
  • TT8 Nesi et al., 2000; At4g09820, accession no. AJ277509
  • TT12 Debeaujon et al., 2001; At3g59030, accession no. AJ294464
  • TT16 Nesi et al., 2002; At5g23260; accession no. NM — 203094.
  • a flower-preferred promoter that may be used is the promoter of the LFY gene (Weigel et al., 1992; At5g61850, accession no. NM — 125579), which can be used to obtain desired flower-specific effects such as reductions in flower opening.
  • a promoter is to be introduced into a plant, a promoter-containing nucleotide sequence of up to 2000 bp would typically be used.
  • genes known or suspected to be involved in modulating cell proliferation There are a number of genes known or suspected to be involved in modulating cell proliferation, either directly or indirectly.
  • Some embodiments of the invention use genes involved in hormone response, biosynthesis, translocation, or other aspects of hormone action. These include but are not restricted to MNT (described above), IPT1 (Takei et al., 2001; At1g68460, accession no. AB062607), and ARGOS (Hu et al., 2003; At3g59900, accession no. AY305869).
  • Other embodiments use core cell cycle genes (Vandepoele et al., 2002).
  • CYCD3;1 (formerly Cyc ⁇ 3; Soni et al., 1995; Vandepoele et al., 2002; At4g34160, accession no. X83371) and CYCB1;l (formerly Cyc1aAt; Ferreira et al., 1994; Vandepoele et al., 2002; At4g37490, accession no. NM — 119913).
  • Other embodiments use transcription factors involved in regulation of the extent or rate of cell proliferation. These include but are not restricted to ANT (Klucher et al., 1996; At4g37750, accession no. NM — 119937).
  • An expression cassette may be used either to enhance or inhibit the function of a gene that modulates cell proliferation.
  • One method of enhancing function is to activate transcription of the gene, resulting in increased levels of mRNA and protein encoded by the gene. This is achieved by linking a plant promoter to the coding region of the gene (either with or without introns) in the sense orientation.
  • Partial or complete inhibition of gene function in order to achieve desirable characteristics in plants such as fertility may be achieved or “engineered” in several ways.
  • One method, which uses ‘antisense technology’ is to link a plant promoter to a nucleic acid segment from the desired gene such that the antisense strand of RNA will be transcribed (see e.g. Branen et al., 2003; Choi et al., 2003).
  • Another method, which uses ‘RNAi technology’ is to link a plant promoter to a nucleic acid segment from the desired gene and place the resulting recombinant nucleic acid into an appropriate vector such that double-stranded RNA is transcribed (Wang and Waterhouse, 2001).
  • mRNA and protein encoded by the endogenous copies of the gene may result in decreased levels of mRNA and protein encoded by the endogenous copies of the gene.
  • levels of mRNA may be reduced by at least 20%, preferably by at least 50% so as to achieve usefully large seeds without compromising fertility compared to wildtype.
  • a nucleic acid fragment for antisense or RNAi technology may also be designed to decrease levels of mRNA and protein encoded by homologues or orthologues of the gene.
  • a third method of inhibiting gene function is to link a plant promoter to a ‘dominant negative’ allele of the gene, which interferes negatively with the function of the gene product (see e.g. Hemerly et al., 1995; Nahm et al., 2003).
  • RNAi RNAi
  • the inserted polynucleotide sequence need not be identical, but may be only “substantially identical” to a sequence of the gene from which it was derived. Inhibition of gene function may be achieved by reduction of expression of the gene through a feedback loop acting on that expression.
  • the nucleic acid sequence is a mutant form of an auxin response factor encoding gene, or a construct that inhibits expression or function of an auxin response factor.
  • the auxin response factor gene may be MNT in the case of Arabidopsis thaliana or its orthologues in other species.
  • the gene in the case of Brassica napus the gene may be BnARF2 as used in Example 2 below.
  • the gene In the case of rice the gene may be OsARF2.
  • the mnt-1 mutant phenotype shows that the wild-type function of the MNT gene is to repress cell division in the integuments. Therefore inhibition of endogenous MNT expression or function may result in larger integuments and a larger seed.
  • enhancement of MNT expression or function may result in a smaller seed.
  • overexpressing the MNT gene may, in fact, result in a larger seed size possibly due to a feedback loop on the expression of the MNT gene.
  • cell division in the integuments/seed coat will be increased, resulting in a larger seed compared to wild type. This may be achieved by enhancing function of a gene or gene product that promotes cell division, or inhibiting function of a gene or gene product that represses cell division. In other embodiments, cell division in the integuments/seed coat will be decreased, resulting in a smaller seed. This may be achieved by enhancing function of a gene or gene product that represses cell division, or inhibiting function of a gene or gene product that promotes cell division. In these embodiments the gene or gene product may be MNT or an orthologue of MNT; alternatively it may be another gene or gene product that affects cell division.
  • a plant may be further modified to maintain desirable characteristics may have been otherwise lost as a result of the transformation step.
  • the desirable characteristic may be fertility.
  • the plant may be engineered or bred further to maintain or introduce desirable characteristics.
  • the plant may be bred so that it is heterozygous for the modulated gene which directly or indirectly modifies cell proliferation.
  • plants heterozygous for the mnt mutation have normal flowers and normal fertility, but that their seeds that are consistently significantly heavier than wild-type (typically about 10-20%), though not as heavy as seeds from mnt homozygous mutants. In other words, if MNT function is reduced by about 50% rather than abolished completely, the plants produce desirable heavier seeds without compromising fertility.
  • plants may be engineered as described above in order to reduce mRNA/protein levels for the cell proliferation gene.
  • MNT function is restored to petals and stamens of an mnt mutant such that seeds have the enlarged mnt-1 mutant phenotype but fertility is not impaired.
  • This may be achieved by operably linking the promoter of a gene that directs expression in petals and stamens but not carpels (which contain the ovules), such as AP3 (Jack et al., 1992), to the wild-type MNT gene.
  • AP3 which contain the ovules
  • different wild type genes may be supplied.
  • MNT function may be restored to sepals and petals of an mnt mutant such that seeds have the enlarged mnt-1 mutant phenotype but fertility is not impaired.
  • This may be achieved by operably linking the promoter of a gene that directs expression in sepals and petals but not carpels, such as AP1 (Mandel et al., 1992), to the wild-type MNT gene.
  • AP1 Mondel et al., 1992
  • different wild-type genes may be supplied.
  • a plant which includes a nucleic acid molecule comprising at least one regulatory sequence capable of directing expression within the integuments and/or seed coat of at least one nucleic acid sequence whose expression or transcription product is capable of directly or indirectly modulating cell proliferation, whereby, on expression of that sequence, cell proliferation is modified.
  • the plant may have been obtained by a method in accordance with the invention and will have the resulting features in terms of genetic structures and phenotype as set out in any of claims 76 to 156 and/or described above.
  • reproducible or propagatable plant material including a nucleic acid molecule comprising at least one regulatory sequence capable of directing expression within integuments and/or seed coat and at least one nucleic acid sequence whose expression or transcription product is capable of directly or indirectly modulating cell proliferation, whereby on expression of that nucleic acid sequence cell proliferation is modified.
  • a method of modifying cell proliferation in a plant which comprises the step of modulating the response of the plant to an auxin whereby the overall cell number of the integuments/seed coat of the plant is modified.
  • the response to an auxin may be modified by altering the expression of an auxin response factor.
  • the auxin response factor is ARF2.
  • the function of a gene encoding the auxin response factor may be modulated so as to affect the function of the factor.
  • the gene may be MNT.
  • the gene may be BnARF2.
  • the gene may be OsARF2. Orthologues of these genes may be used in other species.
  • an endogenous auxin response factor encoding gene is modulated for example by RNAi technology as described above. Most preferably, the function of that gene in the integuments/seed coat is affected.
  • the seeds may be at least 10% or 20% larger than wild type. This may be achieved by breeding a plant that is heterozygous for a mutation in the gene such as MNT or an orthologue of that gene in other species.
  • the plant has a partial loss-of-function mutation in a gene the function of which affects cell proliferation, such as MNT or an orthologue in other species.
  • the level of the RNA of that gene and protein is reduced in a wild-type plant by 30%, 40%, 50%, or 60%, for example by operably linking a promoter, such as the constitutive 35S promoter, to a nucleic acid fragment from the gene to form a recombinant nucleic acid molecule such that an antisense strand of RNA will be transcribed; or to nucleic acid fragments of the gene in an appropriate vector such that double-stranded RNA is transcribed.
  • a promoter such as the constitutive 35S promoter
  • the present invention provides a method of modifying cell proliferation in a plant which comprises the step of transforming a plant or plant propagating material with a nucleic acid molecule comprising at least one regulatory sequence, typically a promoter sequence, capable of directing expression within the stem of at least one nucleic acid sequence whose expression or transcription product is capable of directly or indirectly modulating cell proliferation, whereby, on expression of that sequence, cell proliferation is modified.
  • a nucleic acid molecule comprising at least one regulatory sequence, typically a promoter sequence, capable of directing expression within the stem of at least one nucleic acid sequence whose expression or transcription product is capable of directly or indirectly modulating cell proliferation, whereby, on expression of that sequence, cell proliferation is modified.
  • the overall size of the stems in the plant is modified.
  • the stem may be at least 10%, 20%, 30%, or 40% greater in diameter than wild-type.
  • MNT function is at least partially inhibited in the stem, for example by operably linking a promoter such as the constitutive 35S promoter to a nucleic acid fragment from the MNT gene or MNT orthologue to form a recombinant nucleic acid molecule such that an antisense strand of RNA will be transcribed; or to nucleic acid fragments of the MNT gene or MNT orthologue in an appropriate vector such that double-stranded RNA is transcribed.
  • a promoter such as the constitutive 35S promoter
  • a nucleic acid fragment from the MNT gene or MNT orthologue to form a recombinant nucleic acid molecule such that an antisense strand of RNA will be transcribed
  • nucleic acid fragments of the MNT gene or MNT orthologue in an appropriate vector such that double-stranded RNA is transcribed.
  • the function of a gene that directly or indirectly modulates cell proliferation such as MNT or an orthologue thereof is restored to flowers of an mnt mutant such that stems have the enlarged mnt-1 mutant phenotype but fertility is not impaired.
  • This may be achieved for example by operably linking the promoter of a gene that directs expression in flowers but not stems, such as LEAFY (LFY) (Weigel et al., 1992; At5g61850, accession no. NM — 125579), to the wild-type MNT gene.
  • LEAFY LEAFY
  • LFY Weigel et al., 1992; At5g61850, accession no. NM — 125579
  • different wild type genes may be supplied.
  • FIGS. 1 to 25 in which:
  • FIG. 1A Top: Confocal micrographs of seeds with globular stage embryos from mnt-1 (left) and wild-type (right) seed parents; Bottom: Mature seeds and embryos from mnt-1 mutants and wild-type plants, photographed at the same scale;
  • FIG. 1B is a scatter plot of number of seeds in each pod produced by mnt-1 mutants vs mean seed weight in that pod, following controlled pollinations;
  • FIG. 1C shows seeds from manually pollinated mnt-1 and wild-type plants, and reciprocal crosses between them, photographed at the same scale;
  • FIG. 2A shows light micrographs of mature unfertilized ovules, stage 3-VI (staging as in Schneitz et al., 1995), from wild-type (left) and mnt-1 (right) plants;
  • FIG. 2B shows graphs showing number of cells, total length, and mean cell length for several integument layers in mnt-1 and wild-type stage 3-VI ovules. (The width is also shown for layer ii1′).
  • FIG. 3 shows micrographs of the chalazal endosperm in developing seeds of Arabidopsis thaliana , all at the same scale.
  • the mnt-1 seed is an example of integument-led growth while the 2x ⁇ 6x seed provides an example of endosperm-led growth;
  • FIG. 4A shows micrographs of Arabidopsis thaliana seeds illustrating endosperm-led seed growth illustrated by interploidy crosses in the C24 accession of Arabidopsis thaliana (see also Scott et al., 1998);
  • FIG. 4B shows micrographs illustrating integument-led seed growth illustrated by the mnt-1 mutant in the Columbia accession of Arabidopsis thaliana
  • FIG. 4C is a micrograph of a seed illustrating the ‘big bag’ hypothesis
  • FIG. 5A-C is a photograph illustrating a comparison of floral phenotype and seed size in wild-type Col-3 ( 5 A), mnt-1 mutants ( 5 B), and a Salk insertion mutant (Salk line 108995) homozygous for an insertion in the ARF2 gene ( 5 C);
  • FIG. 5D is a photograph of a gel showing PCR-based scoring of segregants for the T-DNA insertion in Salk line 108995;
  • FIG. 5E is a photograph of a gel showing scoring of presence of the insertion (top) and presence of homozygotes (bottom) in F1 progeny of the cross between an mnt-1 homozygous mutant seed parent and the Salk 108995 homozygous pollen parent. All F1 progeny have a single copy of the insertion;
  • FIG. 5F is a photograph illustrating floral and seed phenotype in an F1 hybrid plant resulting from a cross between a homozygous mnt-1 mutant and a homozygous Salk insertion mutant (Salk line 108995);
  • FIG. 6 is an alignment of wild-type MNT and mutant mnt-1 cDNAs from translational start to stop;
  • FIG. 7 is an alignment of wild-type MNT and mutant mnt-1 predicted proteins
  • FIG. 8 is an alignment of Arabidopsis thaliana a MNT cDNA with its orthologue in Brassica napus, BnARF 2;
  • FIG. 9 is an alignment of Arabidopsis thaliana MNT predicted protein with its orthologues in Brassica napus (oilseed rape) (BnARF2) and Oryza sativa (rice) (OsARF2);
  • FIG. 10 illustrates the BJ60, BJ40, pFGC5941, pART7, and BJ36 vectors used for the cloning strategies described in the following examples;
  • FIG. 11 illustrates a cloning strategy for constructing reporter vectors (Example 3). In this and following figures, only restriction sites significant to the strategy are shown on the diagrams;
  • FIG. 12 is a micrograph of a globular stage seed from a plant containing the TT12::uidA construct assayed for GUS expression; the inner layer of the inner integument is stained (arrow);
  • FIG. 13A illustrates a cloning strategy for constructing an RNAi vector to constitutively decrease MNT expression (Example 4);
  • FIG. 13B is a series of photographs illustrating inflorescence and stem phenotypes (top) and seed sizes and weights (bottom) from independently transformed lines containing the 35S::MNT RNAi expression cassette compared with a wild-type control. Inflorescences and stems were photographed at the same scale, and seeds were photographed at the same scale;
  • FIG. 14 illustrates a cloning strategy for constructing an RNAi vector to constitutively decrease BnARF2 expression (Example 5);
  • FIG. 15 illustrates a cloning strategy for constructing RNAi vectors to decrease MNT expression primarily in the integuments/seed coat (Example 6);
  • FIG. 16 illustrates a cloning strategy for constructing RNAi vectors to decrease BnARF2 expression primarily in the integuments/seed coat (Example 7);
  • FIG. 17A illustrates a cloning strategy for constructing vectors for constitutive expression of MNT (Example 8) or BnARF2 (Example 9);
  • FIG. 17B is a series of photographs illustrating seed sizes and weights from independently transformed lines containing the 35S::MNT expression cassette compared with a wild-type control. Seeds were photographed at the same scale;
  • FIG. 18 illustrates a cloning strategy for constructing vectors for expression of MNT in the integuments/seed coat (Example 10);
  • FIG. 19 illustrates a cloning strategy for constructing vectors for expression of BnARF2 in the integuments/seed coat (Example 11);
  • FIG. 20 illustrates a cloning strategy for constructing vectors for expression of genes promoting cell division in the integuments/seed coat (Examples 12, 13);
  • FIG. 21A is a series of photographs illustrating seed sizes and weights from individual primary transformants containing expression cassettes designed to increase seed size (TT8::CYCD3;l and TT8::IPT1) compared with controls (TT8::uidA). Data is taken from Table 2A. Seeds were photographed at the same scale;
  • FIG. 21B is a series of photographs illustrating seed sizes and weights from transformed plants containing expression cassettes designed to increase seed size compared with wild-type controls. Data is taken from Table 2B. Seeds were photographed at the same scale;
  • FIG. 22 illustrates a cloning strategy for constructing a vector for expression of MNT in petals and stamens (Example 14);
  • FIG. 23 illustrates a cloning strategy for constructing a vector for expression of MNT in sepals and petals (Example 15);
  • FIG. 24A shows a wild-type Col-3 (left) and mnt-1 (right) plant, illustrating the stem phenotype
  • FIG. 24B shows transverse sections of the inflorescence stem between nodes 2 and 3 as counted from the base of a wildtype from a wild-type (top) and mnt-1 (bottom) plant. Each pair of images (low magnification, left; high magnification, right) was photographed at the same scale; and
  • FIG. 25 illustrates a cloning strategy for constructing a vector for expression of MNT in flowers (Example 18).
  • BJ36, BJ40, BJ60 gifts of Bart Janssen, Horticultural & Food Research Institute of New Zealand
  • Plant transformation protocols are based on Clough and Bent (1998) for Arabidopsis thaliana and Moloney et al. (1989) for Brassica.
  • FIG. 1A shows that mnt-1 mutants produce larger seeds with more cells in the seed coat (counts are for ii1, the outer layer of the inner integument).
  • Seeds collected from self-pollinated mnt-1 mutant plants are up to twice the weight of wild-type Col-3 seeds (Table 1A).
  • mnt-1 mutant plants are self-sterile until late in development due to floral abnormalities (see below), raising the possibility that the mutant produces large seeds because there are few seeds requiring maternal resources. Therefore we also conducted controlled pollinations in which only three siliques (seed pods) were allowed to set seed per plant, for both mnt-1 and wild-type (Table 1B).
  • the mnt-1 mutation has a maternal effect on seed size. That is, an mnt-1 homozygous mutant seed parent yields large seeds regardless of whether it is pollinated by an mnt-1 or wild-type plant, while a wild-type parent yields normal seeds even if pollinated by an mnt-1 plant ( FIG. 1C ).
  • FIG. 1C seeds produced by mnt-1 seed parents are shown on top and seeds from wild-type seed parents are below.
  • H fertilization products (embryo and endosperm) are heterozygous for the mnt-1 mutation. This shows that seed size in mnt-1 mutants depends on the genotype of the seed parent, not the fertilization products.
  • mnt-1 and wild-type seeds contain more cells in the seed coat.
  • Comparison of ovule development in mnt-1 and wild-type plants shows that mnt-1 ovules are of normal size and morphology until they are near maturity, at which time we observe that both the inner and outer integuments of mnt-1 ovules are significantly longer than in wild-type, primarily due to a significantly greater number of cells ( FIG. 2 ).
  • FIG. 2 In relation to the results depicted in FIG. 2 , in Arabidopsis thaliana and other members of the Brassicacea most cell division and expansion occurs in the integuments on the abaxial side of the ovule (marked on wild-type ovule in FIG.
  • ii1, ii1′, and ii2 are the three cell layers of the inner integument and oil and oi2 are the two layers of the outer integument.
  • the cells of layer ii1′ which does not completely span the embryo sac, significantly expand in width after fertilization as part of seed growth (Beeckman et al., 2000).
  • mnt-1 ovules have longer integuments with extra cells and in some cases an extra layer (arrow), as well as a larger seed cavity ( FIG. 2A ).
  • peripheral endosperm in mnt-1 mutant seeds also generates more nuclei than in wild-type seeds.
  • the mean number of peripheral endosperm nuclei in mnt-1 seeds at the heart stage is 1150, compared with 550 for a wild-type Col-3 seed at a comparable stage; see Scott et al. (1998) for a description of endosperm morphology and the counting method.
  • the chalazal region of the endosperm which becomes greatly enlarged in endosperm-led seeds (e.g.
  • the size difference between mnt-1 and wild-type seeds follows from differences existing before fertilization i.e. before the endosperm has been created. The overproliferation of peripheral endosperm may follow from the larger seed volume created by enlarged integuments/seed coat.
  • the seed cavity i.e. the space within the post-fertilization embryo sac
  • the seed cavity is larger than normal, giving the embryo more space to grow ( FIG. 4A , 4 B).
  • endosperm-led seed growth is illustrated by interploidy crosses in the C24 accession of Arabidopsis thaliana (see also Scott et al., 1998).
  • extra paternal genomes produce seeds with a large cavity (top left, 2x ⁇ 6x cross), and ultimately large seeds with large embryos (2x ⁇ 4x cross, bottom left).
  • extra maternal genomes generate seeds with small cavities (top right, 6x ⁇ 2x cross), and ultimately small seeds with small embryos (4x ⁇ 2x cross, bottom right).
  • the control 2x ⁇ 2x cross is shown in the middle.
  • the seeds In contrast in integument-led seed growth as illustrated in FIG. 4B the seeds also have a large seed cavity (top left) compared with wild-type (top right). Mature seeds and embryos are compared below.
  • the mnt-1 mutation affects floral morphology as well as seed size. Most flowers fail to open; this is associated with a deviation from the normal ratio of sepal to petal length, so that the petals are shorter than the sepals. Specifically, mnt mutant sepals are about 60% longer than wild-type. This deviation is mainly due to overgrowth of the sepals caused by extra cell division, although under some conditions the petals also fail to expand normally. This characteristic may be commercially useful in some crop species. A smaller increase in sepal length may be sufficient to prevent flower opening whilst allowing self-fertilization. Additionally, pollen is shed from the anthers on to the sides of the carpel rather than the stigma. This is associated with overgrowth of the gynoecium caused by extra cell division, although under some conditions the stamen filaments do not extend normally.
  • Germination frequency of mnt-1 seeds is normal, and the seedlings are vigorous.
  • mnt-1 mutants have thick inflorescence stems compared with wild-type plants ( FIG. 24).
  • Transverse sections show that cells are of normal size in mnt-1 mutant stems but many more cells are formed.
  • FIG. 5D Genotypic scoring of segregants from the Salk 108995 family, including one heterozygote and the homozygote, is shown in FIG. 5D .
  • FIG. 5D Top: Scoring for presence of an insertion in the ARF2 gene. Primers used were 5′ TGG TTC ACG TAG TGG GCC ATC G 3′, and 5′ GAG TGG GTG GAG TGT GTT TG 3′.
  • Lanes M and O show presence of the insertion. Bottom: Scoring for homozygous insertion mutants. Primers used were 5′ GAG TGG GTG GAG TGT GTT TG 3′ and 5′ AGT TGG TTT TCG TTT GAG CAT 3′. PCR conditions are set so that the gene will only amplify if there is no insertion: therefore PCR products will be amplified from DNA extracted from wild-type plants and also those hemizygous for the insertion, but not homozygous plants. Lane M shows no amplification, indicating this plant is homozygous for the insertion. An allelism test was conducted by crossing a seed parent homozygous for the mnt-1 mutation with the Salk 108995 homozygote as pollen parent. F1 progeny were hemizygous for the insertion ( FIG. 5E ) and had the mnt-1 mutant phenotype ( FIG. 5F ), confirming that MNT is the ARF2 gene.
  • MNT/ARF2 will be referred to as MNT in the remainder of this document.
  • the genomic DNA for MNT including the coding region plus 4371 bases of 5′ and 525 bases of 3′ flanking region, is shown in SEQ ID NO. 1.
  • SEQ ID NO. 2 is the complete cDNA, and SEQ ID NO. 3, the predicted protein.
  • ARFs form part of the system for responding to auxin, a hormone known to be involved in many plant developmental processes including cell division and expansion (Stals and Inzé, 2001; Leyser, 2002).
  • ARFs are transcription factors that in general are not induced by auxin themselves but which regulate expression of auxin-inducible genes, such as members of the Aux/IAA class (Liscum and Reed, 2002).
  • ARFs have been shown to bind to Auxin Response Elements (AREs) containing the motif TGTCTC in the promoters of auxin-inducible genes (Ulmasov et al., 1999a).
  • AREs Auxin Response Elements
  • ARFs predicted to be functional have been annotated in the Arabidopsis thaliana genome (Hagen and Guilfoyle, 2002). ARFs contain two conserved domains—an N-terminal DNA binding domain and a C-terminal dimerization domain—and a variable middle region. An ARF may activate or repress transcription of its targets and this is thought to depend on the sequence of the middle region (Ulmasov et al., 1999b). Evidence so far suggests that ARF2 is likely to be a repressor (Tiwari et al., 2003).
  • genomic DNA of the mnt-1 allele was sequenced with 4371 bases of the 5′ and 525 bases of the 3′ flanking regions (this genomic sequence is shown in SEQ ID NO. 4).
  • the mnt-1 cDNA from translational start to stop consisting of the 837 directly sequenced bases plus the remainder of the cDNA coding region as predicted from the sequenced mnt-1 genomic DNA, is shown in SEQ ID NO. 5. Wild-type MNT and mutant mnt-1 cDNA sequences are aligned in FIG. 6 .
  • the predicted mnt-1 protein (SEQ ID NO. 6) has a frameshift from amino acid position 123 and an early stop codon at position 167. Wild-type MNT and mutant mnt-1 predicted protein sequences are aligned in FIG. 7 . The frameshift and early stop codon are both within the DNA binding domain and therefore the mnt-1 allele is likely to cause a complete loss of MNT function.
  • the mnt mutant seed phenotype demonstrates that there is a correlation between the size of integuments before fertilization and the size of the mature seed in Arabidopsis thaliana ( FIGS. 1 , 2 ). Due to the similarities in seed structure among even distantly related groups of flowering plants, this leads to the expectation that modification to integument/seed coat size in other species, and certainly in members of the Brassicaceae such as Brassica napus , will also result in changes to seed size.
  • TILLING Targeting induced local lesions in genomes
  • chemically mutagenized populations are screened for presence of a point mutation in a nucleic acid sequence of interest; this can be done as a high-throughput procedure and is applicable to many species (Till et al., 2003).
  • TILLING could be applied to the Brassica napus or rice orthologues of MNT in order to modify seed size in these crop species.
  • BnARF2 putative Brassica napus orthologue
  • SEQ ID NO 7, 8 primers (SEQ ID NO 7, 8) based on the MNT sequence and on publicly available Brassica oleracea sequence.
  • the BnARF2 cDNA was amplified from total RNA isolated from seedlings of Brassica napus var. Westar .
  • the BnARF2 cDNA from translational start to stop is shown in SEQ ID NO. 9 and is aligned with Arabidopsis thaliana MNT cDNA in FIG. 8 .
  • the BnARF2 predicted protein (SEQ ID NO. 10) has 85% identity to Arabidopsis thaliana MNT.
  • FIG. 9 shows an alignment of the predicted protein sequences of MNT ( Arabidopsis thaliana ARF2), BnARF2, and OsARF2.
  • Orthologues of MMT may be determined for other species using similar techniques.
  • promoters are suitable for driving integument/seed coat-specific or -preferred expression of nucleic acids such as MNT antisense or RNAi constructs, or other genes modifying cell proliferation.
  • FIG. 10 Diagrams of the BJ60, BJ40, pFGC5941, pART7, and BJ36 vectors used in the cloning strategies described in this and following examples are shown in FIG. 10 .
  • the cloning strategy is shown in FIG. 11 .
  • a reporter vector based on the promoter of the TT8 gene (Nesi et al., 2000; At4g09820, accession no. AJ277509) is constructed as described below.
  • a 1.7 kb fragment including the TT8 promoter is amplified by the polymerase chain reaction (PCR) from Arabidopsis thaliana genomic DNA 5′ to translational start of the TT8 gene using the primers TT8F and TT8R which introduce an NdeI and a PstI site at the 5′ and 3′ ends of the TT8 PCR fragment respectively.
  • PCR polymerase chain reaction
  • the TT8 PCR fragment is A-tailed and ligated into pGEMT, then excised with NdeI and PstI and ligated into the NdeI and PstI sites of BJ60, 5′ to the uidA reporter which includes a terminator signal, forming the vector TT8-BJ60.
  • a reporter vector based on the promoter of the TT12 gene (Debeaujon et al., 2000; At3g59030, accession no. AJ294464) is constructed as described below.
  • a 1.7 kb fragment including the TT12 promoter is amplified by PCR from Arabidopsis thaliana genomic DNA 5′ to translational start of the TT12 gene using the primers TT12F and TT12R which introduce an NdeI and a PstI site at the 5′ and 3′ ends of the TT12 PCR fragment respectively.
  • the TT12 PCR fragment is A-tailed and ligated into pGEMT, and then excised with NdeI and PstI and ligated into the NdeI and PstI sites of BJ60, 5′ to the uidA reporter gene forming TT12-BJ60.
  • Reporter cassettes are excised with NotI from the following vectors:
  • the binary vectors are transformed into Agrobacterium tumefaciens and then into Arabidopsis thaliana.
  • the uidA gene encodes ⁇ -glucuronidase (GUS), which is assayed using standard protocols (e.g. Jefferson, 1987).
  • GUS ⁇ -glucuronidase
  • FIG. 12 the following assay was used. Seeds were dissected from siliques into GUS staining buffer (100 mM Tris-HCl pH 7.2, 50 mM NaCl, 0.1% Triton-X-100, 2 mM 5-bromo-4-chloro-3-indolyl-beta-D-glucoronic acid (X-Gluc), 2 mM K 3 Fe(CN 6 ), 2 mM K 4 Fe(CN) 6 ) and incubated overnight at 37° C.
  • GUS staining buffer 100 mM Tris-HCl pH 7.2, 50 mM NaCl, 0.1% Triton-X-100, 2 mM 5-bromo-4-chloro-3-indolyl-beta-D-glucoronic acid
  • FIG. 12 shows a globular stage seed from a plant containing the TT12::uidA construct assayed for GUS expression; the inner layer of the inner integument is stained (arrow), indicating activity of the TT12 promoter fragment in that integument.
  • RNAi Cassette that Decreases MNT Expression In Arabidopsis thaliana , Including Decreased Expression in the Integuments/Seed Coat
  • the cloning strategy is shown in FIG. 13A .
  • RNAi vector based on the MNT gene is constructed as described below.
  • a 0.57 kb fragment of the MNT cDNA (‘MNTi’) is amplified by PCR from Arabidopsis thaliana cDNA using the primers FARF2 i and RARF2inew which introduce XbaI and AscI sites at the 5′ end of the MNTi PCR fragment, and BamHI and SwaI sites at the 3′ end of the PCR fragment.
  • the MNTi PCR fragment is A-tailed and ligated into pGEMT, and then excised with AscI and SwaI and ligated into the AscI and Swal sites of the pFGC5941 RNAi vector 3′ to the 35S promoter and 5′ to the CHSA intron, which places the fragment in forward orientation. This forms the vector 35S-MNTi-pFGC5941.
  • the MNTi PCR fragment is then excised from pGEMT with BamHI and XbaI and ligated into the BamHI and XbaI sites of the 35S-MNTi-pFGC5941 vector, 3′ to the CHSA intron and 5′ to the ocs terminator signal, which places the fragment in inverse orientation. This forms the vector 35S-MNTi-inv MNTi-pFGC5941.
  • Vector 35S-MNTi-inv MNTi-pFGC5941 is transformed into Agrobacterium tumefaciens and then into Arabidopsis thaliana.
  • Wild-type plants transformed with the 35S::MNT RNAi vector described in Example 4a, b have the mnt mutant phenotype, including closed flowers for most of the plant's life cycle ( FIG. 13B top left), inflorescence stems with increased diameter ( FIG. 13B top right), and large seeds ( FIG. 13B , bottom). Seeds from four independently transformed lines, along with wild-type plants grown under the same conditions, are shown in FIG. 13B (bottom). The mean weight for these four lines was 35.3 ⁇ g, compared with 13.8 ⁇ g for the wild-type control.
  • the cloning strategy is shown in FIG. 14 .
  • RNAi vector based on the B ARF2 gene (Example 2, above) is constructed as described below.
  • a 0.56 kb fragment of the BnARF2 cDNA (BnARF21) is amplified by PCR from Brassica napus cDNA using the primers FBnARF2 i and RBnARF2 i which introduce XbaI and AscI sites at the 5′ end of the BnARF2 i PCR fragment, and BamHI and SwaI sites at the 3′ end of the PCR fragment.
  • the BnARF21 PCR fragment is A-tailed and ligated into pGEMT and then excised with AscI and SwaI and ligated into the AscI and SwaI sites of the pFGC5941 RNAi vector 3′ to the 35S promoter and 5′ to the CHSA intron using the enzymes Ascl and SwaI, which places the fragment in forward orientation. This forms the vector 35S-BnARF21-pFGC5941.
  • the BnARF21 PCR fragment is then excised from pGEMT with BamHI and XbaI and ligated into the BamHI and XbaI sites of the 35S-BnARF21-pFGC5941 vector 3′ to the CHSA intron and 5′ to the ocs terminator, which places the fragment in inverse orientation. This forms the vector 35S-BnARF21-inv BnARF21-pFGC5941.
  • Vector 35S-BnARF21-inv BnARF21-pFGC5941 is transformed into Agrobacterium tumefaciens and then into Brassica napus.
  • the cloning strategy is shown in FIG. 15 .
  • RNAi vector in which the TT8 promoter (Nesi et al., 2000; At4g09820, accession no. AJ277509) drives an inverted repeat of an MNT nucleic acid fragment (see Example 4, above) is constructed as described below.
  • a 1.7 kb fragment including the TT8 promoter is amplified by PCR from Arabidopsis thaliana genomic DNA 5′ to translational start of the TT8 gene using the primers TT8 EcoRI F and TT8 NcoI R which introduce an EcoRI and an NcoI site at the 5′ and 3′ ends of the TT8 PCR fragment respectively.
  • the TT8 PCR fragment is A-tailed and ligated into pGEMT, and then excised with EcoRI and NcoI and exchanged for the 35S promoter in the vector 35S-MNT-inv M1MNTi-pFGC5941 (Example 4, above), forming the vector TT8-MNT-inv MNTi-pFGC5941.
  • RNAi vector in which the INO promoter (Villanueva et al., 1999; At1g23420, accession no. AF195047) drives an inverted repeat of an MNT nucleic acid fragment (see Example 4, above) is constructed as described below.
  • a 1.5 kb fragment including the INO promoter is amplified by PCR from Arabidopsis thaliana genomic DNA 5′ to translational start of the INO gene using the primers FINOi and RINOi_which introduce an EcoRI and an NcoI site at the 5′ and 3′ ends of the INO PCR fragment respectively.
  • the INO PCR fragment is A-tailed and ligated into pGEMT, and then excised with EcoRI and NcoI and exchanged for the 35S promoter in the vector 35S-MNT-inv MNTi-pFGC5941 (Example 4, above), forming the vector INO-MNT-inv MNTi-pFGC5941.
  • the TT8-MNT-inv MNTi-pFGC5941 and INO-MNT-inv MNTi-pFGC5941 vectors are transformed into Agrobacterium tumefaciens and then into Arabidopsis thaliana.
  • the cloning strategy is shown in FIG. 16 .
  • RNAi vector in which the TT8 promoter (Nesi et al., 2000; At4g09820, accession no. AJ277509) drives an inverted repeat of a BnARF2 nucleic acid fragment (see Example 5, above) is constructed as described below.
  • a 1.7 kb fragment including the TT8 promoter with EcoRI and NcoI linkers is amplified by PCR from Arabidopsis thaliana genomic DNA as described in Example 6a(i) above.
  • the TT8 PCR fragment is A-tailed and ligated into pGEMT, and then excised with EcoRI and NcoI and exchanged for the 35S promoter in the vector 35S-BnARF2-inv BnARF21-pFGC5941 (Example 5, above), forming the vector TT8-BnARF2-inv BnARF21-pFGC5941.
  • RNAi vector in which the INO promoter (Villanueva et al., 1999; At1g23420, accession no. AF195047) drives an inverted repeat of a BnARF2 nucleic acid fragment (see Example 5, above) is constructed as described below.
  • a 1.5 kb fragment including the INO promoter with EcoRI and NcoI linkers is amplified by PCR from Arabidopsis thaliana genomic DNA as described in Example 6a(ii) above.
  • the INO PCR fragment is A-tailed and ligated into pGEMT, and then excised with EcoRI and NcoI and exchanged for the 35S promoter in the vector 35S-BnARF2-inv BnARF21-pFGC5941 (Example 5, above), forming the vector INO-BnARF2-inv BnARF21-pFGC5941.
  • the TT8-BnARF2-inv BnARF21-pFGC5941 and INO-BnARF2-inv BnARF21-pFGC5941 vectors are transformed into Agrobacterium tumefaciens and then into Brassica napus.
  • the cloning strategy is shown in FIG. 17 .
  • the MNT cDNA including the translational start and stop is amplified by PCR from Arabidopsis thaliana cDNA using the primers 35S Xho new and 35S Bam new which introduce a XhoI and a BamHI site at the 5′ and 3′ ends of the A4NT PCR fragment respectively.
  • the MNT PCR fragment is A-tailed and ligated into pGEMT, and then excised with XhoI and BamHI and ligated into the XhoI and BamHI sites of pART7, 3′ to the 35S promoter and 5′ to the ocs terminator, forming the vector 35S-MNT-pART7.
  • the 35 S::MNT expression cassette (including the ocs terminator signal) is excised from 35S-MNT-pART7 with NotI and ligated into the NotI sites of the binary vector BJ40, forming the vector 35S-MNT-BJ40.
  • the binary vector is transformed into Agrobacterium tumefaciens and then into Arabidopsis thaliana.
  • Wild-type plants transformed with the 35S::MNT cassette described in Example 8a, b have the mnt mutant phenotype, including closed flowers for most of the plant's life cycle ( FIG. 17B , top), and large seeds. Seeds from three independently transformed lines, along with wild-type plants grown under the same conditions, are shown in FIG. 17B , middle. The overall mean weight for these three lines was 25.5 ⁇ g, compared with 15.0 ⁇ g for the wild-type control. Expression of MNT/ARF2 was assayed in transformed and wild-type plants by semiquantitative RT-PCR ( FIG.
  • the cloning strategy is shown in FIG. 17 .
  • BnARF2 cDNA from translational start to stop is amplified by PCR from Brassica napus cDNA using the primers BnARF2 XhoI F and BnARF2 BamHI R which introduce a XhoI and a BamHI site at the 5′ and 3′ ends of the BnARF2 PCR fragment respectively.
  • the BnARF2 PCR fragment is A-tailed and ligated into pGEMT, and then excised with XhoI and BamHI and ligated into the XhoI and BamHI sites of pART7, 3′ to the 35S promoter and 5′ to the ocs terminator, forming the vector 35S-BnARF2-pART7.
  • the 35S::BnARF2 expression cassette (including the ocs terminator signal) is excised from 35S-BnARF2-pART7 with NotI and cloned into the NotI sites of the binary vector BJ40, forming the vector 35S-BnARF2-BJ40.
  • the binary vector is transformed into Agrobacterium tumefaciens and then into Brassica napus .
  • Constitutive expression of the BnARF2 gene (such as achieved under control of the 35S promoter) provides a further method for producing large seeds.
  • the cloning strategy is shown in FIG. 18 .
  • An expression vector based on the TT8 promoter (Nesi et al., 2000; At4g09820, accession no. AJ277509) is constructed as described below.
  • a 1.7 kb fragment including the TT8 promoter with NdeI and PstI linkers is amplified by PCR from Arabidopsis thaliana genomic DNA as described in Example 3a(i), above.
  • the TT8 PCR fragment is A-tailed and ligated into pGEMT, and then excised with NdeI and PstI and ligated into the NdeI and PstI sites of BJ36, 5′ to the ocs terminator signal, forming the vector TT8-BJ36.
  • An expression vector based on the promoter of the INO gene (Villanueva et al., 1999; At1g23420, accession no. AF195047) is constructed as described below.
  • a 1.5 kb fragment including the INO promoter is amplified by PCR from Arabidopsis thaliana genomic DNA 5′ to translational start of the INO gene using the primers INOF and INOR which introduce an NdeI and a PstI site at the 5′ and 3′ ends of the INO PCR fragment respectively.
  • the INO PCR fragment is A-tailed and ligated into pGEMT, and then excised with NdeI and PstI and ligated into the NdeI and PstI sites of BJ36, 5′ to the ocs terminator signal, forming the vector INO-BJ36.
  • the MNT cDNA with XhoI and BamHI linkers is amplified by PCR from Arabidopsis thaliana cDNA and ligated into pGEMT as described in Example 8a, above.
  • the MNT PCR fragment is excised from pGEMT with XhoI and BamHI and ligated into the XhoI and BamHI sites of the TT8-BJ36 vector, 3′ to the TT8 promoter, forming the vector TT8-MNT-BJ36.
  • the MNT PCR fragment is excised from pGEMT with XhoI and BamHI and ligated into the XhoI and BamHI sites of the INO-BJ36 vector, 3′ to the INO promoter, forming the vector INO-MNT-BJ36.
  • the TT8::MNT expression cassette (including the ocs terminator signal) is excised from TT8-MNT-BJ36 with NotI and cloned into the NotI sites of the binary vector BJ40, forming the vector TT8-MNT-BJ40.
  • the INO::MNT expression cassette (including the ocs terminator signal) is excised from INO-MNT-BJ36 with NotI and cloned into the NotI sites of the binary vector BJ40, forming the vector INO-MNT-BJ40.
  • the TT8-MNT-BJ40 and INO-MNT-BJ40 binary vectors are transformed into Agrobacterium tumefaciens and then into Arabidopsis thaliana.
  • the cloning strategy is shown in FIG. 19 .
  • An expression vector based on the promoter of the TT8 gene (Nesi et al., 2000; At4g09820, accession no. AJ277509) is constructed as described below.
  • a 1.7 kb fragment including the TT8 promoter is amplified by PCR from Arabidopsis thaliana genomic DNA 5′ to translational start of the TT8 gene using the primers TT8F and TT8 MluI R which introduce an NdeI and an MluI site at the 5′ and 3′ ends of the TT8 PCR fragment respectively.
  • the TT8 PCR fragment is A-tailed and ligated into pGEMT, and then excised with NdeI and MluI and ligated into the NdeI and MluI sites of BJ36, 5′ to the ocs terminator signal, forming the vector TT8 (NdeI MluI)-BJ36.
  • An expression vector based on the promoter of the INO gene (Villanueva et al., 1999; At1g23420, accession no. AF195047) is constructed as described below.
  • a 1.5 kb fragment including the INO promoter is amplified by PCR from Arabidopsis thaliana genomic DNA 5′ to translational start of the INO gene using the primers INOF and INO MluI R which introduce an NdeI and an MluI site at the 5′ and 3′ ends of the INO PCR fragment respectively.
  • the INO PCR fragment is A-tailed and ligated into pGEMT, and then excised with NdeI and MluI and ligated into the NdeI and MluI sites of BJ36, 5′ to the ocs terminator signal, forming the vector INO (NdeI MluI)-BJ36.
  • the BnARF2 cDNA with XhoI and BamHI linkers is amplified by PCR from Brassica napus cDNA and ligated into pGEMT as described in Example 9a, above.
  • the BnARF2 PCR fragment is excised from pGEMT with XhoI and BamHI and ligated into the XhoI and BamHI sites of the TT8 (NdeI MluI)-BJ36 vector, 3′ to the TT8 promoter, forming the vector TT8-BnARF2-BJ36.
  • the BnARF2 PCR fragment is excised from pGEMT with XhoI and BamHI and ligated into the XhoI and BamHI sites of the INO (NdeI MluI)-BJ36 vector, 3′ to the INO promoter, forming the vector INO-BnARF2-BJ36.
  • the TT8-BnARF2 expression cassette (including the ocs terminator signal) is excised from TT8-BnARF2-BJ36 with NotI and ligated into the NotI sites of the binary vector BJ40, forming the vector TT8-BnARF2-BJ40.
  • the INO-BnARF2 expression cassette (including the ocs terminator signal) is excised from INO-BnARF2-BJ36 with NotI and ligated into the NotI sites of the binary vector BJ40, forming the vector INO-BnARF2-BJ40.
  • the binary vectors TT8-BnARF2-BJ40 and INO-BnARF2-BJ40 are transformed into Agrobacterium tumefaciens and then into Brassica napus.
  • the cloning strategy is shown in FIG. 20 .
  • An expression vector based on the TT8 promoter (Nesi et al., 2000; At4g09820, accession no. AJ277509) is constructed as described below.
  • a 1.7 kb fragment including the TT8 promoter with NdeI and PstI linkers is amplified by PCR from Arabidopsis thaliana genomic DNA as described in Example 3a(i), above.
  • the TT8 PCR fragment is A-tailed and ligated into pGEMT, and then excised with NdeI and PstI and ligated into the NdeI and PstI sites of BJ36, 5′ to the ocs terminator signal, forming the vector TT8-BJ36.
  • An expression vector based on the TT12 promoter (Debeaujon et al., 2000; At3g59030, accession no. AJ294464) is constructed as described below.
  • a 1.7 kb fragment including the TT12 promoter with NdeI and PstI linkers is amplified by PCR from Arabidopsis thaliana genomic DNA as described in Example 3a(ii), above.
  • the TT12 PCR fragment is A-tailed and ligated into pGEMT, and then excised with NdeI and PstI and ligated into the NdeI and PstI sites of BJ36, 5′ to the ocs terminator signal, forming the vector TT12-BJ36.
  • An expression vector based on the INO promoter (Villanueva et al., 1999; At1g23420, accession no. AF195047) is constructed as described below.
  • a 1.5 kb fragment including the INO promoter with NdeI and PstI linkers is amplified by PCR from Arabidopsis thaliana genomic DNA as described in Example 10a(ii), above.
  • the INO PCR fragment is A-tailed and ligated into pGEMT, and then excised with NdeI and PstI and ligated into the NdeI and PstI sites of BJ36, 5′ to the ocs terminator signal, forming the vector INO-BJ36.
  • An expression vector based on the promoter of the BAN gene (Devic et al., 1999; At1g61720, accession no. AF092912) is constructed as described below.
  • a 0.4 kb fragment including the BAN promoter is amplified by PCR from Arabidopsis thaliana genomic DNA 5′ to translational start of the BAN gene using the primers BANF and BANR which introduce an NdeI and a PstI site at the 5′ and 3′ ends of the BAN PCR fragment respectively.
  • the BAN PCR fragment is A-tailed and ligated into pGEMT, and then excised with NdeI and PstI and ligated into the NdeI and PstI sites of BJ36, 5′ to the ocs terminator signal, forming the vector BAN-BJ36.
  • the CYCD3;1 cDNA (formerly Cyc ⁇ 3; Soni et al., 1995; Vandepoele et al., 2002; At4g34160, accession no. X83371) is amplified by PCR from Arabidopsis thaliana cDNA using the primers CYCD3F and CYCD3R which introduce a SmaI and a BamHI site at the 5′ and 3′ ends of the CYCD3;1 PCR fragment respectively.
  • the CYCD3;1 PCR fragment is A-tailed and ligated into pGEMT, and then excised with SmaI and BamHI and ligated into the SmaI and BamHI sites of the following vectors:
  • TT8-BJ36 vector 3′ to the TT8 promoter and 5′ to the ocs terminator signal, forming the vector TT8-CYCD3;1-BJ36
  • TT12-BJ36 vector 3′ to the TT12 promoter and 5′ to the ocs terminator signal, forming the vector TT12-CYCD3;1-BJ36
  • INO-BJ36 vector 3′ to the INO promoter and 5′ to the ocs terminator signal, forming the vector INO-CYCD3;1-BJ36
  • BAN-BJ36 vector 3′ to the BAN promoter and 5′ to the ocs terminator signal, forming the vector BAN-CYCD3;1-BJ36
  • the IPT1 gene (Takei et al., 2001; At1g68460, accession no. AB062607) is amplified by PCR from Arabidopsis thaliana genomic DNA (the IPT1 gene contains no introns) using the primers IPT1F and IPT1R which introduce a SmaI and a BamHI site at the 5′ and 3′ ends of the IPT1 PCR fragment respectively.
  • the IPT1PCR fragment is A-tailed and ligated into pGEMT, and then excised with SmaI and BamHI and ligated into the SmaI and BamHI sites of the following vectors:
  • TT8-BJ36 vector 3′ to the TT8 promoter and 5′ to the ocs terminator signal, forming the vector TT8-IPT1-BJ36
  • TT12-BJ36 vector 3′ to the TT12 promoter and 5′ to the ocs terminator signal, forming the vector TT12-IPT1-BJ36
  • INO-BJ36 vector 3′ to the INO promoter and 5′ to the ocs terminator signal, forming the vector INO-IPT 1-BJ36
  • BAN-BJ36 vector 3′ to the BAN promoter and 5′ to the ocs terminator signal, forming the vector BAN-IPT1-BJ36
  • the ANT gene (Klucher et al., 1996; At4g37750, accession no. NM — 119937) is amplified by PCR from Arabidopsis thaliana cDNA using the primers ANTF and ANTR which introduce a SmaI and a BamHI site at the 5′ and 3′ ends of the ANT PCR fragment respectively.
  • the ANT PCR fragment is A-tailed and ligated into pGEMT, and then excised with SmaI and BamHI and ligated into the SmaI and BamHI sites of the following vectors:
  • TT8-BJ36 vector 3′ to the TT8 promoter and 5′ to the ocs terminator signal, forming the vector TT8-ANT-BJ36
  • TT12-BJ36 vector 3′ to the TT12 promoter and 5′ to the ocs terminator signal, forming the vector TT12-ANT-BJ36
  • INO-BJ36 vector 3′ to the INO promoter and 5′ to the ocs terminator signal, forming the vector INO-ANT-BJ36
  • BAN-BJ36 vector 3′ to the BAN promoter and 5′ to the ocs terminator signal, forming the vector BAN-ANT-BJ36
  • the CYCB1;1 gene (formerly Cyc1aAt; Ferreira et al., 1994; Vandepoele et al., 2002; At4g37490, accession no. NM — 119913) is amplified by PCR from Arabidopsis thaliana cDNA using the primers CYCB1F and CYCB1R which introduce a SmaI and a BamHI site at the 5′ and 3′ ends of the CYCB1;1 PCR fragment respectively.
  • the CYCB1;1 PCR fragment is A-tailed and ligated into pGEMT, and there excised with SmaI and BamHI and ligated into the SmaI and BamHI sites of the following vectors:
  • TT8-BJ36 vector 3′ to the TT8 promoter and 5′ to the ocs terminator signal, forming the vector TT8-CYCB1;1-BJ36
  • TT12-BJ36 vector 3′ to the TT12 promoter and 5′ to the ocs terminator signal, forming the vector TT12-CYCB1;1-BJ36
  • INO-BJ36 vector 3′ to the INO promoter and 5′ to the ocs terminator signal, forming the vector INO-CYCB1;1-BJ36
  • BAN-BJ36 vector 3′ to the BAN promoter and 5′ to the ocs terminator signal, forming the vector BAN-CYCB1;1-BJ36
  • Expression cassettes (including the ocs terminator) are excised with NotI from the following vectors
  • the binary vectors are transformed into Agrobacterium tumefaciens and then into Arabidopsis thaliana.
  • Results from some primary transformants using the TT8 promoter are shown in Table 2A and FIG. 21A .
  • the histogram shows that seeds from TT8::CYCD3;1 and TT8::IPT1 plants have a broader distribution and higher peak of weights than the controls.
  • TT8::uidA lines were used as controls, as expression of the uidA gene is not found to affect plant growth and development.
  • Individual TT8::CYCD3;1 plants produced seeds up to 97% heavier than controls, with a mean increase over 27 lines of 37%.
  • TT8::IPT1 plants produced seeds up to 107% heavier, with a mean increase over 24 lines of 28%.
  • TT8::CYCD3;1 and TT8::IPT1 seeds were compared with the controls using t-tests and found to be significantly different from the controls with P ⁇ 0.000. It should be noted that some of the TT8::IPT1 lines, including the highest weighing line, also had a vegetative phenotype including dwarfing, serrated leaves, and extremely low fertility, most likely due to the TT8 promoter driving vegetative expression of IPT1 in some lines. However lines with normal vegetative development also produced large seeds. It is likely that vegetative expression of TT8 could be prevented if required by the technique of promoter dissection (e.g. Chandrasekharan et al., 2003).
  • Table 2B and FIG. 21B Further results from plants transformed with expression vectors to increase seed size are shown in Table 2B and FIG. 21B .
  • For two of the lines below we weighed seeds produced by T3 plants, confirming the heritability of the large seed trait.
  • weights of controls in this case the controls were wild-type Col-0 are shown alongside transformants where the controls and transformants were grown together.
  • BAN::CYCD3;1 seeds were 35% heavier than controls grown under the same conditions, and INO::ANT seeds were 53% heavier.
  • INO::ANT seeds were also misshapen ( FIG. 21B ), suggesting that the expression cassette indeed affects seed coat development.
  • Example 12c The binary vectors described in Example 12c (above) are transformed into Brassica napus.
  • the cloning strategy is shown in FIG. 22 .
  • An expression vector based on the promoter of the AP3 gene (Jack et al., 1992; At3g54340, accession no. AY142590) is constructed as described below.
  • a 1 kb fragment including the AP3 promoter is amplified by PCR from Arabidopsis thaliana genomic DNA 5′ to translational start of the AP3 gene using the primers AP3F and AP3R which introduce an NdeI and a PstI site at the 5′ and 3′ ends of the AP3 PCR fragment respectively.
  • the AP3 PCR fragment is A-tailed and ligated into pGEMT, and then excised with NdeI and PstI and ligated into the NdeI and PstI sites of the BJ36 vector, 5′ to the ocs terminator signal, forming the vector AP3-BJ36.
  • the MNT cDNA with XhoI and BamHI linkers is amplified by PCR from Arabidopsis thaliana cDNA and ligated into pGEMT as described in Example 8a, above.
  • the M1T PCR fragment is excised with XhoI and BamHI and ligated into the XhoI and BamHI sites of the AP3-BJ36 vector, 3′ to the AP3 promoter and 5′ to the ocs terminator, forming the vector AP3-MNT-BJ36.
  • the AP3::MNT expression cassette (including the ocs terminator signal) is excised from AP3-MNT-BJ36 with NotI and cloned into the NotI sites of the binary vector BJ40, forming the vector AP3-MNT-BJ40.
  • the binary vector is transformed into Agrobacterium tumefaciens and then into Arabidopsis thaliana.
  • the cloning strategy is shown in FIG. 23 .
  • An expression vector based on the promoter of the AP1 gene (Mandel et al., 1992; At1g69120, accession no. NM — 105581) is constructed as described below.
  • a 1.7 kb fragment including the AP1 promoter is amplified by PCR from Arabidopsis thaliana genomic DNA 5′ to translational start of the AP1 gene using the primers AP1F and AP1R which introduce an NdeI and a PstI site at the 5′ and 3′ ends of the AP3 PCR fragment respectively.
  • the AP1 PCR fragment is A-tailed and ligated into pGEMT, and then excised with NdeI and PstI and ligated into the NdeI and PstI sites of the BJ36 vector, 5′ to the ocs terminator signal, forming the vector AP1-BJ36.
  • the MNT cDNA with XhoI and BamHI linkers is amplified by PCR from Arabidopsis thaliana cDNA and ligated into pGEMT as described in Example 8a, above.
  • the MNT PCR fragment is excised with XhoI and BamHI and ligated into the XhoI and BamHI sites of the AP1-BJ36 vector, 3′ to the AP1 promoter and 5′ to the ocs terminator, forming the vector AP1-MNT-BJ36.
  • the AP1::MNT expression cassette (including the ocs terminator signal) is excised from AP1-MNT-BJ36 with NotI and cloned into the NotI sites of the binary vector BJ40, forming the vector AP1-MNT-BJ40.
  • the binary vector is transformed into Agrobacterium tumefaciens and then into Arabidopsis thaliana.
  • mnt-1 mutants have thick inflorescence stems compared with wild-type plants.
  • the increased diameter of mnt-1 stems is caused by extra cell divisions ( FIG. 24B ). Therefore it is expected that stem thickness may be increased in other species by altering expression of an MNT orthologue and thereby increasing the number of cells in the stem.
  • the cloning and transformation strategy is described in Example 4.
  • the cloning strategy is shown in FIG. 13A .
  • the stem phenotype of transformed plants compared with wild-type plants is shown in FIG. 13B .
  • the cloning strategy is shown in FIG. 25 .
  • An expression vector based on the promoter of the LFY gene (Weigel et al., 1992; At5g61850, accession no. NM — 125579) is constructed as described below.
  • a 2.1 kb fragment including the LFY promoter is amplified by PCR from Arabidopsis thaliana genomic DNA 5′ to translational start of the LFY gene using the primers LFYF and LFYR which introduce an NdeI and a PstI site at the 5′ and 3′ ends of the AP3 PCR fragment respectively.
  • the LFY PCR fragment is A-tailed and ligated into pGEMT, and then excised with NdeI and PstI and ligated into the NdeI and PstI sites of the BJ36 vector, 5′ to the ocs terminator signal, forming the vector LFY-BJ36.
  • the MNT cDNA with XhoI and BamHI linkers is amplified by PCR from Arabidopsis thaliana cDNA and ligated into pGEMT as described in Example 8a, above.
  • the MNT PCR fragment is excised with XhoI and BamHI and ligated into the XhoI and BamHI sites of the LFY-BJ36 vector, 3′ to the LFY promoter and 5′ to the ocs terminator, forming the vector AP1-LFY-BJ36.
  • the LFY::MNT expression cassette (including the ocs terminator signal) is excised from AP1-LFY-BJ36 with NotI and cloned into the NotI sites of the binary vector BJ40, forming the vector AP1-LFY-BJ40.
  • the binary vector is transformed into Agrobacterium tumefaciens and then into Arabidopsis thaliana.

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EP1723244A2 (fr) 2006-11-22
EP1723244B1 (fr) 2012-05-30
ES2389934T3 (es) 2012-11-05
AU2011201954B2 (en) 2014-10-02
AU2005219644A1 (en) 2005-09-15
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CA2558084C (fr) 2019-11-26
US9534228B2 (en) 2017-01-03
WO2005085453A3 (fr) 2006-04-20

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