WO2007028979A1 - Plant transformation - Google Patents

Plant transformation Download PDF

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Publication number
WO2007028979A1
WO2007028979A1 PCT/GB2006/003288 GB2006003288W WO2007028979A1 WO 2007028979 A1 WO2007028979 A1 WO 2007028979A1 GB 2006003288 W GB2006003288 W GB 2006003288W WO 2007028979 A1 WO2007028979 A1 WO 2007028979A1
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Prior art keywords
plant
transgenic
hirsuta
cardamine
polynucleotide
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PCT/GB2006/003288
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French (fr)
Inventor
Miltos Tsiantis
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Isis Innovation Limited
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Priority claimed from GB0518058A external-priority patent/GB0518058D0/en
Priority claimed from GB0611563A external-priority patent/GB0611563D0/en
Application filed by Isis Innovation Limited filed Critical Isis Innovation Limited
Publication of WO2007028979A1 publication Critical patent/WO2007028979A1/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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation

Definitions

  • This invention relates to a model plant.
  • the model plant can be used to reveal new aspects of gene function and in the validation of genes intended for use in crop modification.
  • Arabidopsis th ⁇ liana has been widely used as a model organism for carrying out plant molecular genetics. Important reasons for the choice of Arabidopsis as a model plant include the ease with which it can be grown indoors in large numbers and the fact that it produces thousands of offspring per plant after 8 to 10 weeks.
  • Arabidopsis also has the advantage for molecular analysis of having a genome which is one of the smallest known (7 x 10 7 nucleotide pairs) and which has been sequenced in its entirety. Furthermore, cell culture and genetic transformation methods have been established and large numbers of interesting mutants have been isolated. Like many flowering plants, Arabidopsis can reproduce as a hermaphrodite because a single flower produces both eggs and the pollen that can fertilize them. Therefore, when a flower that is heterozygous for a recessive lethal mutation is self-fertilized, one-fourth of its seeds will display the homozygous embryonic phenotype.
  • Arabidopsis has been used as a model for crop species more out of necessity than because it is truly representative.
  • genes which are candidates for transfer to crop species to be "tested" first in Arabidopsis, prior to the more labourious validation in a particular crop species.
  • the present invention is based on the identification of a new model plant species.
  • the species Cardamine hirsuta shares many of the properties that make Arabidopsis genetically tractable.
  • C. hirsuta can be transformed according to a highly convenient technique, a technique which previously was limited to the transformation of Arabidopsis. This is surprising because the technique has been shown to be unsuccessful in plants more closely related to Arabidopsis than C. hirsuta.
  • C. hirsuta is a member of the genus Cardamine, a group of plants which is more closely related to the important crop plants, the Brassicas, than Arabidopsis. Therefore, transgenic constructs aimed at the improvement of agricultural performance in Brassicas could first be tested in C. hirsuta.
  • C. hirsuta plants which ectopically express the homeobox gene KNOTTEDl display severe aberrations in floral organ development which are not observed in Arabidopsis plants expressing KNOTTEDl .
  • Floral development is disrupted in the transgenic C. hirsuta plants such that floral organs exhibit carpelloid transformations and ectopic ovules appear to be present on sepals.
  • the transgenic 35S::KN1 plants demonstrate a complete failure to elaborate the 4 whorls of floral organs present in the wild type flower.
  • Cardamine represents a better model for the Brassicas than Arabidopsis does.
  • the expression of a transgene which has no apparent effect on the development of Arabidopsis may well have an effect on the development of Brassicas.
  • the use of Cardamine is much more likely to reveal such an effect and will allow the testing of transgenes before the time-consuming step of gene transfer into a Brassica is carried out.
  • the use of Cardamine may therefore allow certain effects of transgene expression to be identified which would not be identified by the use of Arabidopsis.
  • a method for obtaining a first- generation transgenic plant of the genus Cardamine comprises: (i) contacting a floral meristem of a plant of the genus Cardamine with a ⁇ iAgrobacterium cell, which Agrobacterium cell comprises a heterologous polynucleotide; (ii) allowing the plant to set seeds;
  • the invention also provides: a transgenic plant or transgenic seed of the genus Cardamine, optionally obtainable according to a method of the invention; a method for evaluating the function of a polynucleotide sequence, which method comprises:
  • heterologous polynucleotide encodes a polypeptide
  • heterologous polynucleotide comprises a nucleic acid molecule from which an antisense nucleic acid is expressed.
  • heterologous polynucleotide comprises a nucleic acid molecule from which an interfering RNA is expressed.
  • KNOX proteins accumulate in the dissected leaf of C. hirsuta but are excluded from the simple leaf of A. thaliana.
  • C. hirsuta leaves are dissected into leaflets, each of which is borne on a petiolule attached to the rachis (c), whereas A. thaliana leaves are simple (d).
  • hirsuta shows that the youngest leaf primordium (1) initiates on the flanks of the SAM (*) with a simple shape, and leaflets initiate at the leaf margins 1—2 plastochrons later (arrowheads, 3,4; the distal leaflet of P4 has been removed) in a basipetal manner.
  • leaf primordia in A thaliana (h) initiate at the SAM (*) and continue to develop with a simple shape.
  • Scale bars 2 cm (a,b), 0.5 cm (c,d), 50 ⁇ m (e,f), 100 ⁇ m (g,h).
  • FIG. 1 C. hirsuta STM expression in young leaf primordia is required for leaflet initiation.
  • C. hirsuta STM expression is restricted to the SAM of embryos (longitudinal section, a) and vegetative shoot apices (transverse section, b) but absent from most cells that comprise leaf primordia. However, expression is observed throughout the outer cell layers of PO to P2 leaf primordia (arrowheads, 0, 1, 2) and in some cells of P3 leaf primordia (arrows, 3).
  • thaliana shoot apex shows expression is restricted to the SAM and absent from leaf primordia (0-3).
  • BP::GUS expression is observed only in the SAM of wild-type (d); ectopic expression is observed in asl leaves (arrow, e), and this ectopic expression is repressed in asl-1 ;35S::cASl (f).
  • f In situ localization of C. hirsuta ASl mRNA in the shoot apex of C. hirsuta, showing expression in initiating leaf primordia (arrowhead) on the flanks of the SAM (*) and in leaflets (arrow),
  • BP::GUS expression is restricted to the SAM in A. thaliana (h) and C. hirsuta (i).
  • ChBP :GUS is expressed in both the SAM and leaves in A. thaliana (j) and C. hirsuta (k). Two leaves have been removed for clarity in i and k.
  • Figure 4 C hirsuta ASl delimits C. hirsuta BP expression and controls leaflet positioning by regulating growth
  • (a-c) Silhouettes of rosette (left) and cauline (right) leaves of 35S::KN1 ⁇ GR induced once with 10 ⁇ 6 M dexamethasone (a), wild type (b), and chasl-1 (c). Arrows denote the length of the leaf rachis and arrowheads indicate extra leaflets.
  • (d,e) In situ localization of C. hirsuta BP mRNA.
  • VISTA analysisl (70% identity and 100 base sliding window) identified conserved regions with >70% identity shared by Arabidopsisan ⁇ C. hirsuta, shown in pink.
  • A Five conserved regions were identified in the 5' upstream regions of STM (see below for sequences). Region 1: 146 bp at 71.2%, region 2:93bp at 81.7%, region 3: 308bp at 80.8%, region 4: 99bp at 72.7%, and region 5: 754bp at 80.1 %, giving a total of 1400bp at 78.9%.
  • B Two conserved regions were identified in the 5' upstream regions of BP (see below for sequences).
  • Region 1 89bp at 89.9%
  • region 2 778bp at 76.6%, giving a total of 867bp at 78.0%.
  • Regions of divergent sequence are good candidates for conferring leaf expression in C. hirsuta. It will be interesting to determine whether such regions reside within conserved islands of sequence or within the more rapidly evolving non- 5 (2 of 3)
  • FIG. 1 A: conserved sequences of 5' upstream regions of STM fcovn. Arabidopsis and C. hirsuta
  • B conserveed sequences of 5 ! upstream regions of BP from Arabidopsis and C. hirsuta
  • FIG. 9 Increased KNOX protein accumulation in ChASl RNAi leaves, a-d. Immunolocalisation of class I KNOX proteins. Transverse sections through shoot apices of wild type (a) and ChASl RNAi (b) show nuclear expression of KNOX proteins in the SAM (*) and young leaf primordia (outlined), with increased KNOX accumulation evident, particularly in the adaxial side of the developing ChASl RNAi leaf, closest to the SAM. Transverse sections through plastochron 4 leaves of wild type (c) and ChASl RNAi (d) show increased accumulation of KNOX proteins in vascular associated cells of ChASl RNAi leaves, particularly on the adaxial side, closest to the xylem pole. Abbreviations: x; xylem, p; phloem. Scale bars: 20 ⁇ m (a-d ). 5 (3 of 3)
  • Amplification of ChGAPDH shows the relative amount of cDNA in each sample.
  • the invention provides a method for the preparation of a transgenic plant of the genus
  • the transgenic plant is of the species C. hirsuta.
  • the method of the invention comprises dipping at least one floral meristem of a plant of the genus Cardamine into a solution of Agrobacterium, wherein the Agrobacteriiim comprises a heterologous polynucleotide which is to be transferred to the plant.
  • the dipped plants are then grown under conditions such that they set seed.
  • the seed is then harvested and screened to identify seeds which comprise the heterologous polynucleotide. Seeds identified as comprising the heterologous polynucleotide may be grown to give rise to a transgenic plant.
  • plant includes reference to whole plants, plant organs (for example leaves, stems, roots etc.), seeds, plant cells and progeny plants.
  • Plant cell as used herein includes reference to seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.
  • transgenic plant refers to a plant which comprises a heterologous polynucleotide.
  • the heterologous polynucleotide is stably integrated within the genome so that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide may be integrated into the genome alone or incorporated as part of a larger polynucleotide construct.
  • transgenic may be applied to any cell, cell line, tissue, plant part or plant, the genotype of which has been altered as well as those created by sexual crosses or asexual propagation from an initial transgenic.
  • transgenic should not be taken to encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally-occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.
  • a heterologous polynucleotide is one mat is to be introduced into a plant or has been introduced into a plant, thus giving rise to a transgenic plant.
  • a heterologous polynucleotide may comprise sequences which originate from a species other than that to be transformed.
  • heterologous polynucleotide may comprise sequences which are not of plant origin.
  • Such sequences may originate from, for example, a virus, a bacterium, a fungus or an animal.
  • a heterologous polynucleotide may comprise, or consist essentially of, sequences which originate from the species to be transformed.
  • a transgenic plant of the invention may thus comprise more than one copy of a polynucleotide which occurs naturally in the species in question.
  • a heterologous polynucleotide may comprise sequences which originate from the species to be transformed and sequences which do not originate from the species to be transformed.
  • a heterologous polynucleotide which is to be introduced in to a plant or has been introduced into a plant, giving rise to a transgenic plant, is sometimes known as a "transgene"
  • JKQTtTi 7 although sometimes the term "transgene" is used to refer only to a coding sequence comprised by a heterologous polynucleotide or to a coding sequence in combination with its control sequences. The precise nature of the meaning of the term will be clear to one skilled in the art in the context of its use.
  • a heterologous polynucleotide suitable for use in the invention is typically a DNA or an analog thereof. Suitable analogs have the essential nature of a natural DNA in that they hybridise, under stringent hybridisation conditions, to substantially the same nucleotide sequence comprising only naturally-occurring nucleotides.
  • Typical stringent hybridisation conditions include from about 0.1 to about 0.2 x SSC at about 6O 0 C to about 65 0 C.
  • the heterologous polynucleotide comprises at least one, often two, or even three or more, coding sequences.
  • a coding sequence is a nucleotide sequence that is capable of being transcribed by the transcriptional mechanisms endogenous to the recipient cell.
  • a coding sequence may encode, for example, an mRNA, a tRNA, an rRNA, a ribozyme, or any other form of RNA known in the art.
  • the invention also includes an heterologous polynucleotide comprising an interfering RNA (siRNA) or antisense RNA.
  • siRNA interfering RNA
  • antisense RNA antisense RNA
  • the siRNA molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double stranded RNA molecule.
  • the siRNA molecule is typically derived from exons of the gene which is to be ablated.
  • RNA interference The mechanism of RNA interference is being elucidated. Many organisms respond to the presence of double stranded RNA by activating a cascade that leads to the formation of siRNA.
  • the presence of double stranded RNA activates a protein complex comprising RNase III which processes the double stranded RNA into smaller fragments (siRNAs, approximately 21-29 nucleotides in length) which become part of a ribonucleoprotein complex.
  • the siRNA acts as a guide for the RNase complex to cleave mRNA complementary to the antisense strand of the siRNA thereby resulting in destruction of the mRNA.
  • RNA which is complementary to normal mKNA transcribed from the "sense" strand of the target gene. See, for example, Rothstein et al, 1987; Smith et al, (1998),
  • the coding sequence encodes an mRNA. More preferably, the mRNA is capable of being translated by the translation mechanisms endogenous to the recipient cell.
  • the mRNA may be monocistronic or polycistronic. If the mRNA is polycistronic, for example encoding two or three polypeptides, it may contain an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • a polypeptide encoded by a heterologous polynucleotide of the invention may have any function.
  • the naturally-occurring form of the protein encoded by the coding sequence may be, for example, typically an extracellular protein (e.g. a secreted protein), an intracellular protein (e.g. cytosolic, organellar, plastidic, nuclear or membrane protein) or a protein present in the cell membrane.
  • the naturally- occurring form of the encoded polypeptide may be constitutively expressed or be tissue specific.
  • Functions of polypeptides encoded by the coding region may include herbicide, insecticide or disease resistance.
  • Preferred herbicide resistance polypeptides may be responsible for tolerance to, for example: glyphosate (e.g.
  • EPSP synthase e.g. EP-A-O 293,358 or a glyphosate oxidoreductase (e.g. WO 92/000377)
  • fosametin e.g. using a phosphinothrycin acetyl transferase (PAT) or a glutamine synthase (e.g. EP-A-O 242,236)
  • PAT phosphinothrycin acetyl transferase
  • glutamine synthase e.g. EP-A-O 242,236)
  • asulam e.g. using a dihydropteroate synthase (EP-A-O 369,367)
  • Insect resistance may be introduced using coding sequences encoding, for example,
  • a coding sequence may encode a polypeptide which confers insect resistance on the recipient plant, e.g. as in WO91/02701 or WO95/06128.
  • a coding sequence of a heterologous polynucleotide of the invention may encode a polypeptide with a role in a metabolic pathway, for example a chloroplastic metabolic pathway such as the photosynthetic metabolic pathway.
  • a chloroplastic metabolic pathway such as the photosynthetic metabolic pathway.
  • the expression of the encoded polypeptide will alter flux in the pathway and thus the encoded polypeptide may alter the photosynthetic ability of the transformed plant.
  • a polypeptide encoded by a heterologous polynucleotide of the invention may allow increased uptake of a nutrient, for example leading to increased production of a carbohydrate such as starch.
  • a polypeptide encoded by a heterologous polynucleotide of the invention may allow a transgenic plant comprising that heterologous polynucleotide to take up a substance which the corresponding wild type plant would not.
  • Such heterologous polynucleotides may be useful where plants are required to carry out bioremediation.
  • Suitable polypeptides include ion channels in the case of bioremediation of heavy metals.
  • Other suitable polypeptides include those conferring resistance to environmental insult, for example drought tolerance or tolerance to high salinity.
  • a polypeptide encoded by a heterologous polynucleotide of the invention may be expressed in order to enable its mass production, and have no particular relation to the biological processes of cell or plant in which it is expressed. It may be any polypeptide known in the art. It may be derived from any organism, for example from a prokaryote, fungus, plant or animal and may perform any function in vivo.
  • a polypeptide encoded by a heterologous polynucleotide of the invention may be an immunoglobulin.
  • a transgenic plant may be capable of expressing more than one immunoglobulin, for example two, three or four immunoglobulins. If the expression of more than one immunoglobulin is required, they may be introduced into a Cardamine plant on the same heterologous polynucleotide, on different heterologous polynucleotides introduced into the same plant, on different heterologous polynucleotides introduced into separate plants which are subsequently sexually crossed or via a combination of these techniques.
  • a transgenic plant of the invention may therefore be capable of expressing an antibody.
  • the term antibody includes fragments of whole antibodies which maintain the binding activity characteristic of the whole antibody. Such fragments include Fv, F(ab') and F(ab) 2 fragments, as well as single chain antibodies. Furthermore, the antibodies or fragments thereof may be chimeric antibodies, CDR-grafted antibodies or humanised antibodies.
  • the polypeptide may be an enzyme, such as a catabolic or anabolic enzyme.
  • the enzyme may be, for example, a metabolic (e.g. glycolysis, Calvin cycle or Krebs cycle) enzyme or a cell signalling enzyme.
  • the enzyme may make, break down or modify lipids, fatty acids, amino acids, proteins, nucleotides, polynucleotides (e.g. DNA or RNA) or carbohydrates (e.g. starch, amylose, amylopectin, or smaller sugars), and may be a protease, lipase or carbohydrase.
  • the enzyme may be a protein modifying enzyme, such as an enzyme that adds or takes chemical moieties from a protein (e.g. a kinase or phosphatase).
  • the polypeptide may be a transport or binding protein (e.g. which binds and/or transports a vitamin, metal ion, amino acid or lipid, such as cholesterol ester transfer protein or phospholipid transfer protein).
  • the polypeptide may be cytotoxic.
  • the polypeptide may be a cytochrome.
  • the polypeptide may be useful for industrial purposes. For example, xylanases, enzymes involved in plant cell wall modification, can be useful, for example in paper manufacture. Alternatively, the polypeptide may be useful for therapeutic purposes.
  • the polypeptide may thus be useful in a method of treatment of the human or animal body, or as an antigenic polypeptide for use as an edible or extractable vaccine.
  • the polypeptide may be able to cause the replication, growth or differentiation of cells.
  • the polypeptide may aid transcription or translation of a gene or may regulate transcription or translation (e.g. a transcription factor or a protein that binds a transcription factor or polymerase).
  • the polypeptide may be a signalling molecule, such as an intracellular or extracellular signalling molecule (e.g. a hormone).
  • Preferred polypeptides are those which are known to affect the development of plants.
  • heterologous polynucleotides for use in the present invention may comprise a coding sequence for a developmentally significant polypeptide which originates from a plant other than a Cardamine plant.
  • a heterologous polynucleotide for use in the present invention may comprise a coding sequence for a developmentally significant polypeptide which originates from a Cardamine plant itself.
  • polypeptides which affect the development of the plant embryo, endosperm or seed and any of those polypeptides may be used according to the invention.
  • genes affecting seed development include a number of chromatin modifying genes, for example the polycomb genes FIE and MEDEA (MEA) (Yadegari et ah,
  • chromatin remodelling genes may affect the overall size of organs.
  • SERRATE Plant Cell 13(6), 1263-1279, 2001
  • PICKLE Ogas et al, Proc.
  • polypeptides are known which affect the development of roots and any of those polypeptides may be used according to the invention.
  • a number of polypeptides are known which affect the development of leaves and any of those polypeptides may be used according to the invention, for example the polypeptide encoded by the KNOTTEDl and other KNOTTEDl-like homeobox (KNOX) genes.
  • a number of polypeptides are known which affect the development of flowers and any of those polypeptides may be used according to the invention. There are four classes of genes affecting floral development.
  • the polypeptide may be one encoded by a flowering time gene, mutations in which cause early or late flowering.
  • Flowering time genes can be divided into distinct classes, based on their differential responses to a number of environmental conditions, such as day length and vernalization (for a review see Weigel et al. Ann. Rev. Genet. 29, 19-39, 1995).
  • the polypeptide could be encoded by a late flowering or an early flowering gene, for example.
  • the polypeptide may be one encoded by a meristem identity gene.
  • meristem identity genes There are two types of meristem identity genes: those which specify flower meristem identity, for example LEAFY, FLORICAULA, APETALAl, CAULIFLOWER or UNUSUAL FLORAL ORGANS; or those which maintain inflorescence meristem identity, for example TERMINAL FLOWER or CENTRORADIALIS.
  • a heterologous polynucleotide of the invention could alternatively encode a polypeptide encoded by a floral organ identity gene. Floral organ identity genes determine the fate of organ primordia and their action has been explained by the "ABC" model of flower development.
  • a polypeptide in the invention could be encoded by: an "A” function gene, such as APETALAl (which is involved in both meristem identity and organ identity ) or
  • APETALA2 APETALA2
  • B B function gene
  • C C
  • AGAMOUS or PLENA AGAMOUS or PLENA
  • a further class of genes which affect flower development includes late-acting genes that control ovule development.
  • a polypeptide encoded by such a gene may be used in the invention.
  • a coding sequence in a heterologous polynucleotide of the invention may encode a polypeptide of unknown function. That is, the heterologous polynucleotide may comprise, for example, an expressed sequence tag (EST).
  • EST expressed sequence tag
  • a transgenic Cardamine plant of the invention may thus be used to try to reveal the function of a polynucleotide sequence of unknown function.
  • the heterologous polynucleotide for use in a method of the invention will comprise a coding sequence of interest operably linked to a control sequence (or control sequences). The control sequence or sequences is/are capable of providing for the
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence such as a promoter "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.
  • the control sequence will typically comprise a promoter and optionally also comprise other types of control sequence, for example an enhancer and/or terminator.
  • a control sequence may be positioned 5', 3' or internal to (for example in an intron) a coding sequence.
  • a coding sequence may be operably linked to more than one control sequence, for example two, three, four or five control sequences. Such multiple control sequences may be positioned, for example, entirely 5' to the coding sequence. However, more typically control sequences will be located both 5' and 3' to the coding sequence, with optional internal control sequences.
  • a promoter is a nucleotide sequence capable of initiating transcription of a coding sequence.
  • a coding sequence is positioned 3' (i.e. downstream) to a promoter, although promoters may be situated in introns.
  • An enhancer is any polynucleotide sequence capable of increasing the level of transcription initiating from a promoter and may act on a cis or trans basis.
  • a terminator is any polynucleotide sequence capable of promoting dissociation of an RNA polymerase from the said sequence. Control sequences may be derived from any suitable source and may be generated by recombinant techniques or synthetic means.
  • Control sequences may be truncated or comprise sequence alterations, for example, in order to reduce or remove areas of homology to the proposed recipient genome or to introduce or improve sequences essential for function as a regulatory region. Control sequences may be specifically designed de novo to ensure heterology to the recipient genome and to comprise the essential features of functional control sequences.
  • suitable promoter regions will contain well defined -10 and -35 sequence motifs (Tanaka et al, FEBS. LETT. 413, 309-313, 1997; Isono et al, Proc. Natl. Acad. Sci. USA 94, 14948-14953, 1997; Kestermann et al, Nucleic Acids Res. 26, 2747-2753, 1998).
  • a -10 sequence motif will have the sequence 5'-TATAAT-3', and a -35 sequence motif will have the sequence motif 5'-TTGACA-3', although the skilled person will appreciate that variants of these sequence can be used (Grierson and Covey, Plant MoI. Biol. (2 nd
  • Promoters and other regulatory elements may be selected to be compatible with the host cell, i.e. the plant species, for which the expression vector is designed.
  • Promoters suitable for use in plant cells may be derived, for example, from plants or from bacteria that associate with plants or from plant viruses. Thus, promoters from
  • Agrobacterium spp. including the nopaline synthase (nos), octopine synthase (ocs) and
  • mannopine synthase (mas) promoters are preferred.
  • plant promoters such as the ribulose bisphosphate small subunit promoter (rubisco ssu), histone promoters (EP-A-O 507,698), the rice actin promoter (US Patent No. 5,641,876) and the phaseolin promoter.
  • plant viral promoters such as the cauliflower mosaic virus (CAMV) 35S and 19S promoters, and the circovirus promoter (AU-A-689,311).
  • promoters may be constitutive, tissue- or stage-specific, and/or inducible.
  • strong constitutive expression in plants can be obtained with the CAMV 35S, Rubisco ssu, or histone promoters mentioned above.
  • tissue-specific or stage-specific promoters may be used to target expression of polypeptides of the invention to particular tissues in a transgenic plant or to particular stages in its development.
  • seed-specific promoters include those described by Dalta et al (Biotechnology Ann. Rev. (1997), 3, pp.269-296). Particular examples of seed-specific promoters are napin promoters (EP-A-O 255, 378), phaseolin promoters, glutenine promoters, helianthenine promoters (WO92/17580), albumin promoters (WO98/45460), oleosin promoters (WO98/45461) and ATSl and ATS3 promoters (PCT/US98/06798).
  • Chemically inducible promoters such as those activated by herbicide safeners may also be used, for example the maize GST 27 promoter (WO97/11189), the maize In2-1 promoter (WO90/11361), the maize In2-2 promoter (De Veylder et al, Plant Cell Physiology, Vol. 38, pp568-577 (1997).
  • Further examples of inducible promoters known in the art include those modulated by exposure to tetracycline, ecdysteroids, glucocorticoids, plant growth regulators such as abscisic acid, animal hormones, nitrates, metal ions (such as copper), environmental conditions (such as cold, heat, light or dark) and wounding.
  • promoters may be used that are limited to expression in specific tissue or cell types, and/or during specific developmental stages, such as during flowering or senescence.
  • a chimeric gene comprises the following elements in 5' to 3' orientation: a promoter functional in a host, i.e. plant, cell, as defined above, a polynucleotide of the invention and a terminator functional in a said cell, as defined above.
  • Enhancers include the tobacco etch virus (TEV) enhancer and the tobacco mosaic virus (TMV) enhancer (WO87/07644).
  • a heterologous polynucleotide suitable for use in the invention may comprise one or more, for example two or three, nucleotide sequences encoding selectable marker polypeptides operably linked to their own control sequences in addition to that/those encoding a polypeptide(s) of interest.
  • the control sequences may be the same as or different from those operably linked to the coding sequence(s) encoding a polypeptide(s) of interest.
  • a selectable marker polypeptide is one which, for example, allows a cell which expresses that marker polypeptide to survive in the presence of an agent that would kill a similar cell which does not express that marker polypeptide.
  • Any coding sequence encoding a suitable selectable marker polypeptide may be used in a heterologous polynucleotide of the invention.
  • herbicide resistance genes for example as defined above, may be used as selectable markers.
  • Notable examples include polypeptides conferring resistance to herbicides such as bialaphos, glyphosate or an isoxazole herbicide may be used. Particular examples are described in EP-A-O 242,236, EP-A-O 242,246,
  • a coding region that encodes a polypeptide which provides resistance to aminoglycoside antibiotics may be used as a selectable marker, for example, encoded polypeptides that provide resistance to kanamycin (e.g. the nptl or nptll genes) , neomycin, ampicillin or chloramphenicol (the CAT gene).
  • kanamycin e.g. the nptl or nptll genes
  • neomycin eomycin
  • ampicillin or chloramphenicol the CAT gene
  • the encoded polypeptide may confer a morphological alteration on a cultured transformed cell, such as isopentyltransferase.
  • the encoded polypeptide may be a scorable marker, which allows transformed cells to be distinguished from non-transformed cells, typically by alteration of the optical properties of a cell expressing the marker polypeptide.
  • Any scorable marker may be used.
  • Preferred scorable markers include polypeptides which are able to alter the appearance or optical properties of transformed cells, for example: 3-glucuronidase (i.e. the uidA:GOS gene); fluorescent proteins such as green fluorescent protein (GFP), yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP); or luminescent proteins such as luciferase or aequorin.
  • Cells with scorable optical differences can be sorted using techniques such as fluorescence activated cell sorting (FACS).
  • a heterologous polynucleotide of the invention may comprise coding sequences encoding a selectable marker and a scorable marker, for example, the FLARE-S marker genes which comprise aadA and GFP.
  • a coding region of a heterologous polynucleotide of the invention encodes a fusion protein.
  • a fusion protein is a single polypeptide comprising at least two contiguous amino acid sequences that are not naturally found joined together.
  • Il C O ⁇ 15 protein may consist of, for example, two or three contiguous amino acid sequences that are not naturally found in that arrangement or even four, five or more contiguous amino acid sequences.
  • at least one of the sequences represents the sequence of a polypeptide of interest (or at least a fragment of that polypeptide), or a marker polypeptide. That is, the fusion protein consists in part of the sequence of a polypeptide that is desirably expressed in a Cardamine plant.
  • the fusion protein may contain the sequences of two, three or more polypeptides of interest, which may be the same or different.
  • At least one of the polypeptide sequences within the fusion protein provides the fusion protein with a selectable or scorable property. This property can aid in the purification of the fusion protein, by allowing rapid and easy identification of fractions containing the fusion protein.
  • Preferred polypeptides providing scorable properties include GUS or GFP.
  • the fusion protein comprises at least one amino acid sequence that allows for the fusion protein to be readily purified.
  • the number of purification sequences is not more than 5, more typically not more than 2, most typically 1.
  • the purification sequence is a His-Tag.
  • the His-Tag comprises multiple contiguous histidine residues, preferably from 3 to 20, more preferably from 4 to 10 most preferably 6.
  • the His-tag will be positioned at either or both of the N- and C- terminals of the fusion protein.
  • the polypeptide of interest is joined to other sequences by a sequence that can be cleaved to release the polypeptide of interest with substantially the same biological activity, or substantially the same amino acid sequence as the individually expressed protein of interest.
  • the cleavage sequence is IEGR, which is recognised and cleaved by Factor Xa (Nagi et al, 1985, Quinlan et at, 1989, Wearne 1990).
  • a coding region in a heterologous polynucleotide of the invention may include a region encoding a signal sequence capable of targeting the encoded polypeptide to specific locations within a plant cell or of ensuring that the fusion polypeptide is secreted from the cell in which it is expressed. Suitable signal sequences can ensure targeting of the polypeptide to the nucleus, to the nuclear envelope, to a plastid (for example a chloroplast), to a mitochondrion, to the cell membrane or to the endoplasmic reticulum. If the targeting sequence targets the fusion polypeptide to an organelle, a particular region of the organelle may be targeted. For example, in the case of a chloroplast, the fusion polypeptide could be targeted to the stroma, to the inter- membrane space, to the thylakoid membranes or the compartments within the thylakoids.
  • Coding sequences encoding such signal sequences may be derived from any suitable organism,
  • SUBSTITU 16 including the recipient Cardamine plant.
  • signal peptide sequences include: sequences which target proteins to the extracellular matrix of the plant cell, such as the signal sequence of the Nicotiana plumbaginifolia extension gene; signal peptides which target proteins to the vacuole, like those of the sweet potato sporamin gene and the barley lectin gene; signal peptides which cause proteins to be secreted such as that of PRIb; or the barley V-amylase leader sequence; and signal peptides which target proteins to the plastids such as that of rapeseed enoyl-Acp reductase.
  • the heterologous polynucleotide to be introduced into a plant is typically incorporated into a recombinant replicable vector.
  • Such vectors may be used to replicate the heterologous polynucleotide in a compatible host cell by introducing the vector into a compatible host cell and cultivating the host cell under conditions which bring about replication of the vector. The vector may then be recovered from the host cell.
  • the vector will allow replication of the heterologous polynucleotide in both is. coli (for convenience in cloning) and va. Agrobacterium sp. (for transfer to plants).
  • Vectors suitable for the expression of heterologous polynucleotides in higher plants are well known to those skilled in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens (Rogers et ah, Meth. In EnzymoL, 153, 253-277, 1987) and the Ri plasmid of Agrobacterium rhizogenes. These vectors are plant integrating vectors in the sense that, on transformation, the vectors integrate a portion of vector DNA into the genome of the host plant. The heterologous DNA is incorporated into such vectors so that it is located between T-DNA borders (a repeated 24-base pair sequence).
  • the vectors are then introduced into the bacterium Agrobacterium.
  • Agrobacterium tumefaciens is used.
  • Suitable methods for the introduction of polynucleotide vectors into Agrobacterium are well-known to those skilled in the art and include tri-parental mating, heat shock and electroporation techniques.
  • Agrobacterium host will direct the insertion of the heterologous polynucleotide into the plant cell DNA when the cell is infected by the bacteria.
  • Plants used for transformation are of the genus Cardamine, preferably of the species C. hirsuta.
  • the plant selected for transformation is grown under any conditions suitable to induce flowering of the said plant. Germination efficiency of the seeds may be increased by striating the seeds before sowing, for example by keeping the seeds at about 4 0 C for about four to five days. Typically, the plant is grown under long day conditions, for example from about 14 to about 20 hour days.
  • the plant may be grown in any suitable medium, for example a 3:1 mixture of soil and vermiculite. It has been found that the frequency of transformation is higher for the primary inflorescence than for secondary inflorescences. This may be because flowers on the primary inflorescence open for longer than flowers on the secondary inflorescences. Therefore,
  • a solution of Agrobacterium is prepared suitable for carrying out the transformation.
  • the Agrobacterium solution is generally prepared by: growing a culture of Agrobacterium comprising the required heterologous polynucleotide is grown to an optical density of about 1.0; pelleting the cells; and resuspending the cells in half the original culture volume.
  • the plant to be transformed is then contacted with the Agrobacterium solution so that at least one of the floral meristems of the plant comes into contact with that Agrobacterium solution.
  • Agrobacterium solution it will be convenient to immerse more than one and if possible, substantially all floral meristems of a plant in the Agrobacterium solution.
  • the entire primary inflorescence is dipped into the Agrobacterium solution. It is not necessary to apply any kind vacuum whilst the inflorescence is immersed.
  • the contacting step is carried out for from about 1 to 30 minutes, more preferably from about 5 to 20 minutes, most preferably for about 10 minutes.
  • the contacting step is carried out in the absence of a vacuum, i.e. at normal or ambient atmospheric pressure.
  • the contacting step will typically be carried out about 1.01325xl0 5 Nm '2 (1.01325 bar).
  • each plant may optionally be isolated from the other, for example by covering with a paper bag, so that self- fertilization only occurs.
  • Cardamine plants are self-compatible so self-fertilization will occur. In fact, it will typically be desirable to use paper bags, as Cardamine plants show explosive pod shatter and the bags will ensure seed containment.
  • the seeds may be harvested and then screened for the presence of the heterologous polynucleotide.
  • the nature of the screening procedure will depend on nature of the heterologous polynucleotide and whether that heterologous polynucleotide comprises a coding sequence for a marker polypeptide. If the heterologous polynucleotide does comprise a sequence encoding a marker polypeptide, it may be possible to screen large amounts of seed quickly and conveniently. The exact screening strategy used will depend on the marker polypeptide to be detected.
  • an antibiotic resistance marker gene may allow the seed to be 18 plated on selective media such that the only seedlings which develop are those which carry the heterologous polynucleotide (and therefore the marker gene).
  • the marker polypeptide is a polypeptide conferring resistance to kanamycin
  • the seeds may be germinated on media comprising from about 50 ⁇ g/ml to about 150 ⁇ g/ml kanamycin, preferably about lOO ⁇ g/ml kanamycin. All viable seeds should develop, but only those expressing the marker polypeptide will develop beyond the stage at which the cotyledons develop. Those not expressing the marker polypeptide will gradually bleach and then die.
  • seedlings may be germinated on a non-selective media and then sprayed with the relevant herbicide. Seedlings which do not express the marker polypeptide will die and the surviving seedlings should carry the heterologous polynucleotide.
  • Seedlings identified as carrying the heterologous polynucleotide may subsequently be analysed to ensure that they carry both the coding sequence for the marker polypeptide and the sequence encoding the polypeptide of interest. Such analysis may be carried out by any appropriate method, for example by PCR, DNA gel blotting or DNA chip screening. RNA gene blotting and/or protein gel blotting may be carried out to ensure that the polypeptide of interest is expressed.
  • heterologous polynucleotide may be confirmed, for example by the use of PCR (using primers specific to the heterologous polynucleotide) and/or via DNA gel blotting.
  • the procedure set out above provides a method for the generation of a first generation transgenic Cardamine plant, preferably a transgenic C. hirsuta plant.
  • the invention also provides a method for obtaining transgenic Cardamine plants of further generations from this first generation transgenic plant. Such plants can conveniently be referred to as progeny transgenic Cardamine plants.
  • Progeny transgenic Cardamine plants of second, third, fourth, fifth, sixth and further generations may be obtained from a first generation transgenic Cardamine plant by any means known in the art.
  • the invention provides a method of obtaining a transgenic Cardamine progeny plant comprising obtaining a second-generation transgenic Cardamine progeny plant from a first-generation transgenic Cardamine plant of the invention, and optionally obtaining transgenic
  • Cardamine plants of one or more further generations from the second-generation progeny plant thus obtained are Cardamine plants of one or more further generations from the second-generation progeny plant thus obtained.
  • Such progeny plants are desirable because the first generation plant may not have all the characteristics required. For example, it may be desired to introduce further characteristics in one or more subsequent generations of progeny plants before a transgenic plant carrying all of the characteristics required is produced.
  • Progeny plants may be produced from their predecessors of earlier generations by any known technique.
  • progeny plants may be produced by:
  • transgenic seed from a transgenic plant of the invention belonging to a previous generation, then obtaining a transgenic progeny plant of the invention belonging to a new generation by growing up the transgenic seed;
  • transgenic progeny plants of one or more further generations from the progeny plant thus obtained are obtained.
  • clonal propagation and sexual propagation may be used at different points in a process that gives rise to a transgenic plant suitable for cultivation.
  • repetitive back-crossing with a plant with desirable characteristics may be undertaken.
  • Further steps of removing cells from a plant and regenerating new plants therefrom may also be carried out.
  • the invention thus provides a transgenic plant of the genus Cardamine, preferably a transgenic plant of the species C. hirsuta.
  • further desirable characteristics may be introduced by transforming the cells, plant tissues, plants or seeds, at any suitable stage in the above process, to introduce desirable coding sequences other than those contained in a heterologous polynucleotide of the invention.
  • This may be carried out by conventional breeding techniques, e.g. fertilizing a transgenic plant of the invention with pollen from a plant with the desired additional characteristic.
  • the characteristic can be added by further transformation of the plant obtained by the method of the invention, using the techniques described herein for further transformation, or by nuclear transformation using techniques well known in the art such as, inter alia, electroporation of plant protoplasts, transformation by Agrobacterium or particle bombardment.
  • each coding sequence to be introduced into a plant is linked to different selectable or scorable marker. That will allow individual selection to be applied for the presence of each coding sequence.
  • Selection, 20 regeneration and breeding techniques for nuclear transformed plants are known in the art. Techniques along the lines of those described may be used.
  • the invention also provides a method for evaluating the function of a polynucleotide in a Cardamine plant by disrupting that polynucleotide and determining the effect(s) of such disruption.
  • Disruption of a polynucleotide in a Cardamine plant may be carried out according to any method known in the art. Broadly speaking, there are two main methods of carrying out polynucleotide disruptions: radiation mutagenesis, chemical mutagenesis; or insertion mutagenesis.
  • Radiation mutagenesis may be earned out using, for example irradiation with X-rays or (-rays.
  • a population of seeds is irradiated, grown to maturity and allowed to set seed. Mutations typically only take place in one chromosome at a particular locus therefore a second generation of plants is grown to reveal the phenotype of any mutations induced by the irradiation.
  • Chemical mutagenesis is typically carried out by contacting a population of seeds of a
  • Cardamine plant with a suitable mutagen A number of other chemical mutagens may be used, with mutagens which are alkylators being preferred. Suitable alkylators are, for example, ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), diethylsulfate (DES) or nitrosoguanidine (NTG, NG, MNNG). Such mutagens do not require active DNA replication to occur to be effective (although DNA replication is required in order for a mutation to be "fixed”) and are thus suitable for use with seeds. Again, a second generation of plants is grown to reveal the phenotype of any mutations induced by the mutagen.
  • EMS ethyl methane sulfonate
  • MMS methyl methane sulfonate
  • DES diethylsulfate
  • NVG, NG, MNNG nitrosoguanidine
  • Insertion mutagenesis relies on disruption of the expression of a coding sequence in a plant with another polynucleotide sequence. This may be effected by the introduction of a heterologous polynucleotide at a locus, whereby the said heterologous polynucleotide disrupts the expression of a coding sequence at that locus.
  • the heterologous polynucleotide may, for example, insert within the coding sequence itself or may disrupt the function of a control sequence.
  • the plant in question comprises endogenous transposable polynucleotide elements, it may be possible to induce transposition so that a transposable element "jumps" to a new locus.
  • Insertion mutagenesis may be carried out in Cardamine using T-DNA tagging. It is not clear whether Cardamine has a population of endogenous transposons and therefore whether transposition using such transposons is applicable to Cardamine. However, insertion mutagenesis could be carried out by the use of heterologous transposition systems. T-DNA tagging screens and heterologous transposition screens may be carried out in
  • Cardamine plants identified in such screens which display phenotypes of interest may be selected for further analysis. Because the sequence of the T-DNA or the heterologous transposon are known it is relatively easy to identify sequence flanking the said T-DNA or heterologous transposon. The flanking sequence will either represent coding sequence or control sequence of the polynucleotide disrupted. Therefore, it may be relatively easy to link the phenotype shown by a mutant with the sequence of the polynucleotide which has been disrupted.
  • Cardamine plants are more related to the important crop plants, the Brassicas, than the commonly used model plant Arabidopsis and therefore may represent a better model for such plants.
  • the phylogenetic relationships of nine genera in the family of the Brassicaceae have been estimated from the sequences of the internal transcribed spacer region (ITS) of the 18S-25S nuclear ribosomal DNA (Weng et al., Molecular Phylogenetics and Evolution .13, 455-462. 1999).
  • the invention provides a method for evaluating the suitability of a polynucleotide which is a candidate for transfer into a crop species, which method comprises: (i) preparing a transgenic Cardamine plant which expresses the candidate polynucleotide; and (ii) determining the effect of the candidate polynucleotide on the transgenic Cardamine plant. It is likely that the effect of the candidate polynucleotide in Cardamine will more closely resemble the effect of that polynucleotide in a crop species, in particular a Brassica species, than the effect of that polynucleotide observed in Arabidopsis would.
  • Cardamine plant as a model may save unnecessary transfer of genes into a crop plant on the basis of data gathered from Arabidopsis only to find that there is no effect (or at least a different effect) of the gene in the crop species.
  • a polynucleotide which shows no effect in Arabidopsis does in fact have a significant effect in a crop species.
  • a polynucleotide which is a candidate for transfer to a crop species, in particular transfer into a Brassica will be introduced into a Cardamine plant according to the method of the invention for the preparation of a transgenic Cardamine plant.
  • Polynucleotides which show an effect of agricultural relevance in Cardamine for example: insect or herbicide resistance; drought, salt or wind tolerance; increased leaf size; increased seed size; bioremediation potential may be subsequently tested in a crop species, in particular in a Brassica.
  • preferred Brassicas include B. napus, B. olerecea, B.
  • a method for obtaining an improved crop plant comprises: (i) identifying a polynucleotide which shows a beneficial effect in Cardamine; and (ii) introducing that polynucleotide into the crop plant.
  • the invention also provides an improved crop plant, in particular an improved Brassica plant, identified by such a method.
  • the improved Brassica may be, for example, a B. napus, B. olerecea, B. Junea or Sinapis alba plant.
  • a further use of plants of the genus Cardamine would be to use them for surveying the genomes of relatives of Arabidopsis (for example Cardamine and Lepidium) in order to identify functional czs-acting elements which are involved in gene expression.
  • This sort of analysis has been shown to be effective in yeast (Hillier et ah, Genome Res. 11(7), 1175-1186).
  • the fact that Cardamine in easily transformable provides a quick way to check the properties of putative exacting elements identified by bioinformatics/genomics approaches. This would of course be possible in Arabidopsis, but the use of Cardamine provides independent verification and will more closely represent what will happen in crops, for example Brassica crops.
  • Plant growth conditions Plants were grown in a greenhouse with supplemental lighting (days: 18 h, 20 0 C; nights: 6 h, 16 0 C).
  • Wild-type C. hirsuta seed was collected from wild populations in Oxford, UK; verified by internal transcribed spacer sequencing; and self-pollinated for seven generations before use (specimen voucher Hay 1 (OXF)). Wild-type C. hirsuta seed was X-ray-irradiated at 16 kR, sown and harvested in pools of five plants. Approximately 100 seed of 150 M2 pools, giving a total of 1,500 plants, were screened. Mutant characterization was performed after backcrossing to wild-type C. hirsuta twice. 23 Transgenic construction. All primers are listed in Figure 12. To construct the C. hirsuta STM
  • RNAi vector a 310-bp fragment was amplified from C. hirsuta shoot cDNA by PCR with the primers ChSTMrnai-F and ChSTMrnai-R. This fragment was cloned in both sense and antisense orientations in the pHANNIBAL vector 18 using the restriction enzyme pairs XballClal and Ec ⁇ SllKpj ⁇ l. This RNAi cassette was transferred as a Notl fragment into the binary vector pMLBART 19 , transformed into the Agrobacterium twnifaciens strain GV3101 and used to transform wild-type C. hirsuta plants by a modified floral dipping protocol. We analyzed 14 independent Tl lines. We constructed a C.
  • RNAi vector in an identical manner using a 343-bp PCR fragment amplified from C. hirsuta shoot cDNA with the primers ChASlrnai-F and ChASlrnai-R, and we used it to transform wild-type C. hirsuta plants as above.
  • ChASl ChASl
  • RNAi vector in an identical manner using a 343-bp PCR fragment amplified from C. hirsuta shoot cDNA with the primers ChASlrnai-F and ChASlrnai-R, and we used it to transform wild-type C. hirsuta plants as above.
  • the pMLBART vector alone was used to transform wild-type C. hirsuta plants as above, and all Tl lines were phenotypically wild-type.
  • 35S::KN1-GR translational fusion 13 was used to transform wild-type C. hirsuta plants as described above.
  • To construct the 35S::ChASl vector we amplified a 1,117-bp fragment of the C. hirsuta ASl coding region by PCR using a proofreading Taq polymerase (Pyrobest, Takara) from a full-length cDNA clone using the primers ChASl-F and ChASl-R. The PCR product was cloned into the pCR Blunt vector (Invitrogen), sequenced to confirm fidelity and cloned as an £coRI fragment behind the CaMV 35S promoter of the ⁇ ART7 vector 19 .
  • the 35S: . ChASl ::ocs cassette was transferred as a Notl fragment into the binary vector pMLBART and transformed into as 1-1 mutant plants by floral dipping.
  • Five T2 lines with a single transgene copy were crossed to asl-l;BP::GUS plants 3 , and GUS expression was analyzed in the Fl.
  • a BAC library of C was made to make transcriptional fusions of C. hirsuta BP and C. hirsuta STM to the uidA (GUS) gene.
  • hirsuta genomic clones was screened (a full description of library construction will be given elsewhere), and ⁇ 6- kb -Ec ⁇ RI and Xbal restriction fragments of C. hirsuta BP and C. hirsuta STM DNA, respectively, were cloned into pBluescript (Stratagene).
  • pBluescript (Stratagene).
  • Transcriptional fusions were generated in a similar manner using ⁇ 5 kb of upstream sequence, including the 5' UTR of BP and STM.
  • AU four promoter-GUS cassettes were transferred as Notl fragments into the binary vector pMLBART, transformed into A. tumefaciens, as above, and
  • Ci jDQrm iTP $MP£T r&ULE 24 used to transform both wild-type A. thaliana (Columbia ecotype) and C. hirsuta.
  • GUS expression was analyzed in 97 independent Tl lines for ChBP::GUS and 96 lines for BP::GUS in A. thaliana, ten lines for ChBP: :GUS and 12 lines for BP::GUS in C. hirsuta, 67 lines for
  • a previously described 35S::BP construct 21 was transformed into A. tumefaciens and used to transform wild-type C. hirsuta plants as described above.
  • PHV BP
  • FIL BP
  • ANT BP lines were generated by constructing pVTOp.-.-BP 22 and transforming PHV::LhG4, FIL::LhG4 (gift from Y. Eshed, Weizmann
  • C. hirsuta ASl full-length cDNA sequence was determined in wild-type lines and chasl-1 mutants by 5' and 3' RACE.
  • cDNA was generated using a SmartRace kit (BD Biosciences) according to manufacturer's protocols. We used 1 ⁇ g of total shoot RNA per reaction. PCR amplification was performed for 5' RACE with the primer ChASl-Rl and for 3' RACE with the primer ChASl-Fl.
  • C. hirsuta ASl -specific products were cloned, and two clones from each RACE reaction were sequenced for each genotype.
  • a premature stop codon at amino acid 170 of ChASl introduces an Accl site that is not present in wild-type plants.
  • This sequence polymorphism was used to generate a cleaved amplified polymorphic sequence marker by amplifying a 600-bp product with primers ChAS 1-F2 and ChASl-R2, which yielded products of 425 bp and 175 bp after Accl digestion of chasl-1 but not after digestion of wild-type amplicons.
  • Leaflet and cell measurements Average number of leaflets per leaf was determined for ten C. hirsuta STM. RNAi plants and ten wild-type plants. Average number of C. hirsuta H4- expressing cells in adjacent longitudinal sections of the two youngest leaf primordia at the shoot apex was determined for C. hirsuta STM RNAi and wild-type plants (as described in ref. 11).
  • Scanning electron microscopy Fixation and dehydration were carried out as previously described 25 . Scanning electron microscopy was performed using a JSM-5510 microscope (Jeol).
  • ChBP5'-F and ChBP5'-R were cloned into the pGEM T-Easy vector (Promega) and sequenced to determine orientation. Antisense and sense probes were transcribed and DIG labeled as previously described 10 .
  • Leaf silhouettes Leaves were flattened onto clear adhesive, adhered to white paper and digitally scanned.
  • Dexamethasone (Sigma) was dissolved in water and applied at a concentration of 10 ⁇ 6 M with 0.02% silwet using a paintbrush.
  • GenBank C. hirsuta STM mRNA, complete coding sequence (cds), DQ512732; C. hirsuta ASl mRNA, complete cds, DQ512733; C. hirsuta BP mRNA, complete cds, DQ630764; C. hirsuta BP gene, 5' upstream region, DQ526379; C. hirsuta STM gene, 5' upstream region, DQ526380.
  • A. tumefaciens cultures containing a 35S::KN] construct (Sinha et al, Genes & Dev. 7, 787-795, 1993; a gift from Sarah Hake) were used to carry out the transformation.
  • the A. tumefaciens cells were grown to an optical density of approximately 1 and then resuspended in half their original volume. The primary inflorescences of the Cardamine plants were dipped in the A.
  • KNATl is the C. hirsuta ortholog of KNl and was generated via PCR.
  • Kanamycin resistant plants i.e. transgenic plants
  • sensitive plants i.e. non-transgenic plants
  • Nine lines of independent transformants showing a consistent phenotype were successfully generated.
  • the transgenic plants show an alteration in the characteristic compound leaf morphology shown by wild type C. hirsuta.
  • Leaf form can be described as simple (if the leaf blade is entire or dissected (if the blade is divided into distinct leaflets). Both simple and dissected leaves are initiated at the flanks of a pluripotent structure termed the shoot apical meristem (SAM). In simple-leafed species, such as A. thaliana and maize, ARP myb proteins act in the leaf to confine
  • KNOX transcription factors to the meristem 2"6 .
  • many dissected-leafed species accumulate KNOX proteins in the leaf and ARP proteins in the meristem 7 ' 8 .
  • KNOX activity is required to produce a dissected leaf, or whether differences in ARP function or regulation are responsible for the divergent patterns of KNOX expression seen in different species.
  • C. hirsuta a small crucifer related to the simple-leafed model species A. thaliana (Fig. la-d). Unlike many A. thaliana relatives, C. hirsuta has the distinct advantages of being a diploid, self-compatible plant that can be used for genetic analyses and transformed, thus allowing parallel genetic studies of leaf development to be conducted in species that diverged relatively recently 9 .
  • KNOX expression in the leaf is associated with dissected leaf form
  • FIG. 2 C. hirsuta STM is expressed in the SAM of the embryo and mature plant and is repressed in the majority of cells that comprise initiating leaf primordia (Fig. 2a,b). However, in contrast to STM expression in A. thaliana (Fig. 2c), we also observed C. hirsuta STM expression throughout the outer cell layers at the base of initiating leaf primordia (Fig. 2b). In comparison to wild- type plants (Fig. 2d), strong RNAi lines produced shootless plants with fused cotyledons
  • KNOTTEDl (KNl) protein which is able to rescue A. thaliana stm mutants 12 , in C. hirsuta in a dosage-sensitive manner using a fusion with the rat glucocorticoid receptor (KNl-GR) 13 .
  • KNl-GR rat glucocorticoid receptor 13 .
  • a single induction of KNl activity with 10 ⁇ 6 M dexamethasone resulted in reiteration of a second order of leaflets along the elongated petiolules of first-order leaflets (Fig. 2i).
  • C. hirsuta Elevated KNOX expression in the dissected-leaf tomato plant can also increase leaflet number 14 ' 15 , suggesting that the requirement for KNOX activity in C. hirsuta leaf development may extend to other species where dissected leaf morphology has evolved independently 7 .
  • EXAMPLE 4 We next investigated whether the differences in KNOX gene expression observed between the two species are attributable to differential activity of KNOX gene regulatory sequences. To test this idea, we analyzed 5' upstream regions of the KNOX genes STM and BP ( Figure 5) and performed promoter swap experiments with these regions between C. hirsuta and A. thaliana. We reasoned that if the regulatory information necessary for species-specific expression is contained within the promoter regions, then each promoter should drive reporter gene expression regardless of the species into which it is transformed. If, however, species-specific activity of trans regulatory factors is required for correct KNOX gene expression, then each reporter should reflect the expression pattern of the species into which it is transformed.
  • each reporter reflected the endogenous gene expression pattern of its promoter in both the native and heterologous context (Fig. 3). That is, the A. thaliana BP promoter generated GUS expression in the SAM of both A. thaliana and C. hirsuta (Fig. 3h,i), and the C. hirsuta BP promoter generated GUS expression in both the SAM and leaves of both species (Fig.3j,k). Swapping the C. hirsuta STM promoter region between C. hirsuta and A. thaliana gave similar results: the C. hirsuta promoter generated GUS expression in the SAM and the abaxial side of developing leaves in both species (Fig. 31,m), whereas the A.
  • C. hirsuta ASl activity is required for development of the proximodistal axis of the leaf and for determining number and positioning of leaflets along this axis.
  • C. hirsuta ASl controls dissected leaf form by defining the domain and level of KNOX expression.
  • KNOX protein and mRNA accumulation in C. hirsuta ASl RNAi lines and chasl-1 mutants We observed increased KNOX protein accumulation in C. hirsuta ASl RNAi leaves ( Figure 9) and observed ectopic expression of C. hirsuta BP but not C. hirsuta STM in chasl-1 leaves (data not shown and Fig. 4).
  • the pattern of ectopic C. hirsuta BP expression correlated well with the phenotypic perturbations observed in chasl-1.
  • FIG. 4e correlating with repression of growth and differentiation along the proximodistal axis (Fig.4b,c) and adaxial rachis (Fig.4h,i) of the leaf.
  • the activity of C. hirsuta ASl in controlling differentiation of the adaxial side of the leaf is shared by ARP proteins in other plant species 8 ' 16 ' 17 . Additionally, these results indicate that, at least in C. hirsuta, the roles of ARP proteins in axial patterning and KNOX repression are intimately intertwined.
  • C. hirsuta ASl and A. thaliana ASl act to safeguard leaf fate by maintaining the repression of KNOX expression in leaves.
  • KNOX gene regulatory sequences drive expression in the leaf where KNOX activity is required for dissected leaf development.
  • C. hirsuta ASl constrains the spatiotemporal domain of KNOX expression and hence leaflet number and arrangement.
  • the PHANTASTICA gene encodes a MYB transcription factor involved in growth and dorsoventrality of lateral organs in Antirrhinum. Cell 93, 779-789 (1998).
  • Gleave, A.P. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant MoI. Biol. 20, 1203-1207 (1992).

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Abstract

The invention provides a method for obtaining a first-generation transgenic plant of the genus Cardamine, which method comprises: (i) contacting a floral meristem of a plant of the genua Cardamine with an Agrobacterium cell, which Agrobacterium cell comprises a heterologous polynucleotide; (ii) allowing the plant to set seeds; (iii) harvesting the seeds; (iv) identifying a seed which comprises the heterologous polynucleotide; and (v) regenerating a plant from the seed identified in step (iv). The method may be used in the evaluation of the effects of polynucleotides which are candidates for transfer into crop species.

Description

Plant Transformation
Technical field of the invention
This invention relates to a model plant. The model plant can be used to reveal new aspects of gene function and in the validation of genes intended for use in crop modification.
Background of the invention
Arabidopsis thάliana has been widely used as a model organism for carrying out plant molecular genetics. Important reasons for the choice of Arabidopsis as a model plant include the ease with which it can be grown indoors in large numbers and the fact that it produces thousands of offspring per plant after 8 to 10 weeks.
Arabidopsis also has the advantage for molecular analysis of having a genome which is one of the smallest known (7 x 107 nucleotide pairs) and which has been sequenced in its entirety. Furthermore, cell culture and genetic transformation methods have been established and large numbers of interesting mutants have been isolated. Like many flowering plants, Arabidopsis can reproduce as a hermaphrodite because a single flower produces both eggs and the pollen that can fertilize them. Therefore, when a flower that is heterozygous for a recessive lethal mutation is self-fertilized, one-fourth of its seeds will display the homozygous embryonic phenotype.
The major drawback associated with the use of Arabidopsis is that it is not a commercially important plant; it is neither a crop species nor an ornamental species. However, crop species in particular are difficult to manipulate at the genetic level (they have long life- cycles, large (sometimes polyploid) genomes and are often difficult to transform). Therefore, Arabidopsis has been used as a model for crop species more out of necessity than because it is truly representative. In particular, it is common for genes which are candidates for transfer to crop species to be "tested" first in Arabidopsis, prior to the more labourious validation in a particular crop species.
Of course, such an approach is limited by the fact that many crop species are taxonomically only distantly related to Arabidopsis. In particular, a transgene showing a particular effect in Arabidopsis may show an entirely different effect in a crop species. Such differences are likely to increase with increasing taxonomic divergence of Arabidopsis and the species of interest.
Furthermore, the comparative study of mutant phenotypes shows that loss of function mutations in orthologous genes may have different phenotypic effects in different species (Tsiantis et at, Science 2845 154-156; Timmermans et at, Science 284, 154-153; and Tsiantis et at, Plant Cell 13, 733-8). This suggests that a full understanding of gene function is best achieved by comparing the effects of mutation or overexpression in a number of species. Such comparisons are more valid if the organisms examined are taxonomically related. Arabidopsis is the best characterised system for elucidating gene function in plants. Therefore, the development of another model system that is related to Arabidopsis would constitute a major breakthrough in terms of improving our ability to understand the function of genes identified by the now completed Arabidopsis genome project. Further, it would be desirable if such a system is more closely related to important crops such as the Brassicas (for example, B. napus, B. olerecea, B. junea or Sinapis alba).
There is therefore a need for new model plant species which has the advantages shown by Arabidopsis in terms of tractability with respect to genetic manipulation, analysis and transformation, but which, although related to Arabidopsis, is also more closely related to important crop species than Arabidopsis.
Summary of the invention
The present invention is based on the identification of a new model plant species. Thus, it has now been shown that the species Cardamine hirsuta shares many of the properties that make Arabidopsis genetically tractable. In particular, it has been shown that C. hirsuta can be transformed according to a highly convenient technique, a technique which previously was limited to the transformation of Arabidopsis. This is surprising because the technique has been shown to be unsuccessful in plants more closely related to Arabidopsis than C. hirsuta.
C. hirsuta is a member of the genus Cardamine, a group of plants which is more closely related to the important crop plants, the Brassicas, than Arabidopsis. Therefore, transgenic constructs aimed at the improvement of agricultural performance in Brassicas could first be tested in C. hirsuta.
Indeed, it has now been shown that expression of the same gene gives rise to different effects in Arabidopsis and C. hirsuta. Thus, C. hirsuta plants which ectopically express the homeobox gene KNOTTEDl display severe aberrations in floral organ development which are not observed in Arabidopsis plants expressing KNOTTEDl . Floral development is disrupted in the transgenic C. hirsuta plants such that floral organs exhibit carpelloid transformations and ectopic ovules appear to be present on sepals. In fact, the transgenic 35S::KN1 plants demonstrate a complete failure to elaborate the 4 whorls of floral organs present in the wild type flower. Instead floral structures are much reduced frequently to a single sepal whorl bearing ectopic ovules. This indicates that within the context of Cardamine, overexpression of KNl perturbs floral development at a very early stage before organ identities within the flower are allocated. Such phenotypes have not been observed in Arabidopsis plants overexpressing KNl (Lincoln et al. Plant Cell 6(12). 1859-1876, 1994).
This comparison shows clearly that there are genes that behave differently as transgenes in Cardamine as compared to Arabidopsis. Given that Cardamine is more closely related to
Brassicas than Arabidopsis is, Cardamine represents a better model for the Brassicas than Arabidopsis does. For example, the expression of a transgene which has no apparent effect on the development of Arabidopsis may well have an effect on the development of Brassicas. The use of Cardamine is much more likely to reveal such an effect and will allow the testing of transgenes before the time-consuming step of gene transfer into a Brassica is carried out. The use of Cardamine may therefore allow certain effects of transgene expression to be identified which would not be identified by the use of Arabidopsis.
According to the present invention there is thus provided a method for obtaining a first- generation transgenic plant of the genus Cardamine, which method comprises: (i) contacting a floral meristem of a plant of the genus Cardamine with aτiAgrobacterium cell, which Agrobacterium cell comprises a heterologous polynucleotide; (ii) allowing the plant to set seeds;
(iii) harvesting the seeds; (iv) identifying a seed which comprises the heterologous polynucleotide; and (v) obtaining a plant from the seed identified in step (iv). The invention also provides: a transgenic plant or transgenic seed of the genus Cardamine, optionally obtainable according to a method of the invention; a method for evaluating the function of a polynucleotide sequence, which method comprises:
(i) disrupting that polynucleotide sequence in a plant of the genus Cardamine; and
(ii) determining the effect of disrupting the polynucleotide sequence; - a method for evaluating the suitability of a polynucleotide which is a candidate for transfer into a crop species, which method comprises: (i) preparing a transgenic Cardamine plant which expresses the candidate polynucleotide; and
(ii) determining the effect of the candidate polynucleotide on the transgenic Cardamine plant; a method for obtaining an improved crop plant, which method comprises:
(i) identifying a polynucleotide which shows a beneficial effect in Cardamine; and
(ii) introducing that polynucleotide into the crop plant; and - an improved crop plant obtained by such a method.
In a preferred method of the invention said heterologous polynucleotide encodes a polypeptide.
In an alternative preferred method of the invention said heterologous polynucleotide comprises a nucleic acid molecule from which an antisense nucleic acid is expressed. In a further preferred method of the invention said heterologous polynucleotide comprises a nucleic acid molecule from which an interfering RNA is expressed.
An embodiment of the invention will now be described by example only and with reference to the following figures:
Figure 1 KNOX proteins accumulate in the dissected leaf of C. hirsuta but are excluded from the simple leaf of A. thaliana. (a,b) Mature plants of C. hirsuta (a), and A. thaliana (b). (c,d) Rosette (left) and cauline (right) leaves of C. hirsuta (c) and .4. thaliana (d). C. hirsuta leaves are dissected into leaflets, each of which is borne on a petiolule attached to the rachis (c), whereas A. thaliana leaves are simple (d). (e,f) Immunolocalization of class I KNOX proteins in transverse sections of shoot apices shows nuclear expression of KNOX proteins in the SAM (*) but no expression in initiating leaf cells (arrow) of C. hirsuta (e) and A. thaliana (f). In C. hirsuta (e), nuclear expression of KNOX proteins is seen throughout plastochron (P) 2 (2), localized to the initiating leaflets and vasculature in P3 (arrowheads, 3) and limited to vascular-associated cells in P4 (4). No KNOX expression is seen in leaves of A. thaliana (f). (g,h) Scanning electron micrographs of the shoot apex of C. hirsuta (g) shows that the youngest leaf primordium (1) initiates on the flanks of the SAM (*) with a simple shape, and leaflets initiate at the leaf margins 1—2 plastochrons later (arrowheads, 3,4; the distal leaflet of P4 has been removed) in a basipetal manner. By contrast, leaf primordia in A, thaliana (h) initiate at the SAM (*) and continue to develop with a simple shape. Scale bars: 2 cm (a,b), 0.5 cm (c,d), 50 μm (e,f), 100 μm (g,h).
Figure 2 C. hirsuta STM expression in young leaf primordia is required for leaflet initiation. (a,b) In situ localization of C. hirsuta 5TMmRNA. C. hirsuta STM expression is restricted to the SAM of embryos (longitudinal section, a) and vegetative shoot apices (transverse section, b) but absent from most cells that comprise leaf primordia. However, expression is observed throughout the outer cell layers of PO to P2 leaf primordia (arrowheads, 0, 1, 2) and in some cells of P3 leaf primordia (arrows, 3). (c) In situ localization of STM mRNA. Transverse section through an A. thaliana shoot apex shows expression is restricted to the SAM and absent from leaf primordia (0-3). (d-f) Vegetative C. hirsuta plants, (d) Wild-type, (e) Scanning electron micrograph (SEM) of a strong C. hirsuta 5TMRNAi line with fused cotyledons (arrowhead) and ectopic leaf initiation (arrow), (f) A weak C. hirsuta STM RNAi line with simple leaves, (g-i) Rosette C. hirsuta leaves, (g) wild-type with four lateral leaflets, (h) A weak C. hirsuta STM RNAi line lacking lateral leaflets, (i) 35S::KN1-GR induced with 10"6 M dexamethasone with ectopic leaflets initiated upon leaflets (arrowheads). (j,k) In situ localization of C. hirsuta H4 mRNA. Longitudinal sections through vegetative apices of wild-type (j) and C. hirsuta STM RNAi (k). * indicates meristem. (I,m) SEM of epidermal cells of the terminal leaflet of leaf three
SUBSTITUTE SHEET in wild-type (1) and C. hirsuta STM RNAi (m). Scale bars: 20 μm (a-c,l,m), 1 cm (d, f-i), 500 μm (e), 50 μm (j,k).
Figure 3 ASl function is conserved between A. ihaliana and C. hirsuta, whereas 5' upstream regions of KNOX genes are sufficient to drive species-specific expression, (a-c) Rosettes of A. thaliana wild-type (Columbia) (a), asl-1 showing an asymmetrical leaf lamina (arrowhead) and short, broad petioles (arrow) (b) and asl- l;35S::ChASl transformants showing restoration of leaf shape (arrowhead) and petiole length (arrow) (c). (d-f) Seedlings of the same genotypes stained for BP::GUS expression. BP::GUS expression is observed only in the SAM of wild-type (d); ectopic expression is observed in asl leaves (arrow, e), and this ectopic expression is repressed in asl-1 ;35S::cASl (f). (g) In situ localization of C. hirsuta ASl mRNA in the shoot apex of C. hirsuta, showing expression in initiating leaf primordia (arrowhead) on the flanks of the SAM (*) and in leaflets (arrow), (h-n) Seedlings stained for GUS expression. BP::GUS expression is restricted to the SAM in A. thaliana (h) and C. hirsuta (i). ChBP: :GUS is expressed in both the SAM and leaves in A. thaliana (j) and C. hirsuta (k). Two leaves have been removed for clarity in i and k. ChSTM:: GUS is expressed in both the SAM and the abaxial side of young leaves (arrowheads) in C. hirsuta (1) and A. thaliana (m), whereas STM:: GUS expression is restricted to the SAM in A. thaliana (n). Scale bars: 0.5 cm (a-c), 100 μm (d-f,l-n), 50 μm (g), 1 cm(h-k).
Figure 4 C. hirsuta ASl delimits C. hirsuta BP expression and controls leaflet positioning by regulating growth, (a-c) Silhouettes of rosette (left) and cauline (right) leaves of 35S::KN1~GR induced once with 10~6 M dexamethasone (a), wild type (b), and chasl-1 (c). Arrows denote the length of the leaf rachis and arrowheads indicate extra leaflets. (d,e) In situ localization of C. hirsuta BP mRNA. Longitudinal sections of wild-type (d) and chasl-1 (e) showing ectopic expression in the adaxial side and the base of developing leaves (arrowheads). (f,g) In situ localization of C. hirsuta H4 mRNA. Longitudinal sections of wild-type (f) and chasl-1 (g) showing C. hirsuta /W-expressing cells at the base of developing leaves in chasl-1 but not wild-type (arrowheads). * indicates shoot meristem. (h,i) Scanning electron micrographs of epidermal cells on the adaxial surface of the leaf rachis at the position of leaflet insertion in wild-type (h) and chasl-1 (i). Scale bars: 1 cm (a-c), 20 μm (d- i).
Figure 5. S equence comparisons of 5' upstream regions of STM and BP from Arabidopsis and C. hirsuta.
VISTA analysisl (70% identity and 100 base sliding window) identified conserved regions with >70% identity shared by Arabidopsisanά C. hirsuta, shown in pink. (A) Five conserved regions were identified in the 5' upstream regions of STM (see below for sequences). Region 1: 146 bp at 71.2%, region 2:93bp at 81.7%, region 3: 308bp at 80.8%, region 4: 99bp at 72.7%, and region 5: 754bp at 80.1 %, giving a total of 1400bp at 78.9%. (B) Two conserved regions were identified in the 5' upstream regions of BP (see below for sequences). Region 1: 89bp at 89.9%,region 2: 778bp at 76.6%, giving a total of 867bp at 78.0%. Regions of divergent sequence are good candidates for conferring leaf expression in C. hirsuta. It will be interesting to determine whether such regions reside within conserved islands of sequence or within the more rapidly evolving non- 5 (2 of 3)
conserved regions. Conserved regions are indicated by labeled boxes, the translation start site for each gene is identified by an arrow, and the numbers below the graphs denote base pairs of C. hirsuta sequence. References Loots, G.G., Ovcharenko, L5 Pachter, L., Dύbchak, I. & Rubin, E.M. rVista for comparative sequence-based discovery of functional transcription factor binding sites. Genome Res 12, 832-9 (2002).
Figure 6. A: Conserved sequences of 5' upstream regions of STM fcovn. Arabidopsis and C. hirsuta B: Conserved sequences of 5! upstream regions of BP from Arabidopsis and C. hirsuta
Figure 7. Leaflet formation in response to ectopic KNOX activity in Arabidopsis.
Arabidopsis rosette leaves of the following genotypes: (a) wild type (Columbia ecotype), showing a simple form,
(b) 35S::LhGR»STM showing leaflet formation. In this experiment STM is expressed under the 35S promoter in an inducible fashion by being placed under the control of the Dexamethasone-inducible LhGRproteinl, (c) wild type (Landsberg erecta ecotype), showing a simple form, (d) ANT»BP (e) PHV»BP (f) FIL»BP showing leaflet formation when BP is driven by the AINTEGUMENTA (ANT), PHA VOLUTA (PHV), and FILAMENTOUS FLOWER (FIL) promoters that are all active early in leaf development, note initiation of secondary leaflets initiation in FIL»BP (arrows, g).
References
1. Craft, J. et al. New pOp/LhG4 vectors for stringent glucocorticoid dependent transgene expression in
Arabidopsis. Plant J 41, 899-918 (2005).
Figure 8. Predicted ChASl protein sequence.
ClustalW alignment ofPHAN (Genbank accession number AJ005586), ASl (Genbank accession number NM129319) and ChASl (Genbank accession number DQ512733) predicted amino acid sequences, indicating the premature stop codon introduced at position 170 in chasl-1 (*). Southern blot analyses indicate that ChASl is a single copy gene in C. hirsuta (data not shown). ChASl and ASl share 91.4% amino acid sequence identity.
Figure 9. Increased KNOX protein accumulation in ChASl RNAi leaves, a-d. Immunolocalisation of class I KNOX proteins. Transverse sections through shoot apices of wild type (a) and ChASl RNAi (b) show nuclear expression of KNOX proteins in the SAM (*) and young leaf primordia (outlined), with increased KNOX accumulation evident, particularly in the adaxial side of the developing ChASl RNAi leaf, closest to the SAM. Transverse sections through plastochron 4 leaves of wild type (c) and ChASl RNAi (d) show increased accumulation of KNOX proteins in vascular associated cells of ChASl RNAi leaves, particularly on the adaxial side, closest to the xylem pole. Abbreviations: x; xylem, p; phloem. Scale bars: 20 μm (a-d ). 5 (3 of 3)
Figure 10. Loss of adaxial cell differentiation in chasl-1 leaves.
Scanning electron micrographs of the adaxial leaf rachis of (a) wild type, (b) 35S::BP and (c) chasl-1. Note the presence of large, elongated epidermal cells that overlay the midvein in wild type (arrow, a), whereas these cells differentiate only infrequently in 35S::BP (arrow, b), and no such adaxial cell differentiation is observed in chasl-1. Cell size is also reduced in 35S::BP (b) and chasl-1 (c) compared with wild type adaxial leaf rachis (a). Scale bars: lOOμm.
Figure 11. Analysis of transcript levels in RNAi lines.
RT-PCR gel blot analysis of ChASl and ChSTM transcript levels in eight independent T2 ChASl RNAi lines and three independent Tl CASTMRNAi lines respectively, compared with wild-type seedlings. Amplification of ChGAPDH shows the relative amount of cDNA in each sample.
Figure 12. Primer sequences
Detailed Description of the Invention
Throughout the present specification and the accompanying claims the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.
The invention provides a method for the preparation of a transgenic plant of the genus
SUBSTJTUTE SHEET (RULE 26) 6
Cardamine. Preferably, the transgenic plant is of the species C. hirsuta. The method of the invention comprises dipping at least one floral meristem of a plant of the genus Cardamine into a solution of Agrobacterium, wherein the Agrobacteriiim comprises a heterologous polynucleotide which is to be transferred to the plant. The dipped plants are then grown under conditions such that they set seed. The seed is then harvested and screened to identify seeds which comprise the heterologous polynucleotide. Seeds identified as comprising the heterologous polynucleotide may be grown to give rise to a transgenic plant.
As used herein the term "plant" includes reference to whole plants, plant organs (for example leaves, stems, roots etc.), seeds, plant cells and progeny plants. Plant cell as used herein includes reference to seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.
The term "transgenic plant" as used herein refers to a plant which comprises a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome so that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or incorporated as part of a larger polynucleotide construct. The term transgenic may be applied to any cell, cell line, tissue, plant part or plant, the genotype of which has been altered as well as those created by sexual crosses or asexual propagation from an initial transgenic.
The term "transgenic" as used herein should not be taken to encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally-occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.
A heterologous polynucleotide is one mat is to be introduced into a plant or has been introduced into a plant, thus giving rise to a transgenic plant. A heterologous polynucleotide may comprise sequences which originate from a species other than that to be transformed.
Indeed, the heterologous polynucleotide may comprise sequences which are not of plant origin.
Such sequences may originate from, for example, a virus, a bacterium, a fungus or an animal.
Alternatively or additionally, a heterologous polynucleotide may comprise, or consist essentially of, sequences which originate from the species to be transformed. In the latter case, a transgenic plant of the invention may thus comprise more than one copy of a polynucleotide which occurs naturally in the species in question. Of course, a heterologous polynucleotide may comprise sequences which originate from the species to be transformed and sequences which do not originate from the species to be transformed. A heterologous polynucleotide which is to be introduced in to a plant or has been introduced into a plant, giving rise to a transgenic plant, is sometimes known as a "transgene",
JKQTtTi 7 although sometimes the term "transgene" is used to refer only to a coding sequence comprised by a heterologous polynucleotide or to a coding sequence in combination with its control sequences. The precise nature of the meaning of the term will be clear to one skilled in the art in the context of its use. A heterologous polynucleotide suitable for use in the invention is typically a DNA or an analog thereof. Suitable analogs have the essential nature of a natural DNA in that they hybridise, under stringent hybridisation conditions, to substantially the same nucleotide sequence comprising only naturally-occurring nucleotides. Typical stringent hybridisation conditions include from about 0.1 to about 0.2 x SSC at about 6O0C to about 650C. Typically the heterologous polynucleotide comprises at least one, often two, or even three or more, coding sequences. A coding sequence is a nucleotide sequence that is capable of being transcribed by the transcriptional mechanisms endogenous to the recipient cell. A coding sequence may encode, for example, an mRNA, a tRNA, an rRNA, a ribozyme, or any other form of RNA known in the art.
The invention also includes an heterologous polynucleotide comprising an interfering RNA (siRNA) or antisense RNA.
A number of techniques have been developed in recent years which claim to specifically ablate genes and/or gene products. For example, the use of anti-sense nucleic acid molecules to bind to and thereby block or inactivate target mRNA molecules is an effective means to inhibit gene expression. Tin's is typically very effective in plants where anti-sense technology produces a number of striking phenotypic characteristics. A more recent technique to specifically ablate gene function is through the introduction of double stranded RNA, also referred to as small inhibitory or interfering RNA (siRNA), into a cell which results in the destruction of mRNA complementary to the sequence included in the siRNA molecule. The siRNA molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double stranded RNA molecule. The siRNA molecule is typically derived from exons of the gene which is to be ablated.
The mechanism of RNA interference is being elucidated. Many organisms respond to the presence of double stranded RNA by activating a cascade that leads to the formation of siRNA. The presence of double stranded RNA activates a protein complex comprising RNase III which processes the double stranded RNA into smaller fragments (siRNAs, approximately 21-29 nucleotides in length) which become part of a ribonucleoprotein complex. The siRNA acts as a guide for the RNase complex to cleave mRNA complementary to the antisense strand of the siRNA thereby resulting in destruction of the mRNA.
SS SRCJTITf ITP S In using anti-sense nucleic acid molecules to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mKNA transcribed from the "sense" strand of the target gene. See, for example, Rothstein et al, 1987; Smith et al, (1998),
Nature 334, 724-726; Zhang et al (1992) The Plant Cell 4, 1575-1588, English et al. (1996) The Plant Cell 8, 179 188. Antisense technology is also reviewed in Bourque (1995), Plant Science 105, 125-149, and Flavell (1994) PNAS USA 91, 3490-3496.
Preferably, the coding sequence encodes an mRNA. More preferably, the mRNA is capable of being translated by the translation mechanisms endogenous to the recipient cell. The mRNA may be monocistronic or polycistronic. If the mRNA is polycistronic, for example encoding two or three polypeptides, it may contain an internal ribosome entry site (IRES).
A polypeptide encoded by a heterologous polynucleotide of the invention may have any function. The naturally-occurring form of the protein encoded by the coding sequence may be, for example, typically an extracellular protein (e.g. a secreted protein), an intracellular protein (e.g. cytosolic, organellar, plastidic, nuclear or membrane protein) or a protein present in the cell membrane. The naturally- occurring form of the encoded polypeptide may be constitutively expressed or be tissue specific. Functions of polypeptides encoded by the coding region may include herbicide, insecticide or disease resistance. Preferred herbicide resistance polypeptides may be responsible for tolerance to, for example: glyphosate (e.g. using an EPSP synthase (e.g. EP-A-O 293,358) or a glyphosate oxidoreductase (e.g. WO 92/000377)); fosametin; a dihalobenzonitrile; glufosinate, e.g. using a phosphinothrycin acetyl transferase (PAT) or a glutamine synthase (e.g. EP-A-O 242,236); asulam, e.g. using a dihydropteroate synthase (EP-A-O 369,367); or a sulphonylurea
(e.g. using an ALS); diphenyl ethers such as acifiuorfen or oxyfluorfen (e.g. using a protoporphyrogen oxidase); an oxadiazole such as oxadiazon; a cyclic imide such as chlorophthalim; a phenyl pyrazole such as TNP, or a phenopylate or carbamate analogue thereof; or spectinomycin e.g using the aadA nucleotide sequence, as exemplified below. Insect resistance may be introduced using coding sequences encoding, for example,
Bacillus thuringiensis (Bt) toxins. Likewise, a coding sequence may encode a polypeptide which confers insect resistance on the recipient plant, e.g. as in WO91/02701 or WO95/06128.
A coding sequence of a heterologous polynucleotide of the invention may encode a polypeptide with a role in a metabolic pathway, for example a chloroplastic metabolic pathway such as the photosynthetic metabolic pathway. Typically, the expression of the encoded polypeptide will alter flux in the pathway and thus the encoded polypeptide may alter the photosynthetic ability of the transformed plant. Alternatively, expression of the encoded
UTE SHEET RULE 26) 9 polypeptide may introduce a new metabolic step or steps into the recipient plant.
Alternatively, a polypeptide encoded by a heterologous polynucleotide of the invention may allow increased uptake of a nutrient, for example leading to increased production of a carbohydrate such as starch. Alternatively, a polypeptide encoded by a heterologous polynucleotide of the invention may allow a transgenic plant comprising that heterologous polynucleotide to take up a substance which the corresponding wild type plant would not. Such heterologous polynucleotides may be useful where plants are required to carry out bioremediation. Suitable polypeptides include ion channels in the case of bioremediation of heavy metals. Other suitable polypeptides include those conferring resistance to environmental insult, for example drought tolerance or tolerance to high salinity.
Alternatively, a polypeptide encoded by a heterologous polynucleotide of the invention may be expressed in order to enable its mass production, and have no particular relation to the biological processes of cell or plant in which it is expressed. It may be any polypeptide known in the art. It may be derived from any organism, for example from a prokaryote, fungus, plant or animal and may perform any function in vivo.
Alternatively, a polypeptide encoded by a heterologous polynucleotide of the invention may be an immunoglobulin. A transgenic plant may be capable of expressing more than one immunoglobulin, for example two, three or four immunoglobulins. If the expression of more than one immunoglobulin is required, they may be introduced into a Cardamine plant on the same heterologous polynucleotide, on different heterologous polynucleotides introduced into the same plant, on different heterologous polynucleotides introduced into separate plants which are subsequently sexually crossed or via a combination of these techniques.
A transgenic plant of the invention may therefore be capable of expressing an antibody. The term antibody includes fragments of whole antibodies which maintain the binding activity characteristic of the whole antibody. Such fragments include Fv, F(ab') and F(ab)2 fragments, as well as single chain antibodies. Furthermore, the antibodies or fragments thereof may be chimeric antibodies, CDR-grafted antibodies or humanised antibodies.
Typically, the antibody will be one which has a therapeutic use, for example in vaccination. The polypeptide may be an enzyme, such as a catabolic or anabolic enzyme. The enzyme may be, for example, a metabolic (e.g. glycolysis, Calvin cycle or Krebs cycle) enzyme or a cell signalling enzyme. The enzyme may make, break down or modify lipids, fatty acids, amino acids, proteins, nucleotides, polynucleotides (e.g. DNA or RNA) or carbohydrates (e.g. starch, amylose, amylopectin, or smaller sugars), and may be a protease, lipase or carbohydrase. The enzyme may be a protein modifying enzyme, such as an enzyme that adds or takes chemical moieties from a protein (e.g. a kinase or phosphatase).
SUBSTITUTE SHEET RULE 26 10
The polypeptide may be a transport or binding protein (e.g. which binds and/or transports a vitamin, metal ion, amino acid or lipid, such as cholesterol ester transfer protein or phospholipid transfer protein). The polypeptide may be cytotoxic. The polypeptide may be a cytochrome. The polypeptide may be useful for industrial purposes. For example, xylanases, enzymes involved in plant cell wall modification, can be useful, for example in paper manufacture. Alternatively, the polypeptide may be useful for therapeutic purposes. The polypeptide may thus be useful in a method of treatment of the human or animal body, or as an antigenic polypeptide for use as an edible or extractable vaccine. The polypeptide may be able to cause the replication, growth or differentiation of cells.
The polypeptide may aid transcription or translation of a gene or may regulate transcription or translation (e.g. a transcription factor or a protein that binds a transcription factor or polymerase). The polypeptide may be a signalling molecule, such as an intracellular or extracellular signalling molecule (e.g. a hormone). Preferred polypeptides are those which are known to affect the development of plants.
The polypeptide may affect the development of any part of a developing plant, for example the endosperm, embryo, root, shoot, leaf, inflorescence, flower or seed. Many such polypeptides are known from a number of diverse plant species, although Arabidopsis is perhaps the best characterised plant in terms of genes affecting its development. Therefore, heterologous polynucleotides for use in the present invention may comprise a coding sequence for a developmentally significant polypeptide which originates from a plant other than a Cardamine plant. Alternatively, for any given developmentally significant coding sequence from a plant other than a Cardamine plant, it should be possible to identify a corresponding coding sequence from a Cardamine plant. Therefore, a heterologous polynucleotide for use in the present invention may comprise a coding sequence for a developmentally significant polypeptide which originates from a Cardamine plant itself.
A number of polypeptides are known which affect the development of the plant embryo, endosperm or seed and any of those polypeptides may be used according to the invention. Examples of genes affecting seed development include a number of chromatin modifying genes, for example the polycomb genes FIE and MEDEA (MEA) (Yadegari et ah,
Plant Cell 12, 2367-2381); Kiyosue et al., Proc. Natl. Acad Sci. USA 96(7), 4186-91, 1999; and Sorensen et ah, Curr. Biol. 11(4), 277-281, 2001). The polypeptides encoded by these genes may be used in the invention.
Other chromatin remodelling genes may affect the overall size of organs. For example, SERRATE (Prigge and Wagner, Plant Cell 13(6), 1263-1279, 2001), PICKLE (Ogas et al, Proc.
Natl. Acad. Sci. USA 96(24), 13839-13844, 1999) and AINTEGUMENTA (Mizukami and
SUBSTITUTE 11
Fisher, Proc. Natl. Acad. ScL USA 97(2\ 942-947). Any of the polypeptides encoded by those genes may be used in the invention.
A number of polypeptides are known which affect the development of roots and any of those polypeptides may be used according to the invention. A number of polypeptides are known which affect the development of leaves and any of those polypeptides may be used according to the invention, for example the polypeptide encoded by the KNOTTEDl and other KNOTTEDl-like homeobox (KNOX) genes.
A number of polypeptides are known which affect the development of flowers and any of those polypeptides may be used according to the invention. There are four classes of genes affecting floral development.
Thus, the polypeptide may be one encoded by a flowering time gene, mutations in which cause early or late flowering. Flowering time genes can be divided into distinct classes, based on their differential responses to a number of environmental conditions, such as day length and vernalization (for a review see Weigel et al. Ann. Rev. Genet. 29, 19-39, 1995). Thus, the polypeptide could be encoded by a late flowering or an early flowering gene, for example.
Alternatively, the polypeptide may be one encoded by a meristem identity gene. There are two types of meristem identity genes: those which specify flower meristem identity, for example LEAFY, FLORICAULA, APETALAl, CAULIFLOWER or UNUSUAL FLORAL ORGANS; or those which maintain inflorescence meristem identity, for example TERMINAL FLOWER or CENTRORADIALIS.
A heterologous polynucleotide of the invention could alternatively encode a polypeptide encoded by a floral organ identity gene. Floral organ identity genes determine the fate of organ primordia and their action has been explained by the "ABC" model of flower development. Thus, a polypeptide in the invention could be encoded by: an "A" function gene, such as APETALAl (which is involved in both meristem identity and organ identity ) or
APETALA2; a "B" function gene, such as PISTILLATA, APETALAS, DEFICIENS or GLOBOSA; or a "C" function gene such as AGAMOUS or PLENA.
A further class of genes which affect flower development includes late-acting genes that control ovule development. A polypeptide encoded by such a gene may be used in the invention. Alternatively, a coding sequence in a heterologous polynucleotide of the invention may encode a polypeptide of unknown function. That is, the heterologous polynucleotide may comprise, for example, an expressed sequence tag (EST). A transgenic Cardamine plant of the invention may thus be used to try to reveal the function of a polynucleotide sequence of unknown function. Typically, the heterologous polynucleotide for use in a method of the invention will comprise a coding sequence of interest operably linked to a control sequence (or control sequences). The control sequence or sequences is/are capable of providing for the
SUBSTITUTE SHF 12 expression of the coding sequence by the host cell into which the heterologous polynucleotide is transferred. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
A control sequence such as a promoter "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence. The control sequence will typically comprise a promoter and optionally also comprise other types of control sequence, for example an enhancer and/or terminator. A control sequence may be positioned 5', 3' or internal to (for example in an intron) a coding sequence. A coding sequence may be operably linked to more than one control sequence, for example two, three, four or five control sequences. Such multiple control sequences may be positioned, for example, entirely 5' to the coding sequence. However, more typically control sequences will be located both 5' and 3' to the coding sequence, with optional internal control sequences.
A promoter is a nucleotide sequence capable of initiating transcription of a coding sequence. Typically, a coding sequence is positioned 3' (i.e. downstream) to a promoter, although promoters may be situated in introns. An enhancer is any polynucleotide sequence capable of increasing the level of transcription initiating from a promoter and may act on a cis or trans basis. A terminator is any polynucleotide sequence capable of promoting dissociation of an RNA polymerase from the said sequence. Control sequences may be derived from any suitable source and may be generated by recombinant techniques or synthetic means. Control sequences may be truncated or comprise sequence alterations, for example, in order to reduce or remove areas of homology to the proposed recipient genome or to introduce or improve sequences essential for function as a regulatory region. Control sequences may be specifically designed de novo to ensure heterology to the recipient genome and to comprise the essential features of functional control sequences. Typically, suitable promoter regions will contain well defined -10 and -35 sequence motifs (Tanaka et al, FEBS. LETT. 413, 309-313, 1997; Isono et al, Proc. Natl. Acad. Sci. USA 94, 14948-14953, 1997; Kestermann et al, Nucleic Acids Res. 26, 2747-2753, 1998). Typically a -10 sequence motif will have the sequence 5'-TATAAT-3', and a -35 sequence motif will have the sequence motif 5'-TTGACA-3', although the skilled person will appreciate that variants of these sequence can be used (Grierson and Covey, Plant MoI. Biol. (2nd
Edition), Chapman Hall, New York, pρ.64-65, 1988).
Promoters and other regulatory elements may be selected to be compatible with the host cell, i.e. the plant species, for which the expression vector is designed.
Promoters suitable for use in plant cells may be derived, for example, from plants or from bacteria that associate with plants or from plant viruses. Thus, promoters from
Agrobacterium spp. including the nopaline synthase (nos), octopine synthase (ocs) and
SUBSTITUTE SHEET /RULE 26> 13 mannopine synthase (mas) promoters are preferred. Also preferred are plant promoters such as the ribulose bisphosphate small subunit promoter (rubisco ssu), histone promoters (EP-A-O 507,698), the rice actin promoter (US Patent No. 5,641,876) and the phaseolin promoter. Also preferred are plant viral promoters such as the cauliflower mosaic virus (CAMV) 35S and 19S promoters, and the circovirus promoter (AU-A-689,311).
Depending on the pattern of expression desired, promoters may be constitutive, tissue- or stage-specific, and/or inducible. For example, strong constitutive expression in plants can be obtained with the CAMV 35S, Rubisco ssu, or histone promoters mentioned above. Also, tissue- specific or stage-specific promoters may be used to target expression of polypeptides of the invention to particular tissues in a transgenic plant or to particular stages in its development.
Thus, for example seed-specific, root-specific, leaf-specific, flower-specific etc promoters may be used. Seed-specific promoters include those described by Dalta et al (Biotechnology Ann. Rev. (1997), 3, pp.269-296). Particular examples of seed-specific promoters are napin promoters (EP-A-O 255, 378), phaseolin promoters, glutenine promoters, helianthenine promoters (WO92/17580), albumin promoters (WO98/45460), oleosin promoters (WO98/45461) and ATSl and ATS3 promoters (PCT/US98/06798).
Chemically inducible promoters such as those activated by herbicide safeners may also be used, for example the maize GST 27 promoter (WO97/11189), the maize In2-1 promoter (WO90/11361), the maize In2-2 promoter (De Veylder et al, Plant Cell Physiology, Vol. 38, pp568-577 (1997). Further examples of inducible promoters known in the art include those modulated by exposure to tetracycline, ecdysteroids, glucocorticoids, plant growth regulators such as abscisic acid, animal hormones, nitrates, metal ions (such as copper), environmental conditions (such as cold, heat, light or dark) and wounding. Furthermore, promoters may be used that are limited to expression in specific tissue or cell types, and/or during specific developmental stages, such as during flowering or senescence.
Other regulatory signals may also be incorporated in the vector, for example a terminator and/or polyadenylation site. Preferred terminators include the nos terminator and the histone terminator of EP-A-O 633,317 although other terminators functional in plant cells may also be used. Typically, therefore, a chimeric gene comprises the following elements in 5' to 3' orientation: a promoter functional in a host, i.e. plant, cell, as defined above, a polynucleotide of the invention and a terminator functional in a said cell, as defined above.
Other elements, for example enhancers, may also be present in a vector of the invention. Enhancers include the tobacco etch virus (TEV) enhancer and the tobacco mosaic virus (TMV) enhancer (WO87/07644).
Similarly, an origin of replication may be present. Sequences capable of securing
O! IDPTITi !Tr > - - - - - 14 integration into a cell's genome, e.g. Agrobacterium tumefaciens T-DNA sequences may be present.
A heterologous polynucleotide suitable for use in the invention may comprise one or more, for example two or three, nucleotide sequences encoding selectable marker polypeptides operably linked to their own control sequences in addition to that/those encoding a polypeptide(s) of interest. The control sequences may be the same as or different from those operably linked to the coding sequence(s) encoding a polypeptide(s) of interest. A selectable marker polypeptide is one which, for example, allows a cell which expresses that marker polypeptide to survive in the presence of an agent that would kill a similar cell which does not express that marker polypeptide.
Any coding sequence encoding a suitable selectable marker polypeptide may be used in a heterologous polynucleotide of the invention. Typically, herbicide resistance genes, for example as defined above, may be used as selectable markers. Notable examples include polypeptides conferring resistance to herbicides such as bialaphos, glyphosate or an isoxazole herbicide may be used. Particular examples are described in EP-A-O 242,236, EP-A-O 242,246,
GB-A-2,197,653, WO91/02701, WO95/06128, WO96/38567 and WO97/04103.
Alternatively, a coding region that encodes a polypeptide which provides resistance to aminoglycoside antibiotics may be used as a selectable marker, for example, encoded polypeptides that provide resistance to kanamycin (e.g. the nptl or nptll genes) , neomycin, ampicillin or chloramphenicol (the CAT gene).
The encoded polypeptide may confer a morphological alteration on a cultured transformed cell, such as isopentyltransferase.
The encoded polypeptide may be a scorable marker, which allows transformed cells to be distinguished from non-transformed cells, typically by alteration of the optical properties of a cell expressing the marker polypeptide. Any scorable marker may be used. Preferred scorable markers include polypeptides which are able to alter the appearance or optical properties of transformed cells, for example: 3-glucuronidase (i.e. the uidA:GOS gene); fluorescent proteins such as green fluorescent protein (GFP), yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP); or luminescent proteins such as luciferase or aequorin. Cells with scorable optical differences can be sorted using techniques such as fluorescence activated cell sorting (FACS). A heterologous polynucleotide of the invention may comprise coding sequences encoding a selectable marker and a scorable marker, for example, the FLARE-S marker genes which comprise aadA and GFP.
In a further embodiment a coding region of a heterologous polynucleotide of the invention encodes a fusion protein. A fusion protein is a single polypeptide comprising at least two contiguous amino acid sequences that are not naturally found joined together. Thus, a fusion
Figure imgf000018_0001
r\t ιp> r»-rι-rj i-rr- purrt /Dl Il C Oβ\ 15 protein may consist of, for example, two or three contiguous amino acid sequences that are not naturally found in that arrangement or even four, five or more contiguous amino acid sequences. Typically, at least one of the sequences represents the sequence of a polypeptide of interest (or at least a fragment of that polypeptide), or a marker polypeptide. That is, the fusion protein consists in part of the sequence of a polypeptide that is desirably expressed in a Cardamine plant.
The fusion protein may contain the sequences of two, three or more polypeptides of interest, which may be the same or different.
In a further embodiment, at least one of the polypeptide sequences within the fusion protein provides the fusion protein with a selectable or scorable property. This property can aid in the purification of the fusion protein, by allowing rapid and easy identification of fractions containing the fusion protein. Preferred polypeptides providing scorable properties include GUS or GFP.
In a further embodiment the fusion protein comprises at least one amino acid sequence that allows for the fusion protein to be readily purified. Typically, the number of purification sequences is not more than 5, more typically not more than 2, most typically 1. Typically such a sequence enables the fusion protein to be purified by affinity based chromatography methods. In a preferred embodiment, the purification sequence is a His-Tag. Typically the His-Tag comprises multiple contiguous histidine residues, preferably from 3 to 20, more preferably from 4 to 10 most preferably 6. Typically the His-tag will be positioned at either or both of the N- and C- terminals of the fusion protein.
In a further embodiment, the polypeptide of interest is joined to other sequences by a sequence that can be cleaved to release the polypeptide of interest with substantially the same biological activity, or substantially the same amino acid sequence as the individually expressed protein of interest. In a preferred embodiment the cleavage sequence is IEGR, which is recognised and cleaved by Factor Xa (Nagi et al, 1985, Quinlan et at, 1989, Wearne 1990).
A coding region in a heterologous polynucleotide of the invention may include a region encoding a signal sequence capable of targeting the encoded polypeptide to specific locations within a plant cell or of ensuring that the fusion polypeptide is secreted from the cell in which it is expressed. Suitable signal sequences can ensure targeting of the polypeptide to the nucleus, to the nuclear envelope, to a plastid (for example a chloroplast), to a mitochondrion, to the cell membrane or to the endoplasmic reticulum. If the targeting sequence targets the fusion polypeptide to an organelle, a particular region of the organelle may be targeted. For example, in the case of a chloroplast, the fusion polypeptide could be targeted to the stroma, to the inter- membrane space, to the thylakoid membranes or the compartments within the thylakoids.
Coding sequences encoding such signal sequences may be derived from any suitable organism,
SUBSTITU 16 including the recipient Cardamine plant.
Some specific examples of suitable signal peptide sequences include: sequences which target proteins to the extracellular matrix of the plant cell, such as the signal sequence of the Nicotiana plumbaginifolia extension gene; signal peptides which target proteins to the vacuole, like those of the sweet potato sporamin gene and the barley lectin gene; signal peptides which cause proteins to be secreted such as that of PRIb; or the barley V-amylase leader sequence; and signal peptides which target proteins to the plastids such as that of rapeseed enoyl-Acp reductase.
The heterologous polynucleotide to be introduced into a plant is typically incorporated into a recombinant replicable vector. Such vectors may be used to replicate the heterologous polynucleotide in a compatible host cell by introducing the vector into a compatible host cell and cultivating the host cell under conditions which bring about replication of the vector. The vector may then be recovered from the host cell. Typically, the vector will allow replication of the heterologous polynucleotide in both is. coli (for convenience in cloning) and va. Agrobacterium sp. (for transfer to plants). Vectors suitable for the expression of heterologous polynucleotides in higher plants are well known to those skilled in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens (Rogers et ah, Meth. In EnzymoL, 153, 253-277, 1987) and the Ri plasmid of Agrobacterium rhizogenes. These vectors are plant integrating vectors in the sense that, on transformation, the vectors integrate a portion of vector DNA into the genome of the host plant. The heterologous DNA is incorporated into such vectors so that it is located between T-DNA borders (a repeated 24-base pair sequence).
The vectors are then introduced into the bacterium Agrobacterium. Typically, Agrobacterium tumefaciens is used. Suitable methods for the introduction of polynucleotide vectors into Agrobacterium are well-known to those skilled in the art and include tri-parental mating, heat shock and electroporation techniques. The virulence functions of the
Agrobacterium host will direct the insertion of the heterologous polynucleotide into the plant cell DNA when the cell is infected by the bacteria.
Plants used for transformation are of the genus Cardamine, preferably of the species C. hirsuta. The plant selected for transformation is grown under any conditions suitable to induce flowering of the said plant. Germination efficiency of the seeds may be increased by striating the seeds before sowing, for example by keeping the seeds at about 40C for about four to five days. Typically, the plant is grown under long day conditions, for example from about 14 to about 20 hour days. The plant may be grown in any suitable medium, for example a 3:1 mixture of soil and vermiculite. It has been found that the frequency of transformation is higher for the primary inflorescence than for secondary inflorescences. This may be because flowers on the primary inflorescence open for longer than flowers on the secondary inflorescences. Therefore,
SUBSTITUTE SHEPT /Pi n P 9^ 17 it will generally not be beneficial to pinch out the primary shoot/inflorescence to encourage the growth of secondary inflorescences.
A solution of Agrobacterium is prepared suitable for carrying out the transformation. The Agrobacterium solution is generally prepared by: growing a culture of Agrobacterium comprising the required heterologous polynucleotide is grown to an optical density of about 1.0; pelleting the cells; and resuspending the cells in half the original culture volume.
The plant to be transformed is then contacted with the Agrobacterium solution so that at least one of the floral meristems of the plant comes into contact with that Agrobacterium solution. Generally, it will be convenient to immerse more than one and if possible, substantially all floral meristems of a plant in the Agrobacterium solution. Typically, the entire primary inflorescence is dipped into the Agrobacterium solution. It is not necessary to apply any kind vacuum whilst the inflorescence is immersed. The contacting step is carried out for from about 1 to 30 minutes, more preferably from about 5 to 20 minutes, most preferably for about 10 minutes. Typically, the contacting step is carried out in the absence of a vacuum, i.e. at normal or ambient atmospheric pressure. Thus, the contacting step will typically be carried out about 1.01325xl05 Nm'2 (1.01325 bar).
Following contact with the Agrobacterium, it will generally be convenient to dry the plant using an absorbent material, for example tissue paper. The plant is then returned to suitable growth conditions, which are typically the same conditions as were used to grow the plant prior to its being contacted with Agrobacteria. The plant is allowed to grow under conditions so that the plant sets seed. Typically, the plant will be grown for about one to three weeks, preferably about two weeks, after which time no further water is supplied to the plant. If a number of similarly treated plants are being grown in close proximity, each plant may optionally be isolated from the other, for example by covering with a paper bag, so that self- fertilization only occurs. Cardamine plants are self-compatible so self-fertilization will occur. In fact, it will typically be desirable to use paper bags, as Cardamine plants show explosive pod shatter and the bags will ensure seed containment.
Once the plant has set seed, the seeds may be harvested and then screened for the presence of the heterologous polynucleotide. The nature of the screening procedure will depend on nature of the heterologous polynucleotide and whether that heterologous polynucleotide comprises a coding sequence for a marker polypeptide. If the heterologous polynucleotide does comprise a sequence encoding a marker polypeptide, it may be possible to screen large amounts of seed quickly and conveniently. The exact screening strategy used will depend on the marker polypeptide to be detected.
For example, the use of an antibiotic resistance marker gene may allow the seed to be 18 plated on selective media such that the only seedlings which develop are those which carry the heterologous polynucleotide (and therefore the marker gene). Thus, if the marker polypeptide is a polypeptide conferring resistance to kanamycin, the seeds may be germinated on media comprising from about 50μg/ml to about 150μg/ml kanamycin, preferably about lOOμg/ml kanamycin. All viable seeds should develop, but only those expressing the marker polypeptide will develop beyond the stage at which the cotyledons develop. Those not expressing the marker polypeptide will gradually bleach and then die.
Alternatively, if a herbicide resistance marker gene is used, seedlings may be germinated on a non-selective media and then sprayed with the relevant herbicide. Seedlings which do not express the marker polypeptide will die and the surviving seedlings should carry the heterologous polynucleotide.
Seedlings identified as carrying the heterologous polynucleotide may subsequently be analysed to ensure that they carry both the coding sequence for the marker polypeptide and the sequence encoding the polypeptide of interest. Such analysis may be carried out by any appropriate method, for example by PCR, DNA gel blotting or DNA chip screening. RNA gene blotting and/or protein gel blotting may be carried out to ensure that the polypeptide of interest is expressed.
Subsequently, presence of the heterologous polynucleotide may be confirmed, for example by the use of PCR (using primers specific to the heterologous polynucleotide) and/or via DNA gel blotting.
The procedure set out above provides a method for the generation of a first generation transgenic Cardamine plant, preferably a transgenic C. hirsuta plant. The invention also provides a method for obtaining transgenic Cardamine plants of further generations from this first generation transgenic plant. Such plants can conveniently be referred to as progeny transgenic Cardamine plants. Progeny transgenic Cardamine plants of second, third, fourth, fifth, sixth and further generations may be obtained from a first generation transgenic Cardamine plant by any means known in the art.
Thus, the invention provides a method of obtaining a transgenic Cardamine progeny plant comprising obtaining a second-generation transgenic Cardamine progeny plant from a first-generation transgenic Cardamine plant of the invention, and optionally obtaining transgenic
Cardamine plants of one or more further generations from the second-generation progeny plant thus obtained.
Such progeny plants are desirable because the first generation plant may not have all the characteristics required. For example, it may be desired to introduce further characteristics in one or more subsequent generations of progeny plants before a transgenic plant carrying all of the characteristics required is produced.
Qi IRSTΓΓUTE SHEET RULE 26) 19
Progeny plants may be produced from their predecessors of earlier generations by any known technique. In particular, progeny plants may be produced by:
obtaining a transgenic seed from a transgenic plant of the invention belonging to a previous generation, then obtaining a transgenic progeny plant of the invention belonging to a new generation by growing up the transgenic seed; and/or
propagating clonally a transgenic plant of the invention belonging to a previous generation to give a transgenic progeny plant of the invention belonging to a new generation; and/or
crossing a first-generation transgenic plant of the invention belonging to a previous generation with another compatible plant to give a transgenic progeny plant of the invention belonging to a new generation; and optionally
obtaining transgenic progeny plants of one or more further generations from the progeny plant thus obtained.
These techniques set out above may be used in any combination. For example, clonal propagation and sexual propagation may be used at different points in a process that gives rise to a transgenic plant suitable for cultivation. In particular, repetitive back-crossing with a plant with desirable characteristics may be undertaken. Further steps of removing cells from a plant and regenerating new plants therefrom may also be carried out.
The invention thus provides a transgenic plant of the genus Cardamine, preferably a transgenic plant of the species C. hirsuta.
Also, further desirable characteristics may be introduced by transforming the cells, plant tissues, plants or seeds, at any suitable stage in the above process, to introduce desirable coding sequences other than those contained in a heterologous polynucleotide of the invention. This may be carried out by conventional breeding techniques, e.g. fertilizing a transgenic plant of the invention with pollen from a plant with the desired additional characteristic. Alternatively, the characteristic can be added by further transformation of the plant obtained by the method of the invention, using the techniques described herein for further transformation, or by nuclear transformation using techniques well known in the art such as, inter alia, electroporation of plant protoplasts, transformation by Agrobacterium or particle bombardment. Preferably, each coding sequence to be introduced into a plant is linked to different selectable or scorable marker. That will allow individual selection to be applied for the presence of each coding sequence. Selection, 20 regeneration and breeding techniques for nuclear transformed plants are known in the art. Techniques along the lines of those described may be used.
As well as providing methods for the introduction of heterologous polynucleotides into a Cardamine plant, the invention also provides a method for evaluating the function of a polynucleotide in a Cardamine plant by disrupting that polynucleotide and determining the effect(s) of such disruption.
Disruption of a polynucleotide in a Cardamine plant may be carried out according to any method known in the art. Broadly speaking, there are two main methods of carrying out polynucleotide disruptions: radiation mutagenesis, chemical mutagenesis; or insertion mutagenesis.
Radiation mutagenesis may be earned out using, for example irradiation with X-rays or (-rays. A population of seeds is irradiated, grown to maturity and allowed to set seed. Mutations typically only take place in one chromosome at a particular locus therefore a second generation of plants is grown to reveal the phenotype of any mutations induced by the irradiation. Chemical mutagenesis is typically carried out by contacting a population of seeds of a
Cardamine plant with a suitable mutagen. A number of other chemical mutagens may be used, with mutagens which are alkylators being preferred. Suitable alkylators are, for example, ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), diethylsulfate (DES) or nitrosoguanidine (NTG, NG, MNNG). Such mutagens do not require active DNA replication to occur to be effective (although DNA replication is required in order for a mutation to be "fixed") and are thus suitable for use with seeds. Again, a second generation of plants is grown to reveal the phenotype of any mutations induced by the mutagen.
Insertion mutagenesis relies on disruption of the expression of a coding sequence in a plant with another polynucleotide sequence. This may be effected by the introduction of a heterologous polynucleotide at a locus, whereby the said heterologous polynucleotide disrupts the expression of a coding sequence at that locus. The heterologous polynucleotide may, for example, insert within the coding sequence itself or may disrupt the function of a control sequence. Alternatively, if the plant in question comprises endogenous transposable polynucleotide elements, it may be possible to induce transposition so that a transposable element "jumps" to a new locus.
Insertion mutagenesis may be carried out in Cardamine using T-DNA tagging. It is not clear whether Cardamine has a population of endogenous transposons and therefore whether transposition using such transposons is applicable to Cardamine. However, insertion mutagenesis could be carried out by the use of heterologous transposition systems. T-DNA tagging screens and heterologous transposition screens may be carried out in
Cardamine, preferably by use of the transformation technique of the invention. Transgenic
= 21
Cardamine plants identified in such screens which display phenotypes of interest may be selected for further analysis. Because the sequence of the T-DNA or the heterologous transposon are known it is relatively easy to identify sequence flanking the said T-DNA or heterologous transposon. The flanking sequence will either represent coding sequence or control sequence of the polynucleotide disrupted. Therefore, it may be relatively easy to link the phenotype shown by a mutant with the sequence of the polynucleotide which has been disrupted.
Cardamine plants are more related to the important crop plants, the Brassicas, than the commonly used model plant Arabidopsis and therefore may represent a better model for such plants. The phylogenetic relationships of nine genera in the family of the Brassicaceae have been estimated from the sequences of the internal transcribed spacer region (ITS) of the 18S-25S nuclear ribosomal DNA (Weng et al., Molecular Phylogenetics and Evolution .13, 455-462. 1999). Neighbour-joining and parsimony trees suggested that the three groups can be divided into three groups: (I) Arabidopsis, Cardaminopsis, Capsella and Lepidium; (2) Rorippa and Cardamine; and (3) Brassica; Sinapis; and Raphanus. Furthermore, it is apparent that Cardamine is more closely related to Brassica and Sinapis than Arabidopsis is.
We have now shown that Cardamine appears to shares many of the characteristics that make Arabidopsis so amenable to molecular genetic study. At present, the effects of many genes which are candidates for transfer into crop species are first tested in Arabidopsis to determine the sorts of effects they might have. It is not easy to test the effects of a particular polynucleotide directly in a crop species, as many of those species are recalcitrant to transformation techniques.
The drawback is of course that Arabidopsis is only distantly related even to its nearest crop neighbours, the Brassicas.
Accordingly, the invention provides a method for evaluating the suitability of a polynucleotide which is a candidate for transfer into a crop species, which method comprises: (i) preparing a transgenic Cardamine plant which expresses the candidate polynucleotide; and (ii) determining the effect of the candidate polynucleotide on the transgenic Cardamine plant. It is likely that the effect of the candidate polynucleotide in Cardamine will more closely resemble the effect of that polynucleotide in a crop species, in particular a Brassica species, than the effect of that polynucleotide observed in Arabidopsis would. Thus, use of a Cardamine plant as a model may save unnecessary transfer of genes into a crop plant on the basis of data gathered from Arabidopsis only to find that there is no effect (or at least a different effect) of the gene in the crop species. Alternatively, it might be that a polynucleotide which shows no effect in Arabidopsis, does in fact have a significant effect in a crop species.
Typically, a polynucleotide which is a candidate for transfer to a crop species, in particular transfer into a Brassica, will be introduced into a Cardamine plant according to the method of the invention for the preparation of a transgenic Cardamine plant.
Si IRSTf 22
Polynucleotides which show an effect of agricultural relevance in Cardamine, for example: insect or herbicide resistance; drought, salt or wind tolerance; increased leaf size; increased seed size; bioremediation potential may be subsequently tested in a crop species, in particular in a Brassica. In the methods described above, preferred Brassicas include B. napus, B. olerecea, B.
Junea and Sinapis alba.
According to the present invention there is thus provided a method for obtaining an improved crop plant, which method comprises: (i) identifying a polynucleotide which shows a beneficial effect in Cardamine; and (ii) introducing that polynucleotide into the crop plant. Thus, the invention also provides an improved crop plant, in particular an improved Brassica plant, identified by such a method. The improved Brassica may be, for example, a B. napus, B. olerecea, B. Junea or Sinapis alba plant.
A further use of plants of the genus Cardamine would be to use them for surveying the genomes of relatives of Arabidopsis (for example Cardamine and Lepidium) in order to identify functional czs-acting elements which are involved in gene expression. This sort of analysis has been shown to be effective in yeast (Hillier et ah, Genome Res. 11(7), 1175-1186). The fact that Cardamine in easily transformable provides a quick way to check the properties of putative exacting elements identified by bioinformatics/genomics approaches. This would of course be possible in Arabidopsis, but the use of Cardamine provides independent verification and will more closely represent what will happen in crops, for example Brassica crops.
Materials and methods
Unless otherwise indicated, the methods used are standard biochemistry and molecular biology techniques. Examples of suitable methodology textbooks include Sambrook et ah, Molecular
Cloning, A Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley and Sons, Inc.
Plant growth conditions. Plants were grown in a greenhouse with supplemental lighting (days: 18 h, 20 0C; nights: 6 h, 16 0C).
Genetic stocks. Wild-type C. hirsuta seed was collected from wild populations in Oxford, UK; verified by internal transcribed spacer sequencing; and self-pollinated for seven generations before use (specimen voucher Hay 1 (OXF)). Wild-type C. hirsuta seed was X-ray-irradiated at 16 kR, sown and harvested in pools of five plants. Approximately 100 seed of 150 M2 pools, giving a total of 1,500 plants, were screened. Mutant characterization was performed after backcrossing to wild-type C. hirsuta twice. 23 Transgenic construction. All primers are listed in Figure 12. To construct the C. hirsuta STM
RNAi vector, a 310-bp fragment was amplified from C. hirsuta shoot cDNA by PCR with the primers ChSTMrnai-F and ChSTMrnai-R. This fragment was cloned in both sense and antisense orientations in the pHANNIBAL vector18 using the restriction enzyme pairs XballClal and EcόSllKpjϊl. This RNAi cassette was transferred as a Notl fragment into the binary vector pMLBART19, transformed into the Agrobacterium twnifaciens strain GV3101 and used to transform wild-type C. hirsuta plants by a modified floral dipping protocol. We analyzed 14 independent Tl lines. We constructed a C. hirsuta ASl (hereafter, ChASl). RNAi vector in an identical manner using a 343-bp PCR fragment amplified from C. hirsuta shoot cDNA with the primers ChASlrnai-F and ChASlrnai-R, and we used it to transform wild-type C. hirsuta plants as above. We analyzed 16 independent T2 lines. The pMLBART vector alone was used to transform wild-type C. hirsuta plants as above, and all Tl lines were phenotypically wild-type. We used three independent T2 lines as wild-type comparisons for analyses of RNAi lines. Transcript levels were analyzed by RT-PCR (Figure 11). A previously described 35S::KN1-GR translational fusion13 was used to transform wild-type C. hirsuta plants as described above. We analyzed 16 independent T2 lines. To construct the 35S::ChASl vector, we amplified a 1,117-bp fragment of the C. hirsuta ASl coding region by PCR using a proofreading Taq polymerase (Pyrobest, Takara) from a full-length cDNA clone using the primers ChASl-F and ChASl-R. The PCR product was cloned into the pCR Blunt vector (Invitrogen), sequenced to confirm fidelity and cloned as an £coRI fragment behind the CaMV 35S promoter of the ρART7 vector19.
The 35S: . ChASl ::ocs cassette was transferred as a Notl fragment into the binary vector pMLBART and transformed into as 1-1 mutant plants by floral dipping. We analyzed 100 independent Tl lines for recovery of the asl mutant phenotype as described previously for 35S::AS1 (ref. 20). Five T2 lines with a single transgene copy were crossed to asl-l;BP::GUS plants3, and GUS expression was analyzed in the Fl. To make transcriptional fusions of C. hirsuta BP and C. hirsuta STM to the uidA (GUS) gene, a BAC library of C. hirsuta genomic clones was screened (a full description of library construction will be given elsewhere), and ~6- kb -EcøRI and Xbal restriction fragments of C. hirsuta BP and C. hirsuta STM DNA, respectively, were cloned into pBluescript (Stratagene). We amplified 4 kb of upstream sequence, including the 5' UTR, by PCR using a proofreading Taq polymerase (Pyrobest,
Takara) with the primers M13 reverse and ChBP-R with a Pstl restriction site introduced at the ATG, and cSTMpst-F and cSTMbam-R with a BamΗI restriction site introduced at the ATG. These sequences were transferred, as a Pstl fragment for C. hirsuta BP (hereafter 'ChBP') and as a Pstl/BamiU. fragment for C. hirsuta STM (hereafter 'ChSTM), upstream of GUS in the pRITA vector19. Orientation and integrity of the sequence junctions were confirmed by sequencing.
Transcriptional fusions were generated in a similar manner using ~5 kb of upstream sequence, including the 5' UTR of BP and STM. AU four promoter-GUS cassettes were transferred as Notl fragments into the binary vector pMLBART, transformed into A. tumefaciens, as above, and
Ci jDQrm iTP $MP£T r&ULE 24 used to transform both wild-type A. thaliana (Columbia ecotype) and C. hirsuta. GUS expression was analyzed in 97 independent Tl lines for ChBP::GUS and 96 lines for BP::GUS in A. thaliana, ten lines for ChBP: :GUS and 12 lines for BP::GUS in C. hirsuta, 67 lines for
ChSTM::GUS and eight lines for STM::GUS in A. thaliana, and eight lines for ChSTM: :GUS in C. hirsuta. A previously described 35S::BP construct21 was transformed into A. tumefaciens and used to transform wild-type C. hirsuta plants as described above. We analyzed 18 independent
T2 lines. PHV»BP, FIL»BP and ANT»BP lines were generated by constructing pVTOp.-.-BP22 and transforming PHV::LhG4, FIL::LhG4 (gift from Y. Eshed, Weizmann
Institute of Science, Israel) and ANT::LhG423-p\aτAs with this construct. We analyzed 30 independent T2 lines for each construct.
5' and 3' RACE. C. hirsuta ASl full-length cDNA sequence was determined in wild-type lines and chasl-1 mutants by 5' and 3' RACE. cDNA was generated using a SmartRace kit (BD Biosciences) according to manufacturer's protocols. We used 1 μg of total shoot RNA per reaction. PCR amplification was performed for 5' RACE with the primer ChASl-Rl and for 3' RACE with the primer ChASl-Fl. C. hirsuta ASl -specific products were cloned, and two clones from each RACE reaction were sequenced for each genotype. In chasl-1 mutants, a premature stop codon at amino acid 170 of ChASl introduces an Accl site that is not present in wild-type plants. This sequence polymorphism was used to generate a cleaved amplified polymorphic sequence marker by amplifying a 600-bp product with primers ChAS 1-F2 and ChASl-R2, which yielded products of 425 bp and 175 bp after Accl digestion of chasl-1 but not after digestion of wild-type amplicons.
Leaflet and cell measurements. Average number of leaflets per leaf was determined for ten C. hirsuta STM. RNAi plants and ten wild-type plants. Average number of C. hirsuta H4- expressing cells in adjacent longitudinal sections of the two youngest leaf primordia at the shoot apex was determined for C. hirsuta STM RNAi and wild-type plants (as described in ref. 11).
Error shown in all cases is standard error.
Immunocytochemistry. Fixation and hybridization were carried out as previously described23 on 8-μm paraffin sections using a previously described polyclonal antibody to KNOX24 that detects class I KNOX proteins (encoded by a four-member gene family in A. thaliana and C. hirsuta (data not shown)).
Scanning electron microscopy. Fixation and dehydration were carried out as previously described25. Scanning electron microscopy was performed using a JSM-5510 microscope (Jeol).
In situ RNA localization. Fixation and hybridization were carried out as previously described10 on 8-μm paraffin sections using probes for A. thaliana STMW, C. hirsuta STM, C. hirsuta H4, C. hirsuta ASl and C. hirsuta BP. To generate a probe to C. hirsuta ASl, a 373-bp fragment was amplified from C. hirsuta shoot cDNA using the primers ASl-F and ASl-R. To generate a probe
28 25 to C. hirsuta H4, a 297-bp fragment was amplified from C. hirsuta shoot cDNA using the primers H4-F and H4-R. To generate a probe to C. hirsuta STM, a 986-bp fragment was amplified from a cDNA clone using the primers ChSTM-F and ChSTM-R. Three probes were generated to C. hirsuta BP by amplifying 306-bp, 387-bp and 351-bp fragments from a cDNA clone using the primer pairs ChBP457-F and ChBP762-R, ChBP700-F and Chl086-R, and
ChBP5'-F and ChBP5'-R, respectively. All fragments were cloned into the pGEM T-Easy vector (Promega) and sequenced to determine orientation. Antisense and sense probes were transcribed and DIG labeled as previously described10.
Leaf silhouettes. Leaves were flattened onto clear adhesive, adhered to white paper and digitally scanned.
Chemical treatments. Dexamethasone (Sigma) was dissolved in water and applied at a concentration of 10~6 M with 0.02% silwet using a paintbrush.
Accession codes. GenBank: C. hirsuta STM mRNA, complete coding sequence (cds), DQ512732; C. hirsuta ASl mRNA, complete cds, DQ512733; C. hirsuta BP mRNA, complete cds, DQ630764; C. hirsuta BP gene, 5' upstream region, DQ526379; C. hirsuta STM gene, 5' upstream region, DQ526380.
Transformation of Cardamine hirsuta plants is possible by the use of a floral dipping technique
C. hirsuta plants were sown on a 3:1 soil to vermiculite mixture. Seeds were kept at 40C in the dark for 4 days and subsequently grown under a 14h photoperiod. Under these conditions flowering occurred after approximately 4 weeks. A. tumefaciens cultures containing a 35S::KN] construct (Sinha et al, Genes & Dev. 7, 787-795, 1993; a gift from Sarah Hake) were used to carry out the transformation. The A. tumefaciens cells were grown to an optical density of approximately 1 and then resuspended in half their original volume. The primary inflorescences of the Cardamine plants were dipped in the A. tumefaciens cultures for approximately 10 minutes. Plants were subsequently dried on tissue paper and returned to the previous growth conditions. After 2 weeks, plants were not watered further and paper bags were placed on the inflorescence stems. This is critical, because Cardamine plants show explosive pod shatter therefore containment of seeds is essential. Seeds were sown on lOOμg/ml kanamycin. Resistant plants, i.e. transgenic plants, could successfully be grown on the selective media and produced branched roots and true leaves. By contrast, sensitive plants, i.e. non-transgenic plants, did not develop beyond the cotyledon stage and were bleached. This experiment shows that transgenic Cardamine plants may be successfully prepared using a simple, convenient floral dipping technique.
5i iR« 26
This experiment has also been carried out with a 35S::KNAT1 chimeric gene construct. KNATl is the C. hirsuta ortholog of KNl and was generated via PCR. Kanamycin resistant plants, i.e. transgenic plants, could successfully be grown on the selective media and produced branched roots and true leaves. By contrast, sensitive plants, i.e. non-transgenic plants," did not develop beyond the cotyledon stage and were bleached. Nine lines of independent transformants showing a consistent phenotype were successfully generated. The transgenic plants show an alteration in the characteristic compound leaf morphology shown by wild type C. hirsuta.
EXAMPLES
Morphological innovations are often associated with altered expression of key developmental regulators1. However, it is unclear how such divergent expression arises, how it modifies growth to produce differences in form, or how the potentially pleiotropic effects of altered regulatory gene activity are constrained during evolution. Leaves of seed plants provide an attractive system to study the evolution of developmental mechanisms because they present considerable morphological variation. Leaf form can be described as simple (if the leaf blade is entire or dissected (if the blade is divided into distinct leaflets). Both simple and dissected leaves are initiated at the flanks of a pluripotent structure termed the shoot apical meristem (SAM). In simple-leafed species, such as A. thaliana and maize, ARP myb proteins act in the leaf to confine
KNOX transcription factors to the meristem2"6. Conversely, many dissected-leafed species accumulate KNOX proteins in the leaf and ARP proteins in the meristem7'8. However, it is not known whether KNOX activity is required to produce a dissected leaf, or whether differences in ARP function or regulation are responsible for the divergent patterns of KNOX expression seen in different species.
EXAMPLE 1
To answer these questions, we analyzed dissected leaf development in C. hirsuta, a small crucifer related to the simple-leafed model species A. thaliana (Fig. la-d). Unlike many A. thaliana relatives, C. hirsuta has the distinct advantages of being a diploid, self-compatible plant that can be used for genetic analyses and transformed, thus allowing parallel genetic studies of leaf development to be conducted in species that diverged relatively recently9. To understand whether KNOX expression in the leaf is associated with dissected leaf form, we examined KNOX protein accumulation patterns in C. hirsuta and A. thaliana shoot apices. Class I KNOX proteins were expressed in the SAM but were excluded from the cells that comprise an initiating leaf primordium in both A. thaliana and C. hirsuta (Fig. le,f). However, in contrast to what we
Ql !KOTfTI ITP QWPE=T /Ri Il F 9Pft 27 observed in A. thaliana, we observed nuclear expression of KNOX proteins in later leaf primordia of C. hirsuta (Fig. le,fj, associated with leaflet initiation (Fig. lg,h). EXAMPLE 2
To investigate whether KNOX activity is required for leaflet initiation, we reduced expression of the KNOX gene SHOOTMERISTEMLESS (STM) in C. hirsuta by RNA interference (RNAi)
(Fig. 2). C. hirsuta STM is expressed in the SAM of the embryo and mature plant and is repressed in the majority of cells that comprise initiating leaf primordia (Fig. 2a,b). However, in contrast to STM expression in A. thaliana (Fig. 2c), we also observed C. hirsuta STM expression throughout the outer cell layers at the base of initiating leaf primordia (Fig. 2b). In comparison to wild- type plants (Fig. 2d), strong RNAi lines produced shootless plants with fused cotyledons
(Fig. 2e) that often initiated leaves from ectopic positions (Fig. 2e). Thus, as in A. thaliana10, C. hirsuta STM is required for SAM initiation and cotyledon separation in the embryo. Furthermore, in weak RNAi lines that developed a functional SAM, leaflet initiation was severely reduced
(Fig. 2f-h, 0.8 ± 0.2 leaflets per leaf in C. hirsuta SfM RNAi lines compared with 4.4 ± 0.2 in wild-type lines), demonstrating that C. hirsuta STM is required to initiate leaflets.
To investigate whether the control of leaflet initiation by C. hirsuta STM involves regulation of cell division, we assayed the effects of reducing C. hirsuta STM activity on expression of HISTONE 4 (H4), which has previously been used to monitor cell cycle activity in A. thaliana lateral organs11. We observed that in C. hirsuta STMKNAi plants, fewer cells in developing leaf primordia express C. hirsuta H4 (Fig. 2j,k, 23.5 ± 2.1 cells in C. hirsuta STM RNAi lines compared with 76.5 ± 3.2 cells in wild-type plants and the epidermal cells are much larger in these leaves than in wild-type plants (Fig. 21,m). This reduction in C. hirsuta H4 expression and increased cell expansion suggests that C. hirsuta STM prevents the precocious exit of tissues from the cell cycle into differentiation pathways, thus promoting leaflet initiation. To investigate whether KNOX expression is sufficient for leaflet initiation, we expressed the maize
KNOTTEDl (KNl) protein, which is able to rescue A. thaliana stm mutants12, in C. hirsuta in a dosage-sensitive manner using a fusion with the rat glucocorticoid receptor (KNl-GR)13. A single induction of KNl activity with 10~6 M dexamethasone resulted in reiteration of a second order of leaflets along the elongated petiolules of first-order leaflets (Fig. 2i). These results demonstrate that KNOX activity is not only necessary but also sufficient for leaflet initiation in
C. hirsuta. Elevated KNOX expression in the dissected-leaf tomato plant can also increase leaflet number14'15, suggesting that the requirement for KNOX activity in C. hirsuta leaf development may extend to other species where dissected leaf morphology has evolved independently7.
EXAMPLE 3 We have shown that differences in KNOX expression contribute to the different leaf forms observed in A. thaliana and C. hirsuta, indicating that distinct mechanisms of KNOX gene
SUBSTITUTE SHEET RULE 26 28 regulation evolved in these two closely related species. In A. thaliana, ASl represses KNOX gene expression in the leaf; therefore, loss of this regulation could be responsible for KNOX expression in C. hirsuta leaves. This scenario would be consistent with the coexpression of KNOX and ARP proteins observed in the shoot meristems of many dissected-leaf plants8. To investigate tin's possibility, we determined the extent of functional equivalence between C. hirsuta AS 1 and A. thaliana AS 1. Expression of C. hirsuta ASl under the control of the broadly expressed CaMV 35S promoter complemented the A. thaliana asl mutant phenotype (Fig. 3a-c) and repressed expression of the KNOX gene BREVIPEDICELLUS (BP) in asl leaves (Fig. 3d- f), indicating that the function of the two proteins is conserved. Moreover, C. hirsuta ASl mRNA was expressed in leaves and excluded from the SAM (Fig.3g) in an equivalent pattern as ASl in
A. thaliana2. Thus, it is unlikely that changes in either the function or expression of ASl account for the differences in KNOX expression and leaf shape between Λ(. thaliana and C. hirsuta.
EXAMPLE 4 We next investigated whether the differences in KNOX gene expression observed between the two species are attributable to differential activity of KNOX gene regulatory sequences. To test this idea, we analyzed 5' upstream regions of the KNOX genes STM and BP (Figure 5) and performed promoter swap experiments with these regions between C. hirsuta and A. thaliana. We reasoned that if the regulatory information necessary for species-specific expression is contained within the promoter regions, then each promoter should drive reporter gene expression regardless of the species into which it is transformed. If, however, species-specific activity of trans regulatory factors is required for correct KNOX gene expression, then each reporter should reflect the expression pattern of the species into which it is transformed. We found that each reporter reflected the endogenous gene expression pattern of its promoter in both the native and heterologous context (Fig. 3). That is, the A. thaliana BP promoter generated GUS expression in the SAM of both A. thaliana and C. hirsuta (Fig. 3h,i), and the C. hirsuta BP promoter generated GUS expression in both the SAM and leaves of both species (Fig.3j,k). Swapping the C. hirsuta STM promoter region between C. hirsuta and A. thaliana gave similar results: the C. hirsuta promoter generated GUS expression in the SAM and the abaxial side of developing leaves in both species (Fig. 31,m), whereas the A. thaliana promoter generated expression in the SAM only (Fig. 3n). These results indicate that differences in KNOX gene expression between A. thaliana and C. hirsuta are at least in part determined by differential activity of promoter sequences. KNOX activity in developing leaf primordia is sufficient to elicit leaflet formation in the simple leaf of A. thaliana (Figure 7), suggesting that cis regulatory changes may be sufficient to determine the differences in leaf form between A. thaliana and C. hirsuta. However, future work will determine if this is the case or if additional factors facilitate KNOX-dependent leaflet formation in C. hirsuta.
SUBSTITUTE SHEET RULE 29
EXAMPLE 5
To understand how the pleiotropic effects (such as compressed proximodistal axis and supernumerary leaflets; Fig. 4a) of widespread KNOX expression in C. hirsuta leaves are constrained, we conducted a genetic screen to isolate recessive mutants that phenocopy the effects of KNOX overexpression. One such mutant initiated leaflets close together along an extremely compressed proximodistal axis and had additional orders of leaflets (Fig. 4b,c). This mutant phenotype was very similar to that observed in transgenic C. hirsuta lines when we reduced C. hirsuta ASl activity using RNAi (data not shown), suggesting that these phenotypic effects were a consequence of loss of C. hirsuta ASl function. Molecular analysis confirmed that the mutant contained a premature stop codon at amino acid residue 170 in the C. hirsuta ASl coding sequence that cosegregated with the mutant phenotype, and the allele was hence designated chasl-1 (Figure 8). Thus, C. hirsuta ASl activity is required for development of the proximodistal axis of the leaf and for determining number and positioning of leaflets along this axis.
EXAMPLE 6
To investigate whether C. hirsuta ASl controls dissected leaf form by defining the domain and level of KNOX expression, we analyzed KNOX protein and mRNA accumulation in C. hirsuta ASl RNAi lines and chasl-1 mutants. We observed increased KNOX protein accumulation in C. hirsuta ASl RNAi leaves (Figure 9) and observed ectopic expression of C. hirsuta BP but not C. hirsuta STM in chasl-1 leaves (data not shown and Fig. 4). Notably, the pattern of ectopic C. hirsuta BP expression correlated well with the phenotypic perturbations observed in chasl-1. For example, in contrast to the wild-type expression of C. hirsuta BP (Fig. 4d), in chasl-1, we observed intense C. hirsuta BP expression in the adaxial domain and base of developing leaves
(Fig. 4e), correlating with repression of growth and differentiation along the proximodistal axis (Fig.4b,c) and adaxial rachis (Fig.4h,i) of the leaf. The activity of C. hirsuta ASl in controlling differentiation of the adaxial side of the leaf is shared by ARP proteins in other plant species8'16'17. Additionally, these results indicate that, at least in C. hirsuta, the roles of ARP proteins in axial patterning and KNOX repression are intimately intertwined.
EXAMPLE 7
To investigate whether this reduction in growth along the chasl-1 leaf rachis reflects a reduction in cell division or cell expansion, we analyzed C. hirsuta H4 gene expression and cell size in developing leaves. Compared with wild-type (Fig. 4f), a greater proportion of cells in chasl-1 leaf primordia express C. hirsuta H4, particularly at the leaf base (Fig. 4g). In addition, epidermal cells along the adaxial surface of the chasl-1 leaf rachis fail to elongate, or
SUBSTITUTE SHEET (RULE 2m 30 differentiate a striated cell wall, as occurs in wild- type (Fig. 4h,i). Similar defects were observed in the leaves of C. hirsuta plants expressing BP under the control of the 35S promoter (Figure 10). These observations suggest that C. hirsuta ASl regulates C. hirsuta BP expression within the C. hirsuta leaf, thereby defining the correct timing for leaf cells to exit the cell cycle and enter differentiation pathways. Thus, changes in KNOX promoter activity underpin differences in leaf shape between A. thaliana and C. hirsuta by allowing KNOX proteins to become part of the regulatory toolkit that controls leaf growth and differentiation. However, potentially pleiotropic effects arising from KNOX activity in leaves are constrained by the repressive action of C. hirsuta ASl.
We found it striking that although the molecular function of C. hirsuta ASl and A. thaliana ASl to repress KNOX gene expression is conserved, the developmental significance of this repression is different for the two species. InA. thaliana, ASl acts to safeguard leaf fate by maintaining the repression of KNOX expression in leaves. By contrast, in C. hirsuta, KNOX gene regulatory sequences drive expression in the leaf where KNOX activity is required for dissected leaf development. Within this different developmental context of the dissected leaf, C. hirsuta ASl constrains the spatiotemporal domain of KNOX expression and hence leaflet number and arrangement. Our work identifies two processes that underpin the evolution of new morphologies in multicellular eukaryotes. First, changes in the expression domain of key developmental regulators offer the potential to alter morphology by changing tissue growth. Second, conserved molecular interactions of these regulators, within their new expression domains, can acquire new developmental significance and mold morphology to its final state. Differences between C. hirsuta and A thaliana extend to many other aspects of their growth and development, including shoot branching and floral organ morphogenesis. Therefore, future research in these species will test how robustly these principles apply to the evolution of relevant developmental pathways.
References
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3. Ori, N., Eshed, Y., Chuck, G., Bowman, J.L. & Hake, S. Mechanisms that control knox gene expression in the Arabidopsis shoot. Development 127, 5523-5532 (2000).
4. Schneeberger, R., Tsiantis, M., Freeling, M. & Langdale, J.A. The rough sheath2 gene negatively regulates homeobox gene expression during maize leaf development.
Development 125, 2857-2865 (1998).
SUBSTi i "u i £ Srt&isϊ (RUL& 28) 31
5. Tsiantis, M., Schneeberger, R., GoIz, J.F., Freeling, M. & Langdale, J.A. The maize rough sheath2 gene and leaf development programs in monocot and dicot plants. Science 284, 154-156 (1999).
6. Timmermans, M.C., Hudson, A., Becraft, P.W. & Nelson, T. ROUGH SHEATH2: a Myb protein that represses knox homeobox genes in maize lateral organ primordia.
Science 284, 151-153 (1999).
7. Bharathan, G. et al. Homologies in leaf form inferred from KNOXI gene expression during development. Science 296, 1858-1860 (2002).
8. Kim, M., McCormick, S., Timmermans, M. & Sinha, N. The expression domain of PHANTASTICA determines leaflet placement in compound leaves. Nature 424, 438-
443 (2003).
9. Koch, M., Haubold, B. & Mitchell-Olds, T. Molecular systematics of the Brassicaceae: evidence from coding plastidic matK and nuclear Chs sequences. Am. J. Bot. 88, 534- 544 (2001). 10. Long, J.A., Moan, E.I., Medford, J.I. & Barton, M.K. A member of the KNOTTED class of homeodomain proteins encoded by the SHOOTMERISTEMLESS gene of Arabidopsis. Nature 379, 66-69 (1996).
11. Dinneny, J.R., Yadegari, R., Fischer, R.L., Yanofsky, M.F. & Weigel, D. The role of
JAGGED in shaping lateral organs. Development 131, 1101-1110 (2004). 12. Kim, J.Y., Yuan, Z. & Jackson, D. Developmental regulation and significance of
KNOX protein trafficking in Arabidopsis. Development 130, 4351-4362 (2003).
13. Hay, A., Jackson, D., Ori, N. & Hake, S. Analysis of the dompetence to respond to
KNOTTEDl activity in Arabidopsis leaves using a steroid induction system. Plant Physiol. 131, 1671-1680 (2003). 14. Hareven, D., Gutfinger, T., Parnis, A., Eshed, Y. & Lifschitz, E. The making of a compound leaf: Genetic manipulation of leaf architecture in tomato. Cell 84, 735-744 (1996).
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SUBSTITUTE 32
17. Xu, L. et al. Novel asl and as2 defects in leaf adaxial-abaxial polarity reveal the requirement for ASYMMETRIC LEAVESl and 2 and ERECTA functions in specifying leaf adaxial identity. Development 130, 4097-4107 (2003).
18. Wesley, S.V. et al. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 27, 581-590 (2001).
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c E 26 ^

Claims

33 CLAIMS
1. A method for obtaining a first-generation transgenic plant of the genus Cardamine, which method comprises:
(i) contacting a floral meristem of a plant of the genus Cardamine with an
Agrobacterium cell, which Agrobacterium cell comprises a heterologous polynucleotide;
(ii) allowing the plant to set seeds; (iii) harvesting the seeds;
(iv) identifying a seed which comprises the heterologous polynucleotide; and (v) obtaining a plant from the seed identified in step (iv).
2. A method for obtaining a transgenic plant of the genus Cardamine, which method comprises obtaining a second-generation transgenic plant from a first-generation transgenic plant obtained by a method according to claim 1, and optionally obtaining transgenic plants of one or more further generations from the second-generation transgenic plant thus obtained.
3. A method according to claim 2 comprising;
(i) obtaining a transgenic seed from a first-generation transgenic plant obtained by the method according to claim 1, then obtaining a second-generation transgenic plant from the transgenic seed; and/or (ii) propagating clonally a first-generation transgenic plant obtained by the method according to claim 1 to give a second-generation transgenic plant; and/or (iii) crossing a first-generation transgenic plant obtained by a method according to claim 1 with another transgenic plant to give a second-generation transgenic plant; and/or (iv) selfing a first-generation transgenic plant obtained by a method according to claim 1 to give a second-generation transgenic plant; and optionally (v) obtaining transgenic progeny plants of one or more further generations from the transgenic second-generation progeny plant thus obtained.
4. A method for obtaining a transgenic plant seed comprising obtaining a transgenic seed from a plant obtainable by any one of claims 1 to 3. 34
5. A method according to any one of claims 1 to 4 wherein the plant of the genus
Cardamine is a Cardamine hirsuta (C. hirsuta) plant.
6, A method according to any of claims 1-5 wherein the heterologous polynucleotide encodes a polypeptide.
7. A method according to any of claims 1-5 wherein the heterologous polynucleotide comprises a nucleic acid molecule from which an antisense nucleic acid is expressed.
8. A method according to any of claims 1-5 wherein the heterologous polynucleotide comprises a nucleic acid molecule from which an interfering RNA is expressed.
9. A transgenic plant or transgenic seed of the genus Cardamine.
10. A transgenic plant or transgenic seed according to claim 9 which is obtainable by the method according to any one of claims 1 to 8.
11. A transgenic plant or transgenic plant seed according to claim 9 or 10 which is a transgenic C. hirsuta plant or seed.
12. A method for evaluating the function of a polynucleotide sequence, which method comprises:
(i) disrupting or inhibiting the expression of a polynucleotide sequence in a plant of the genus Cardamine; and (ii) determining the effect of disrupting the polynucleotide sequence or inhibiting expression of the polynucleotide sequence.
13. A method according to claim 12, wherein the polynucleotide sequence is disrupted by chemical mutagenesis or by insertion mutagenesis.
14. A method according to claim 13, wherein the insertion mutagenesis is T-DNA tagging.
SUBSTITUTE SHEET (RULE 2 d6£Λ' , 35
15. A method according to claim 12 wherein the inhibition of expression of the polynucleotide is by an antisense nucleic acid molecule.
16. A method according to claim 12 wherein the inhibition of expression of the polynucleotide is by a siRNA molecule.
17. A method according to any of claims 12-16 wherein the insertion mutagenesis or inhibition of expression is carried out using a method according to any one of claims 1 to 3.
18. A method for evaluating the suitability of a polynucleotide which is a candidate for transfer into a crop species, which method comprises:
(i) preparing a transgenic Cardamine plant which expresses the candidate polynucleotide; and
(ii) determining the effect of the candidate polynucleotide on the transgenic Cardamine plant.
19. A method according to claim 18, wherein step (i) is carried out according to a method according to any one of claims 1 to 3.
20. A method for obtaining an improved crop plant, which method comprises:
(i) identifying a polynucleotide which shows a beneficial effect in Cardamine; and
(ii) introducing that polynucleotide into the crop plant.
21. A method according to claim 20, wherein the polynucleotide showing a beneficial effect in Cardamine is identified by a method according to any of claims 12-17.
22. An improved crop plant obtained by a method according to claim 20 or 21.
PCT/GB2006/003288 2005-09-06 2006-09-06 Plant transformation WO2007028979A1 (en)

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CN103651114A (en) * 2012-12-14 2014-03-26 湖北盛硒生物科技有限公司 Tissue culture and rapid propagation method for cardamine hupingshanesis
CN110483619A (en) * 2019-07-31 2019-11-22 恩施德源健康科技发展有限公司 A kind of anti-oxidant selenium polypeptide and preparation method thereof derived from Hairy Bittercress
CN110483619B (en) * 2019-07-31 2022-11-15 恩施德源硒材料工程科技有限公司 Antioxidant selenium polypeptide derived from cardamine hirsute and preparation method thereof
CN113151303A (en) * 2021-05-21 2021-07-23 浙江大学 Cabbage stem cell determination related gene BrWUS1 and application thereof
CN113151303B (en) * 2021-05-21 2022-07-05 浙江大学 Cabbage stem cell determination related gene BrWUS1 and application thereof

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