WO2008119136A1 - Procédés et constructions perfectionnés pour transformation à médiation par agrobacterium sans marqueur - Google Patents

Procédés et constructions perfectionnés pour transformation à médiation par agrobacterium sans marqueur Download PDF

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WO2008119136A1
WO2008119136A1 PCT/AU2008/000478 AU2008000478W WO2008119136A1 WO 2008119136 A1 WO2008119136 A1 WO 2008119136A1 AU 2008000478 W AU2008000478 W AU 2008000478W WO 2008119136 A1 WO2008119136 A1 WO 2008119136A1
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apoptosis
cells
polynucleotide
plant
inhibiting
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PCT/AU2008/000478
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English (en)
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James Langham Dale
Harjeet Khanna
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Queensland University Of Technology
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Priority claimed from AU2007901775A external-priority patent/AU2007901775A0/en
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Publication of WO2008119136A1 publication Critical patent/WO2008119136A1/fr

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202Methods 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
    • C12N15/8205Agrobacterium mediated transformation
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon

Definitions

  • This invention relates generally to improved methods and constructs for stably or transiently transforming plant cells using Agrobacte ⁇ um mediated transformation without selecting for the presence of a marker gene.
  • Agrobacterium-mediated transformation is one of the preferred methods of plant transformation.
  • a large number of plant species, including monocots are recalcitrant to Agrob ⁇ cterium tumef ⁇ ciens, significantly reducing the applicability of Agrobacterium-mediated transformation for many important crop plants.
  • this method is most often limited to certain lines or cultivars and protocols are difficult to extend to other cultivars of the same plant species. This has proved to be a major obstacle in developing transformation protocols that can be easily adapted to a wide range of plant genera and species, leaving this technique of gene transfer very genotype dependent.
  • Agrobacterium triggers many genes in plant cells including the plant defence machinery (Dirt et al., 2001, Proc. Natl. Acad. Sci. USA, 98:10954-10954; Veena et al., 2003, Plant J, 35:219-236). Modification of the tissue culture conditions during transformation can increase the probability of stably transforming some recalcitrant cell types. However, cell death following A. tumefaciens infection of plant cells still remains a limitation, and requires the use of selection markers to identify those plants which have been transformed (Gelvin, 2003, Microbiol. MoI. Biol, Rev, 67:16-37).
  • Tissue browning and subsequent death following exposure to Agrobacterium has previously been reported in many monocot and dicot plants including aspen, poplar (de Block, 1990, Plant Physiol, 93:1110-1116), grape (Deng et al, 1995, MoI. Plant-Microbe. Interact, 8:538-548; Perl et al, 1996, Nat. Biotechnol. 14:624-628; Pu and Goodman, 1992, Physiol. MoI. Plant Pathol. , 41 :245-254), sorghum (Carvalho et. al, 2004, Genet. MoI. Biol, 27:259-269 and; Gao et al, 2005, Plant Biotechnol.
  • Maize calli infected with A. tumefaciens were reported as undergoing a rapid, hypersensitive type of cell death in a study characterizing A. tumefaciens- induced apoptosis in maize (Hansen, 2000, MoI Plant-Microbe.
  • Apoptotic regulators such as those coded by the Ced-91 Bcl-21 Bax gene family of animals can either induce or suppress cell death in transgenic plants exposed to stress (Dickman et al, 2001, supra; Lacomme and Cruz, 1999, Proc. Natl Acad. Sci. USA,
  • Agrobacterium transformation is described in U.S. Patent Application Number 20020088029 (2002, Hansen) in which a plant cell is exposed to Agrobacterium under conditions that inhibit Agrobacterium induced necrosis (AIN) such as in the presence of an AIN-inhibiting agent.
  • the AIN-inhibiting agent is disclosed as a mammalian bcl-1 gene, a coding region of an apoptosis-inhibiting gene from baculovirus such as p35 or pIAP, or a gene capable of suppressing a gene response in plants.
  • the method further comprises selecting transformed plant cells that contain a marker gene that confers resistance or tolerance to a selection agent such as phosphinothricin, hygromycin and mannose.
  • the present invention is predicated in part on the discovery that expression of an anti-apoptotic gene such as Bcl-xL, Ced-9 and the 3' non-translated region o ⁇ Bcl-2 in plant cells improves Agrobacterium transformation efficiency by more than about 100-fold as compared to control transformations in the absence of an anti-apoptosis gene, which permits plant transformations to be carried out without screening for the presence of a co-transformed marker gene.
  • This discovery is highly advantageous as it permits plant transformations to be carried out in the absence of a positive or negative selection step, which is very time-consuming, laborious and expensive.
  • the present invention provides methods for introducing a polynucleotide of interest into a plant cell. These methods generally comprise exposing plant cells to an Agrobacterium that contains the polynucleotide of interest under conditions that inhibit apoptosis in the cells, and identifying a transformed plant cell that contains the polynucleotide of interest without selecting for the presence of a marker gene in that cell.
  • Illustrative apoptosis-inhibiting conditions include the use of apoptosis-inhibiting agents, which are suitably selected from: chemical inhibitors including small molecule compounds such as silver nitrate and ethylene inhibitors; apoptosis-inhibiting polynucleotides that may be stably integrated or transiently operative in the cells to be transformed; and apoptosis-inhibiting polypeptides.
  • the plants cells may be exposed to an apoptosis-inhibiting agent either before or after or at the same time as exposing the cells to the Agrobacterium.
  • the apoptosis-inhibiting agent is an apoptosis- inhibiting polynucleotide, which inhibits apoptosis directly or which encodes an apoptosis-inhibiting RNA or an apoptosis-inhibiting polypeptide.
  • the apoptosis- inhibiting RNA or polypeptide may be of any suitable origin including prokaryotic ⁇ e.g., viral, bacterial) and eukaryotic (e.g., animal and plant) origin.
  • Illustrative apoptosis- inhibiting polypeptides include Bcl-xL, Bcl-2, BI-I, Hsp70, AtILP-I, AtBAGl, AtBAG2, AtBAG3, AtBAG4, AtBAG5, AtBAG ⁇ , AtBAG7, p35, pIAP, DAD-I, CED- 9 and synthetic peptide analogs of caspases capable of triggering apoptosis.
  • the apoptosis-inhibiting polynucleotide may be introduced into the plant cells or may be expressed in a host cell to produce an apoptosis-inhibiting polypeptide to which the plant cells are subsequently exposed.
  • the apoptosis-inhibiting polynucleotide is in the form of a construct, typically a chimeric construct, in which the apoptosis- inhibiting polynucleotide is operably connected to a regulatory element (e.g., a promoter) that modulates its expression (e.g., constitutive, tissue specific or conditional expression).
  • a regulatory element e.g., a promoter
  • the methods of the present invention do not require the step of selecting transformed plant cells on the basis that they express an identifying characteristic (e.g. , antibiotic resistance, antibiotic sensitivity, cell death, enzymatic activity and light emission or absorbance) conferred by a marker gene that they contain.
  • the construct does not contain a marker gene.
  • a marker gene may be present in the construct, provided that it is not used for identifying or selecting plant cells that contain the apoptosis-inhibiting polynucleotide.
  • the apoptosis-inhibiting polynucleotide may be introduced into the plant cells (e.g., by Agrobacterium mediated transformation, microprojectile particle bombardment or electrophoresis) either before or after or at the same time as exposing the plant cells to the Agrobacterium to facilitate introduction of the polynucleotide of interest.
  • the apoptosis-inhibiting polynucleotide is stably introduced into the genome of the plant cells.
  • the apoptosis-inhibiting polynucleotide is transiently present and operable, for example, at or around the time the cell is exposed to the Agrobacterium.
  • Transient expression of the apoptosis-inhibiting polynucleotide can be obtained, for example, using an agroinfection system.
  • a plurality of apoptosis-inhibiting polynucleotides is introduced into the plant cells and these polynucleotides may be the same or different.
  • the apoptosis-inhibiting polynucleotide is expressed by the same Agrobacterium that contains the polynucleotide of interest.
  • the apoptosis-inhibiting polynucleotide and the polynucleotide of interest may be present on the same construct or on different constructs.
  • the one comprising the apoptosis-inhibiting polynucleotide and the other comprising the polynucleotide of interest may be contained on different vectors or on the same vector.
  • the apoptosis- inhibiting polynucleotide is expressed by a different Agrobacterium than the one containing the polynucleotide of interest.
  • the Agrobacterium containing the apoptosis-inhibiting polynucleotide may be the same type of Agrobacterium as the one containing the polynucleotide of interest, or may be a different type.
  • the apoptosis-inhibiting agent is an apoptosis- inhibiting polypeptide.
  • the polypeptide is in the form of a fusion protein that comprises a virulence protein (e.g., an Agrobacterium VirF protein) fused directly or indirectly to the apoptosis-inhibiting polypeptide.
  • the fusion protein may be produced from a construct which comprises a fusion protein coding sequence that is operably connected to a regulatory element.
  • the regulatory element drives expression of the coding sequence in a bacterium to produce the fusion protein and the virulence protein portion of the fusion protein directs the protein to the nucleus of the plant cells.
  • the methods further comprise modulating the level or functional activity of the apoptosis- inhibiting agent in the plant cells.
  • modulating the level or functional activity of an apoptosis-inhibiting polynucleotide or polypeptide after plant transformation which may be desirable for maintaining the well being of plants that are grown or regenerated from the transformed plant cells or from a regulatory standpoint.
  • a modulator gene may be introduced into the plant cells either before or after or at the same time as introducing the apoptosis-inhibiting agent.
  • the modulator gene produces an expression product that has at least one activity selected from: a transcript- degrading activity that degrades a transcript product of the apoptosis-inhibiting polynucleotide; a transcript-interacting activity that inhibits translation of a transcript product of the apoptosis-inhibiting polynucleotide; a polypepti de-interacting activity that inhibits the functional activity of the apoptosis-inhibiting polypeptide; and a nucleic acid-excising activity that mediates excision of at least a portion of the apoptosis- inhibiting polynucleotide.
  • the modulator gene expresses (e.g., constitutively or conditionally) a nucleic acid sequence that encodes a ribozyme or antisense nucleic acid which is specific to the transcript product of the anti-apoptosis polynucleotide employed for the transformation.
  • the modulator gene may cause post-transcriptional gene silencing.
  • the apoptosis-inhibiting polynucleotide and/or the polynucleotide of interest and/or modulator gene as broadly defined herein are replicated and amplified in a plant cell using rolling circle viral replication, as described for example by Dale et al. in International Publication No. WO 01/72996.
  • rolling circle replication systems include the InPACT system developed by the present inventors.
  • the apoptosis-inhibiting agent is a small molecule compound, illustrative examples of which include silver nitrate, ethylene inhibitors, ethylene synthesis inhibitors, gibberellin antagonists and phosphatase inhibitors.
  • the polynucleotide of interest comprises a nucleotide sequence that encodes a polypeptide of interest for commercial manufacture, or encodes a product conferring a beneficial property to the transformed plant cells or a plant generated from the transformed plant cells.
  • the transformed plant cells can be identified using any suitable technique that detects the presence of the polynucleotide of interest in the plant cells, illustrative examples of which include: nucleic acid hybridization (e.g., Southern blotting or northern blotting), nucleic acid amplification (e.g. , polymerase chain reaction (PCR)), and detection of a polypeptide of interest using enzyme assays or antigen- binding molecules that are immuno-interactive with the polypeptide of interest.
  • nucleic acid hybridization e.g., Southern blotting or northern blotting
  • nucleic acid amplification e.g. , polymerase chain reaction (PCR)
  • detection of a polypeptide of interest using enzyme assays or antigen- binding molecules that are immuno-interactive with the polypeptide of interest.
  • the plant cells are derived from or form at least part of an explant, stem, seed, seedling, shoot, root, leaf cutting, stem cutting, root cutting, tuber eye, stolon, flower, pollen or callus to name but a few.
  • the plants cells to be transformed are regenerable plant cells from which differentiated genetically modified plants are producible.
  • the invention provides methods for producing a differentiated genetically modified plant, wherein a polynucleotide of interest is introduced into regenerable plant cells according to the 'marker free' transformation methods broadly described above so as to yield a population of transformed plant cells and regenerating a differentiated genetically modified plant from the population.
  • the regenerable plant cells may be selected from regenerable dicotyledonous cells and regenerable monocotyledonous plant cells (e.g., regenerable graminaceous monocotyledonous plant cells or regenerable non- graminaceous monocotyledonous plant cells).
  • the invention provides transformed plant cells and genetically modified plants resulting from the methods as broadly described above.
  • the invention contemplates the use of a vector or vector system for introducing a polynucleotide of interest in a plant cell by Agrobacterium-mediated transformation under conditions that inhibit apoptosis in the plant cell, wherein the vector or vector system comprises the polynucleotide of interest but lacks a marker gene that confers an identifying characteristic (e.g., antibiotic resistance, antibiotic sensitivity, cell death, enzymatic activity and light emission or absorbance) on the plant cell (i.e., containing the marker gene).
  • an identifying characteristic e.g., antibiotic resistance, antibiotic sensitivity, cell death, enzymatic activity and light emission or absorbance
  • the present invention provides a culture of plant cells for Agrobacterium-media ⁇ ed transformation, wherein the plant cells comprise in their nucleome an apoptosis-inhibiting polynucleotide that is operably connected to a regulatory element.
  • the regulatory element comprises a regulatable promoter that conditionally expresses the apoptosis-inhibiting polynucleotide.
  • the regulatory element is used to control expression of the apoptosis-inhibiting polynucleotide so it is expressed for the transformation but is not expressed at other times (e.g. , before and/or after the transformation).
  • the present invention contemplates the use of a culture of plant cells as broadly described above for introducing a polynucleotide of interest in those cells by Agrob ⁇ cterium-mQdiated transformation.
  • the plant cells of the culture are transformed using the methods broadly described above.
  • the present invention provides a use of a culture of plant cells for marker free Agrob ⁇ cterium-mediated transformation (i.e., transformation in the absence of selecting for the presence of a marker gene in the plant cells), wherein the plant cells comprise in their nucleome an apoptosis-inhibiting polynucleotide that is operably connected to a regulatory element.
  • FIG. 2 is a photographic representation illustrating Agrobacterium induced fragmentation of nuclear DNA in banana suspension cultures that can be inhibited by anti-apoptosis gene expression.
  • Untransformed banana suspension cell cultures are shown in images (a-d) and transformed banana cell cultures are shown in images (e-j).
  • images (a and b) represent banana suspension cells before 48 hours of A. tumefaciens infection and images (c- j) represent banana suspension cells after 48 hours of A. tumefaciens infection.
  • Images a, c, e, g and i show propidium iodide stained nuclei and images b, d, f, h and j show the same nuclei with TUNEL labeling.
  • TUNEL-positive illustrated in images (h and j) after exposure to Agrobacterium strain AGLl at (OD (600nm) 0.5).
  • Figure 3 is a photographic representation of an agarose gel illustrating Agrobacterium-indvLCQd nuclear DNA fragmentation in embryogenic cell suspensions of banana detected by gel electrophoresis of genomic DNA after 12 hours (lanes 2-4), 24 hours (lanes 5-7), 48 hours (lanes 8-10) and 72 hours (lanes 11-13) of Agrobacterium infection; lane 1, is an unexposed control.
  • FIG. 4 is a photographic representation of an agarose gel illustrating inhibition of Agrobacterium induced nuclear DNA fragmentation in embryogenic cell suspensions of banana detected by gel electrophoresis of genomic DNA after 72 hours of exposure to Agrobacterium (AGLl) at an OD 6 oo-
  • the samples loaded into the lanes of the agarose gel are as follows: lane 1, untransformed cells (cv. Grand Nain); lane 2, untransformed cells (cv.
  • Figure 5 is a photographic representation of transformed embryos from banana cell suspensions, wherein anti-apoptosis genes are shown to enhance recovery. Embryogenesis (see images a-d) and regeneration (see image e) on selection media in banana cell suspensions transformed with Bcl-xL (Gl 38A) (image a); BcI- xL(image b); Bcl-2 3' non-translated region (images c and e) and; Ced-9 (image d).
  • FIG. 6 is a photographic representation of a Southern blot of transgenic banana plants. The banana plants were analyzed to detect transgene copy number. Lanes 1-5 indicate independent transgenic plants transformed with (a) Bcl-xL (Gl 38A); (b) Bcl-xL; (c) Bcl-2 3 'non-translated region and (d) Ced-9. Transgenics were digested with restriction enzymes Sad (a, b, c) or EcoRY (d) that digest the binary plasmid in the T-DNA region only once. "N” indicates untransformed plants and, "P” indicates the respective binary plasmid used as a positive control. The probe used corresponds to the coding region of the respective transgene.
  • Figure 7 is a photographic representation of an agarose gel, showing
  • RT PCR detection of transgene specific RNA from banana transgenics using for (a) 5c/- xL(G138A); (b) Bcl-xL; (c) Bcl-2 3 'non-translated region and (d) CW-9.Transgene specific primers and five banana transgenic lines (lanes 1-5) were used for each transgene.
  • the lanes are illustrated as follows: lane N, untransformed control; lane P, PCR amplification product from transgene coding DNA sequence.
  • RNA samples were treated with Dnase and subjected to PCR amplification without reverse transcription to ascertain the absence of genomic DNA contamination that could produce a false positive signal; these samples were then used for RT-PCR analysis.
  • FIG. 8 is a photographic representation of banana root cells.
  • the banana root cells demonstrate that cells transformed with an anti-apoptosis gene are not affected by Agrobacterium induced fragmentation of nuclear DNA.
  • Untransformed cells images a-d
  • transformed cells images e-j
  • banana root cells before Agrobacterium infection images a and b
  • images c-j after 48 hours of Agrobacterium infection
  • Images a, c, e, g and i show propidium iodide stained nuclei and images b, d, f, h and j show the same nuclei with TUNEL labeling.
  • Figure 9 is a photographic representation of an agarose gel illustrating inhibition of Agrobacterium induced nuclear DNA fragmentation in root cells of banana, after 72 hours of exposure to Agrobacterium at an OD 600 .
  • the samples in each lane are set out as follows: lane 1, untransformed banana roots not exposed to
  • Agrobacterium (control); lane 2, Bcl-xL 3' non-translated region transformed root cells exposed to Agrobacterium; lane 3, Ced-9 3' non-translated region transformed root cells exposed to Agrobacterium; lane 4, Bcl-2 3' non-translated region transformed root cells exposed to Agrobacterium; lane 5, Bcl-xL(G ⁇ 38A) transformed roots, not exposed to Agrobacterium; lane 6, untransformed root cells (cv. Grand Nain) exposed to Agrobacterium; lane 7, untransformed root cells (cv. Grand Nain) exposed to Agrobacterium; lane 8, untransformed root cells (cv.
  • Figure 10 is a photographic representation showing the results of a sugarcane tissue culture. Leaf whorl (A), Callus induction (B), regeneration Ql 17 (C) regeneration Q208 (D) and rooting (Ql 17).
  • Figure 11 is a photographic representation showing the results of a sugarcane callus (Ql 17) (34 day old) before (A) and after (B) exposure to Agrobacterium.
  • Figure 12 is a photographic representation showing DNA degradation induced by Agrobacterium in sugarcane suspension cells from cultivar Ql 17. Genomic DNA from unexposed suspension cells (lane 2), Agrobacterium strain LBA4404 exposed (lane 3) and AGLl exposed (lane 4) 48 hours after exposure. Lane 1 : 10kb ladder.
  • Figure 13 is a photographic representation showing that TUNEL labeling (column 1), propidium iodide counterstaining (column 2) and overlay (column 3) of sugarcane suspension cells (Q208) following exposure to Agrobacterium indicate that intra-nucleosomal fragmentation of DNA increases with increase in the inoculum density.
  • Al, A2 and A3: Q208 exposed to Agrobacterium AGLl OD 660 0.1.
  • B4, B5 and B6: Q208 exposed to Agrobacterium LBA4404 OD 660 1.0.
  • -ve,T, -ve,PI and -ve,C control Q208 cells, not exposed to Agrobacterium.
  • Figure 14 is a photographic representation showing that TUNEL labeling (column 1), propidium iodide counterstaining (column 2) and overlay (column 3) of sugarcane suspension cells (Ql 17) following exposure to Agrobacterium indicate that intra-nucleosomal fragmentation of DNA increases with increase in the inoculum density.
  • D4, D5 and D6: Ql 17 exposed to Agrobacterium LBA4404 OD 660 1.0.
  • -ve,T, -ve,PI and -ve,C control Q208 cells, not exposed to Agrobacterium.
  • Figure 15 is a photographic representation illustrating the protective effect of expression of cell death inhibitor genes on Agrobacterium-induced cell death leads to improved embryogenic response from 14 day old Ql 17 explants after Agrobacterium-mediated transformation with LBA4404 (pUGfpnptll).
  • the explants were bombarded with (A) Bcl-xL, (B) Hsp70h (S) (C) AtB ⁇ g4 (D) Ql 17 Bcl-xL G138A and (E) Gfp before exposure to Agrobacterium (F) is untransformed and unexposed control. Photographs were taken two weeks after Agrobacterium exposure. Results for Q208 were identical.
  • Figure 16 is a photographic representation showing the effect of expression of cell death inhibitor genes on survival of 34 day old explants of Q 117 after exposure to Agrobacterium (1) unexposed and untransformed control and (2) untransformed but exposed, 3-9 were transformed with: Bcl-xL (3) Bcl-xL G138A (4) , AtBag4 (5), Hsp70h (S) (6), Hsp70h (M) (7), AtBag4 + Hsp70h (S) (8), AtBag4 + Hsp70h (M) (9) before transformation with Agrobacterium LBA4404 (pUGfpnptll(S). Photographs were taken two weeks later.
  • Figure 17 is a photographic representation showing the effect of expression of cell death inhibitor genes on survival of 34 day old explants of Q208 after exposure to Agrobacterium (1) unexposed and untransformed control and (2) untransformed but exposed, 3-9 were transformed with: Bcl-xL (3) Bcl-xL G 138 A (4), AtBag4 (5), Hsp70h (S) (6), Hsp70h (M) (7), AtBag4 + Hsp70h (S) (8), AtBag4 + Hsp70h (M) (9) before transformation with Agrobacterium LBA4404 (pUGfpnptll(S). Photographs were taken two weeks later.
  • antigen-binding molecule means a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
  • cells "plant cells,” “transformed plant cells,”
  • progenerable plant cells and the like are terms that not only refer to the particular subject cells but to the progeny or potential progeny of the plant cells. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent plant cells, but are still included within the scope of the term as used herein.
  • coding sequence any nucleic acid sequence that contributes to the code for the polypeptide product of a gene.
  • complementary and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
  • nucleic acid strands For example, for the sequence "A-G-T,” is complementary to the sequence “T-C-A.”
  • Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • constitutive promoter refers to a promoter that directs expression of an operably connected transcribable sequence in many or all tissues of a plant.
  • chimeric construct refers to a gene or nucleic acid sequence or segment comprising at least two nucleic acid sequences or segments from species which do not combine those sequences or segments under natural conditions, or which sequences or segments are positioned or linked in a manner which does not normally occur in the native genome or nucleome of the untransformed host.
  • a “chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding or non-coding sequences that are not found together in nature.
  • a chimeric gene may comprise regulatory sequences and coding or non-coding sequences that are derived from different sources, or regulatory sequences and coding or non-coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • a polynucleotide having a nucleotide sequence that is identical or substantially identical (e.g., one displaying at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % sequence identity) or complementary to all or a portion of a reference polynucleotide sequence or (b) encoding an amino acid sequence identical to an amino acid sequence in a peptide or protein.
  • This phrase also includes within its scope a peptide or polypeptide having an amino acid sequence that is identical or substantially identical (e.g., one displaying at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % sequence identity) to a sequence of amino acids in a reference peptide or protein.
  • the term “endogenous” refers to a gene or nucleic acid sequence or segment that is normally found in a host cell of interest.
  • expression refers to a gene or nucleic acid sequence or segment that is normally found in a host cell of interest.
  • expression refers to a gene or nucleic acid sequence or segment that is normally found in a host cell of interest.
  • expression includes genes that are transcribed into messenger RNA (mRNA) and then translated into protein, as well as genes that are transcribed into types of RNA such as transfer RNA (tRNA) and ribosomal RNA (rRNA) that are not translated into protein.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • the term "foreign” or “exogenous” or “heterologous” refers to any molecule (e.g., a polynucleotide or polypeptide) which is introduced into a host by experimental manipulations and may include gene sequences found in that host so long as the introduced gene contains some modification ⁇ e.g., a point mutation, the presence of a selectable marker gene, the presence of a recombination site, etc.) relative to the naturally-occurring gene.
  • the term "gene” as used herein refers to any and all discrete coding regions of the cell's nucleome, as well as associated non-coding and regulatory regions.
  • the gene is also intended to mean the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression.
  • the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene or heterologous control signals.
  • the DNA sequences may be cDNA or genomic DNA or a fragment thereof.
  • the gene may be introduced into an appropriate vector for extra chromosomal maintenance or for integration into a plant cell.
  • growing or “regeneration” as used herein mean growing a whole, differentiated plant from a plant cell, a group of plant cells, a plant part (including seeds), or a plant piece (e.g., from a protoplast, callus, or tissue part).
  • knock-out is meant the inactivation or loss-of-function of a gene, which decreases, abrogates or otherwise inhibits the level or functional activity of an expression product of that gene.
  • a “knock-out” plant or plant cell refers to a genetically modified plant or plant cell in which a gene is inactivated or loses function.
  • marker gene is meant a gene that imparts a distinct phenotype to cells expressing the marker gene and thus allows such transformed cells to be distinguished from cells that do not have the marker.
  • a selectable marker gene confers a trait for which one can 'select' based on resistance to a selective agent (e.g., an herbicide, antibiotic, radiation, heat, or other treatment damaging to untransformed cells).
  • a screenable marker gene confers a trait that one can identify through facile observation or testing, i.e., by 'screening' (e.g., ⁇ -glucuronidase, luciferase, green fluorescent protein or other activity not present in untransformed cells). It shall be understood that the term “marker gene” excludes the anti-apoptosis polynucleotide and the polynucleotide of interest that is introduced into the plant cells.
  • the term "marker free” as used herein refers to Agrobacterium- mediated transformation of plant cells, which is carried out without selecting for the presence of an identifying characteristic that is conferred by a marker gene in the plant cells.
  • negative selection refers to the act of selecting against cells through the implementation of methodologies which allow for the killing of those cells.
  • negative selection encompasses the situation in which a host cell grown in the presence of a negative selective agent such as acyclovir, ganciclovir, or 5- fluoro-2'-deoxyuridine (F2dU) dies if the cell containing a suicide gene, such as the herpes simplex virus (HSV) thymidine kinase (TK) gene, is expressed within the cell.
  • non-coding sequence refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene.
  • 5' non-coding region is used herein in its broadest context to include all nucleotide sequences which are derived from the upstream region of an expressible gene, other than those sequences which encode amino acid residues which comprise the polypeptide product of the gene, wherein 5' non-coding region confers or activates or otherwise facilitates, at least in part, expression of the gene.
  • nucleome is meant the total nucleic acid complement and includes the genome, extrachromosomal nucleic acid molecules and all RNA molecules such as mRNA, heterogenous nuclear RNA (hnRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small cytoplasmic RNA (scRNA), ribosomal RNA (rRN A), translational control RNA (tcRNA), transfer RNA (tRNA), eRNA, messenger- RNA-interfering complementary RNA (micRNA) or interference RNA (iRNA), chloroplast or plastid RNA (cpRNA) and mitochondrial RNA (mtRNA).
  • mRNA mitochondrial RNA
  • nucleic acid extract obtained from a sample such as, for example, a nucleic acid extract is isolated from, or derived from, a particular source of a plant.
  • the nucleic acid extract may be obtained from tissue isolated directly from a plant.
  • oligonucleotide refers to a polymer composed of a multiplicity of nucleotide units (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof).
  • An oligonucleotide is typically rather short in length, generally from about 10 to 30 nucleotides, but the term can refer to molecules of any length, although the term “polynucleotide” or "nucleic acid” is typically used for large oligonucleotides.
  • operably connected or “operably linked” and the like is meant a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid is
  • operably linked when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the nucleic acid sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • a coding sequence is "operably linked to" another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from both coding sequences.
  • the coding sequences need not be contiguous to one another so long as the expressed sequences are ultimately processed to produce the desired protein.
  • operably connecting a promoter to a transcribable polynucleotide is meant placing the transcribable polynucleotide (e.g. , protein encoding polynucleotide or other transcript) under the regulatory control of a promoter, which then controls the transcription and optionally translation of that polynucleotide.
  • a promoter or variant thereof it is generally preferred to position a promoter or variant thereof at a distance from the transcription start site of the transcribable polynucleotide, which is approximately the same as the distance between that promoter and the gene it controls in its natural setting; i.e. : the gene from which the promoter is derived.
  • a regulatory sequence element e.g., an operator, enhancer etc
  • a transcribable polynucleotide to be placed under its control is defined by the positioning of the element in its natural setting; i.e. the genes from which it is derived.
  • plant and “differentiated plant” refer to a whole plant or plant part containing differentiated plant cell types, tissues and/or organ systems. Plantlets and seeds are also included within the meaning of the foregoing terms. Plants included in the invention are any plants amenable to transformation techniques, including angiosperms, gymnosperms, monocotyledons and dicotyledons.
  • plant cell refers to any plant cell or cell line including protoplasts, gamete-producing cells, and cells which regenerate into whole plants. Plant cells also include cells in plants as well as protoplasts in culture.
  • plant tissue is meant differentiated and undifferentiated tissue derived from roots, shoots, pollen, seeds, tumor tissue, such as crown galls, and various forms of aggregations of plant cells in culture, such as embryos and calluses.
  • polypeptide polypeptide
  • peptide protein
  • proteins proteins
  • amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • polynucleotide or “nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators and modified linkages (e.g., ⁇ -anomeric nucleic acids, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc
  • RNA forms of the genetic molecules of the present invention are generally mRNA or iRNA including siRNAs.
  • the genetic form may be in isolated form or integrated with other genetic molecules such as vector molecules and particularly expression vector molecules.
  • nucleotide sequence used herein interchangeably and encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.
  • positive selection refers to a selection method that enables only those cells that carry a DNA insert integrated at a specific chromosomal location to grow under particular conditions.
  • promoter and "expression modulating sequence” are used interchangeably herein and are meant as a region of DNA, which controls at least in part the initiation and level of transcription of a polynucleotide.
  • Reference herein to a "promoter” or an “expression modulating sequence” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including a TATA box and CCAAT box sequences, as well as additional regulatory elements (i.e., activating sequences, enhancers and silencers) that alter gene expression in response to developmental and/or environmental stimuli, or in a tissue- specific or cell-type-specific manner.
  • a promoter is usually, but not necessarily, positioned upstream or 5', of a transcribable sequence (e.g., a coding sequence or a sequence encoding a functional RNA), the expression of which it regulates.
  • promoter also includes within its scope inducible, repressible and constitutive promoters as well as minimal promoters.
  • inducer stimulus refers to hormonal, environmental, chemical and physical means by which a promoter is expressible, “switched on”.
  • repressor stimulus refers to hormonal, environmental, chemical and physical means by which a promoter is not-expressible, “switched off.
  • Minimal promoters typically refer to minimal expression control elements that are capable of initiating transcription of a selected DNA sequence to which they are operably connected. In some examples, a minimal promoter is not capable of initiating transcription in the absence of additional regulatory elements (e.g., enhancers or other cw-acting regulatory elements) above basal levels. A minimal promoter frequently consists of a TATA box or TATA-like box. Numerous minimal promoter sequences are known in the literature. For example, minimal promoters may be selected from a wide variety of known sequences, including promoter regions from CaMV 35S promoter and SV40 among many others.
  • regulatory promoter and “regulatable expression modulating sequence” refers to polynucleotide sequences that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and include both tissue-specific and inducible promoters. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered. Since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.
  • regulatory sequences refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non- coding sequences) of a transcribable sequence, including a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated transcribable sequence.
  • Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.
  • stringency refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency, the higher will be the degree of complementarity between immobilized nucleotide sequences and the labeled polynucleotide sequence.
  • stringent conditions refers to temperature and ionic conditions under which only nucleotide sequences having a high frequency of complementary bases will hybridize.
  • the stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization.
  • stringent conditions are selected to be about 10 to 20° C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe.
  • transcribable nucleic acid sequence or "transcribed nucleic acid sequence” includes nucleic acid that is transcribed by cellular machinery to produce a transcript and excludes the non-transcribed regulatory sequence that drives transcription.
  • the transcribable sequence may be derived in whole or in part from any source known to the art, including a plant, a fungus, an animal, a bacterial nucleome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA or chemically synthesized DNA.
  • the transcribable sequence may contain an uninterrupted coding sequence or it may include one or more introns, bound by the appropriate splice junctions.
  • the transcribable sequence may also encode a fusion protein. In other embodiments, the transcribable sequence comprises non- coding regions only.
  • transformation means alteration or manipulation of the genotype of a plant cell by the introduction of an expression system or exogenous sequence according to the invention.
  • transformed and transgenic refer to a cell, tissue, organ or organism into which a foreign or exogenous nucleic acid, such as a chimeric construct or recombinant vector, has been introduced, including progeny thereof in which foreign or exogenous nucleic acid is present.
  • transgene is used herein to describe genetic material that has been or is about to be artificially introduced into the nucleome, especially the genome, of a plant cell and that is transmitted to the progeny of the plant. The transgene is used to transform a plant cell, meaning that a permanent or transient genetic change, especially a permanent genetic change, is induced in a plant cell following incorporation of one or more nucleic acid components of the expression system as defined herein.
  • vector is meant a nucleic acid molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned.
  • a vector typically contains one or more unique restriction sites and may be capable of autonomous replication in a plant cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the nucleome of the defined host such that the cloned sequence is reproducible.
  • the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one which, when introduced into the plant cell, is integrated into the nucleome and replicated together with the chromosome(s) into which it has been integrated.
  • a vector system may comprise a single vector or plasmid, or two or more vectors or plasmids, which contain the total DNA (e.g.
  • a polynucleotide of interest an apoptosis-inhibiting polynucleotide and optionally a modulator polynucleotide, as defined herein
  • the choice of vector will typically depend on the compatibility of the vector with the plant cell into which the vector is to be introduced.
  • the vector from which the polynucleotide of interest is expressed lacks a marker gene for selecting transformants that contain the polynucleotide of interest.
  • the vector includes a marker gene but the step of identifying or screening for the marker gene is excluded from the method of the invention.
  • wild type refers to an untransformed plant, plant cell, plant part or plant tissue, i.e., one where the nucleome, especially the genome, has not been altered by the presence of one or more nucleic acid components of an expression system as defined herein.
  • the present invention is based in part on the determination that the efficiency of Agrobacterium-medialed transformation of plant cells can be increased substantially (by more than about 100-fold in banana embryogenic cells) by exposing the plant cells during the transformation to conditions that inhibit apoptosis in those cells.
  • the inventors have surprisingly discovered that this increased efficiency permits identification of transformed plant cells that contain an exogenous polynucleotide or encoded polypeptide (also referred to herein as polynucleotide of interest or polypeptide of interest) without screening for the presence of a co-transformed marker gene (also referred to herein as "marker free" transformation.
  • the present invention provides methods for introducing a polynucleotide of interest into a plant cell, wherein the methods comprise exposing plant cells to an Agrob ⁇ cterium that contains the polynucleotide of interest under conditions that inhibit apoptosis in the cells, and identifying a transformed plant cell that contains the polynucleotide of interest without selecting for the presence of a marker gene in those cells.
  • Any method of inhibiting apoptosis is contemplated by the present invention, including the use of apoptosis- inhibiting agents such as small molecule compounds and apoptosis-inhibiting polynucleotides and polypeptides.
  • the apoptosis-inhibiting agent is either present in the plant cells at the time of transformation or introduced into the plant cells prior to transformation or at the same time as transforming the cells with the polynucleotide of interest.
  • Small molecule inhibitors of apoptosis are either present in the plant cells at the time of transformation or introduced into the plant cells prior to transformation or at the same time as transforming the cells with the polynucleotide of interest.
  • the apoptosis-inhibiting agent is a chemical inhibitor, for example, a small typically organic compound, which has a molecular weight of more than about 50 and less than about 2,500 Dalton.
  • the small molecule compound is suitably selected from ethylene inhibitors (e.g., 2,5- norbornadiene, norbornene, silver thiosulfate, and silver nitrate), ethylene synthesis inhibitors (e.g., aminoethoxyvinylglycine (AVG), cobalt salts, acetyl salicylic acid, or salicylic acid), gibberellin antagonists (e.g., abscisic acid (ABA)) and phosphatase inhibitors (e.g., okadaic acid).
  • ethylene inhibitors e.g., 2,5- norbornadiene, norbornene, silver thiosulfate, and silver nitrate
  • ethylene synthesis inhibitors e.g., aminoethoxyvin
  • a small molecule compound is suitably present in an effective concentration, e.g. , for silver nitrate in a concentration of from 0.1 to 20 mg/L, especially 1 to 10 mg/L.
  • the plant cells to be transformed are contacted with the small molecule compound for a time and under conditions sufficient to inhibit apoptosis of the plant cells that would otherwise occur in response to an apoptotic signal (e.g. , Agrobacterium infection).
  • the apoptosis-inhibiting agent is an apoptosis- inhibiting polynucleotide, which inhibits apoptosis directly or which encodes an apoptosis-inhibiting RNA or an apoptosis-inhibiting polypeptide.
  • apoptosis-inhibiting polypeptides include but are not restricted to: Bcl-xL; CED-9; Bcl-2; BI-I (a Bax inhibitor with known anti-apoptosis activity); Hsp70; AtILP-I (an IAP-like protein; IAPs from baculovirus are known anti- apoptosis genes (Higashi et al, 2005); AtBAGl (in mammals, BAGl enhances the anti- apoptotic effects of Bcl-2 and is a Hsp70 co-chaperone), AtBAG2, AtBAG3, AtBAG4, AtBAG5, AtBAG ⁇ , AtBAG7; CED-9; IAP-I and; mcl-1.
  • apoptosis-inhibiting genes from one organism have been shown to function in other disparate organisms (e.g., apoptosis-inhibiting genes from animal and bacteria have been shown to function in plants and vice versa)
  • the present invention contemplates the use of any apoptosis- inhibiting gene and encoded product from any source provided that it is operable in plant cells.
  • Table 1 Listed below in Table 1 are illustrative examples of apoptosis-inhibiting genes and their corresponding sequences.
  • the nucleotide sequence of the apoptosis-inhibiting polynucleotide may by modified using codons which are preferred by the host plant cells and avoiding nucleotide sequences, e.g., polyadenylation signals or splice sites within the coding region, which may affect optimal expression in the host plant, e.g., analogously to the methods described in U.S. Pat. No. 5,380,831 or U.S. Pat. No. 5,610,042.
  • the apoptosis-inhibiting polynucleotide comprises a nucleic acid sequence encoding an inhibitory RNA molecule ⁇ e.g., a ribozyme, an antisense nucleic acid or RNAi molecule) that is specific to a transcript product of an apoptosis gene whose expression product ⁇ e.g., protease, kinase, phosphatase or regulatory protein) stimulates or otherwise advances apoptosis.
  • an inhibitory RNA molecule e.g., a ribozyme, an antisense nucleic acid or RNAi molecule
  • the apoptosis-inhibiting polynucleotide comprises a nucleic acid sequence encoding a RNA molecule that directly or indirectly attenuates or otherwise disrupts the expression of an apoptosis gene.
  • the apoptosis-inhibiting polynucleotide comprises a nucleic acid sequence, which when expressed in the host cell produces a RNA molecule that comprises a targeting region having sequence identity with a nucleotide sequence of the apoptosis gene and that attenuates or otherwise disrupts the expression of that gene.
  • the targeting region may have sequence identity with the sense strand or the anti-sense strand of the apoptosis gene.
  • RNA molecules are discussed in more detail below in Section 3 and although these methods are discussed in the context of controlling the expression of an apoptosis-inhibiting polynucleotide or the level or functional activity of an apoptosis-inhibiting polypeptide, they can be used analogously for controlling the expression of an apoptosis gene or the level or functional activity of its gene product.
  • apoptosis-inhibiting polynucleotide or the level or functional activity of an apoptosis-inhibiting polypeptide, they can be used analogously for controlling the expression of an apoptosis gene or the level or functional activity of its gene product.
  • the apoptosis-inhibiting polynucleotide is usually in the form of a construct, which is typically chimeric in nature. These constructs will usually comprise regulatory elements that control expression of the apoptosis-inhibiting polynucleotide or that process its expression product, as described infra. [0092]
  • the apoptosis-inhibiting polynucleotide may be stably incorporated into the nucleome ⁇ e.g., genome) of the plant cells to be transformed or may be only transiently present and operable, e.g., at or around the time the cell is exposed to the Agrobacterium.
  • Transient expression can be obtained, e.g., using an agroinfection system, in which, for example, the T-DNA carries two geminiviruses in tandem such that a viral replicon that carries the apoptosis-inhibiting polynucleotide may replicate in the cell.
  • the virus typically will not integrate into the plant genome but will replicate to a high copy number and provide a high level of transient expression.
  • Cells thus primed to be resistant to apoptosis can then be transformed using Agrobacterium having Ti plasmids comprising the polynucleotide of interest, which will be incorporated into the nucleome, while the virus is diluted through regeneration and will not be transmitted to the seed.
  • the progeny and descendants of the infected plant cells are stably transformed with the gene of interest but not with the apoptosis- inhibiting polynucleotide.
  • Transient expression may alternatively be obtained by introducing short apoptosis-inhibiting oligonucleotide sequences into the plant, e.g., antisense sequences.
  • the apoptosis-inhibiting agent is an apoptosis- inhibiting polypeptide, which may be produced from an apoptosis-inhibiting polynucleotide that is expressed in the plant cells into which it is introduced, or which may be delivered independently of an introduced polynucleotide.
  • the apoptosis-inhibiting polypeptide may be delivered from Agrobacte ⁇ um into plant cells in the form of a fusion protein comprising a targeting moiety that targets the fusion protein to a specific subcellular location.
  • an apoptosis-inhibiting polypeptide may be fused to a virulence (Vir) fusion protein in Agrobacterium, wherein the polypeptide is delivered to plant cells using Agrobacterium-mediated transformation.
  • the apoptosis-inhibiting polypeptide will generally be localized to the nucleus of the plant cell. However, other sub-cellular localization could be targeted within the cell.
  • Agrobacterium fusion proteins are constructed to retain the functional properties of the Vir protein so that the selected protein as well as the transgene can be delivered.
  • an apoptosis-inhibiting polynucleotide or the level or functional activity of an apoptosis-inhibiting polypeptide is desirably controllable to reduce or abrogate anti-apoptotic activity post-transformation.
  • high levels of expression of Bcl-xL or CED-9 polynucleotides in transgenic tomato plants has been shown to affect both plant growth and seed development Xu et al. , (2004, Proc Natl AcadSci USA, 101(44):15805-15810) and there is now growing evidence that apoptosis-like events are involved in a range of plant development processes.
  • apoptosis-inhibiting polynucleotide it is possible therefore that continued expression of the apoptosis-inhibiting polynucleotide and ensuing production of the apoptosis-inhibiting polypeptides could have an effect on normal plant development and may negate the advantage of increased transformation efficiency. Accordingly, in certain advantageous embodiments, once an apoptosis- inhibiting polynucleotide has been transformed into a plant cell, its expression is suitably disrupted or the level or functional activity of its expression product is abrogated or reduced.
  • RNA-mediated inhibitory techniques e.g., sense or antisense suppression techniques as disclosed for example by Waterhouse et ⁇ /.,1998, Proc Natl Acad Sci USA, 95(23): 13959- 13964.
  • An illustrative approach may incorporate constitutive expression of the anti-apoptosis polynucleotide and conditional expression of the RNA inhibitory molecule under the control of an inducible promoter. In this approach, expression of the apoptosis-inhibiting polynucleotide would be silenced upon chemical induction of the RNA inhibitory molecule.
  • an inhibitor of the encoded apoptosis-inhibiting polypeptide can be employed to reduce the level or functional activity of the polypeptide.
  • a modulator polynucleotide is employed in the methods and constructs of the invention to abrogate or otherwise disrupt the expression of an apoptosis-inhibiting polynucleotide that has been introduced into a plant cell by Agrobacterium-mediated transformation.
  • the modulator polynucleotide comprises a nucleic acid sequence encoding an antisense RNA molecule that directly blocks the translation of mRNA transcribed from the apoptosis-inhibiting polynucleotide by binding to the mRNA and preventing protein translation.
  • antisense RNAs should be at least about 10-20 nucleotides or greater in length, and be at least about 75% complementary to their target genes or gene transcripts such that expression of the targeted homologous sequence is precluded.
  • the modulator polynucleotide comprises a nucleic acid sequence encoding a ribozyme that functions to inhibit the translation of the mRNA of the anti-apoptosis polynucleotide.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of target gene RNA sequences.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the anti-apoptosis polynucleotide containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable.
  • ribozymes When employed, ribozymes may be selected from the group consisting of hammerhead ribozymes, axehead ribozymes, newt satellite ribozymes, Tetrahymena ribozymes and RNAse P, and are designed according to methods known in the art based on the sequence of the target polynucleotide (for instance, see U.S. Pat. No. 5,741,679). The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
  • the modulator polynucleotide comprises a nucleic acid sequence encoding an antigen-binding molecule that is interactive with a protein product of the apoptosis-inhibiting polynucleotide.
  • the antigen-binding molecule may be selected from immunoglobulin molecules (e.g., whole polyclonal antibodies and monoclonal antibodies)and fragments thereof (e.g., Fv, Fab, Fab' and F(ab') 2 immunoglobulin fragments) as well as synthetic antigen-binding molecules(e.g., stabilized Fv fragments, single variable region domains (also known as a dAbs), minibodies and the like as known in the art).
  • immunoglobulin molecules e.g., whole polyclonal antibodies and monoclonal antibodies
  • fragments thereof e.g., Fv, Fab, Fab' and F(ab') 2 immunoglobulin fragments
  • synthetic antigen-binding molecules e.g.,
  • the modulator polynucleotide comprises a nucleic acid sequence encoding a RNA molecule that directly or indirectly attenuates or otherwise disrupts the expression of the apoptosis-inhibiting polynucleotide by post- transcriptional gene silencing (PTGS).
  • PTGS post- transcriptional gene silencing
  • the PTGS conferred by the RNA molecules is sometimes referred to as RNA interference (RNAi).
  • RNAi refers to interference with or destruction of the product of a target gene by introducing a single stranded or double stranded RNA (dsRNA) that is homologous to a transcript of the apoptosis-inhibiting polynucleotide. Absolute homology is not required for RNAi, with a lower threshold being described at about 85% homology for a dsRNA of about 200 base pairs (Plasterk and Ketting, 2000, Current Opinion in Genetics and Dev, 10:562-
  • the RNAi-encoding nucleic acids can vary in the level of homology they contain toward the apoptosis-inhibiting polynucleotide, e.g., with dsRNAs of 100 to 200 base pairs having at least about 85% homology with the apoptosis-inhibiting polynucleotide, and longer dsRNAs, i.e., 300 to 100 base pairs, having at least about 75% homology to the apoptosis-inhibiting polynucleotide.
  • RNA-encoding constructs that express a single RNA transcript designed to anneal to a separately expressed RNA, or single constructs expressing separate transcripts from convergent promoters are suitably at least about 100 nucleotides in length.
  • RNA-encoding constructs that express a single RNA designed to form a dsRNA via internal folding are desirably at least about 200 nucleotides in length.
  • expression of the nucleic acid sequence in plant cells produces a RNA molecule that comprises a targeting region having sequence identity with a nucleotide sequence of the apoptosis-inhibiting polynucleotide and that attenuates or otherwise disrupts the expression of that polynucleotide.
  • the targeting sequence displays at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identity to a nucleotide sequence of the apoptosis-inhibiting polynucleotide.
  • the targeting sequence hybridizes to a nucleotide sequence of the apoptosis-inhibiting polynucleotide under at least low stringency conditions, more suitably under at least medium stringency conditions and even more suitably under high stringency conditions.
  • Reference herein to low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C, and at least about 1 M to at least about 2 M salt for washing at 42° C.
  • Low stringency conditions also may include 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO 4 (pH 7.2), 5% SDS for washing at room temperature.
  • BSA bovine serum albumin
  • Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.5 M to at least about 0.9 M salt for washing at 42° C.
  • Medium stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO 4 (pH 7.2), 5% SDS for washing at 42° C.
  • High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization at 42° C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42° C.
  • High stringency conditions also may include 1% BSA, 1 niM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, ImM EDTA, 40 mM NaHPO 4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C.
  • the targeting sequence hybridizes to a nucleotide sequence of the anti-apoptosis polynucleotide under physiological conditions.
  • Other stringent conditions are well known in the art. A skilled artisan will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see Ausubel et ah, supra at pages 2.10.1 to 2.10.16 and Sambrook et al. ("Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press, 1989) at sections 1.101 to 1.104.
  • the targeting region has sequence identity with the sense strand or antisense strand of the apoptosis-inhibiting polynucleotide.
  • the RNA molecule is unpolyadenylated, which can lead to efficient reduction in expression of the anti-apoptosis polynucleotide, as described for example by Waterhouse et al., in U.S. Patent No. 6,423,885.
  • the length of the targeting region may vary from about 10 nucleotides up to a length equaling the length (in nucleotides) of the apoptosis- inhibiting polynucleotide.
  • the length of the targeting region is at least 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nts, usually at least about 50 nts, more usually at least about 100 nts, especially at least about 150 nts, more especially at least about 200 nts, even more especially at least about 500 nts. It is expected that there is no upper limit to the total length of the targeting region, other than the total length of the apoptosis-inhibiting polynucleotide. However for practical reason (such as e.g. stability of the constructs described herein) it is expected that the length of the targeting region should not exceed 5000 nts, particularly should not exceed 2500 nts and could be limited to about 1000 nts.
  • the RNA molecule may further comprise one or more other targeting regions (e.g., from about 1 to about 10, or from about 1 to about 4, or from about 1 to about 2 other targeting regions) each of which has sequence identity with a nucleotide sequence of the apoptosis-inhibiting polynucleotide.
  • the targeting regions are identical or share at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with each other.
  • PTGS of the apoptosis-inhibiting polynucleotide is achieved using the strategy of Glassman et al., described in U.S. Patent Application Publication No 2003/0036197.
  • suitable nucleic acid sequences and their reverse complement can be used to alter the expression of any homologous, endogenous target RNA (e.g., a target RNA defining an expression product of the apoptosis-inhibiting polynucleotide) which is in proximity to the suitable nucleic acid sequence and its reverse complement.
  • the suitable nucleic acid sequence and its reverse complement can be either unrelated to any endogenous RNA in the plant cell or can be encoded by any nucleic acid sequence in the nucleome of the plant cells provided that nucleic acid sequence does not encode any target RNA or any sequence that is substantially similar to the target RNA.
  • the RNA molecule further comprises two complementary RNA regions which are unrelated to any endogenous RNA in the plant cells and which are in proximity to the targeting region.
  • the RNA molecule further comprises two complementary RNA regions which are encoded by any nucleic acid sequence in the nucleome of the plant cells provided that the sequence does not have sequence identity with the nucleotide sequence of the apoptosis-inhibiting polynucleotide, wherein the regions are in proximity to the targeting region.
  • one of the complementary RNA regions can be located upstream of the targeting region and the other downstream of the targeting region.
  • both the complementary regions can be located either upstream or downstream of the targeting region or can be located within the targeting region itself.
  • the proposed apoptosis-inhibiting polynucleotides of the invention can be "knocked out” using targeted homologous recombination. See, for example, Kempin et al., 1997, Nature, 389:802; Smithies et al., 1985, Nature, 317:230 234; Thomas and Capecchi, 1987, Cell, 51:503 512; and Thompson et al., 1989, Cell, 5:313 321.
  • the modulator polynucleotide comprises a nucleic acid sequence encoding a site specific recombinant protein.
  • the site-specific recombinase protein may include components of any recombinant system that mediates nucleic acid rearrangements in a specific nucleic acid locus, including site specific recombinases of the integrase or resolvase/invertase classes Abremski et ah, (1992, Protein Engineering, 5:87-91) and site-specific recombination mediated by intron-encoded endonucleases Perrin et al., (1993, EMBO J. 12:2939-2947).
  • Suitable site-specific recombinase proteins may be selected from the group comprising FLP recombinase, Cre recombinase, R recombinase from the Zygosaccharomyces rouxii plasmid pSRl, a recombinase from the Kluyveromyces drosophilarium plasmid pKDl, a recombinase from the Kluyveromyces waltii plasmid pKWl, any component of the Gin recombination system, the specific DNA binding component of the recombinase complex or the enzyme component of the recombinase complex.
  • the site specific recombinase protein is produced from a separate construct from which a modulator polynucleotide that encodes the recombinase protein is regulatably expressed.
  • the expression of the recombinase-encoding polynucleotide is regulated using a regulatable or inducible promoter as described for example below or a polynucleotide that attenuates or otherwise disrupts the expression of the recombinase-encoding polynucleotide by PTGS.
  • the site-specific recombinase protein is a fusion protein, comprising the recombinase protein or a component of the recombinase complex, fused to part or all of a nuclear receptor, such that the amino acids that encode the ligand- binding regions of the nuclear receptor are included.
  • the site-specific recombinase fusion protein comprises Cre recombinase flanked by two loxP sites (i.e., a Cre/loxP expression system).
  • Such an expression system comprises the DNA recombinase enzyme derived from E. coli Pl phage, which is flanked by two loxP sites in direct orientation.
  • the loxP DNA sequence is derived from E. coli Pl phage, which has the following 34 bp DNA nucleotide sequence: 5'- ATAACTTCGTATAGCATA CATTATACGAAGTTAT-3'.
  • the Cre recombinase fusion protein ligand-binding domain allows the activity of expressed Cre recombinase to be inhibited in the absence of ligand binding to the ligand binding domain, and wherein the inhibition of the Cre recombinase activity in relation to the loxP sites is relieved by binding of the ligand to the ligand-binding domain.
  • An alternative to post-transcriptional gene silencing may involve transcriptional gene silencing using a disaggregated ssDNA (geminivirus or nanovirus) silencing system.
  • Plant single stranded DNA (ssDNA) viruses replicate by rolling circle replication where the virus encodes a Replication initiation protein (Rep protein) which directs the replication of ssDNA.
  • the plant host provides the DNA polymerase machinery.
  • the Rep protein recognizes its cognate intergenic region to which it binds, initiating replication within the highly conserved loop sequence of the stem/loop structure. This initiation can occur in either the cis or trans orientation and, furthermore, all viral sequences other than the intergenic region(s) and the Rep protein can be dispensed with for replication and can be replaced by heterologous sequences of virtually any origin.
  • a system in which an expression cassette that comprises an apoptosis-inhibiting polynucleotide operably connected to a regulatory element, is flanked by intergenic regions of a geminivirus (e.g., the intergenic regions of tobacco yellow dwarf virus (TYDV)) to form a chimeric construct that is stably integrated into the nucleome of plants cells.
  • a geminivirus e.g., the intergenic regions of tobacco yellow dwarf virus (TYDV)
  • TYDV tobacco yellow dwarf virus
  • episomes expressing the apoptosis-inhibiting polynucleotide of the invention can be replicated for up to 20 days, which is considered to provide sufficient time to inhibit apoptosis of the plant cells, which is caused, for example, by infection with A.
  • the polynucleotide of interest may be an endogenous polynucleotide that is found naturally in the genome of the host plant cells.
  • the polynucleotide of interest is a recombinant or artificial nucleic acid that has been or is about to be introduced into the nucleome of the host plant cells.
  • the polynucleotide of interest is selected from 1) genes that are both transcribed into mRNA and translated into polypeptides as well as (2) genes that are only transcribed into RNA ⁇ e.g., functional RNA molecules such as rRNA, tRNA, RNAi, ribozymes and antisense RNA).
  • the polynucleotide of interest encodes a polypeptide for commercial manufacture, where the polypeptide is extracted or purified from the host plant, host plant cell or host plant part.
  • polypeptides include, but are not limited to, polypeptides involved in the biosynthesis of antibiotics or secondary metabolites, immunogenic molecules for use in vaccines, cytokines and hormones.
  • the polynucleotide of interest encodes a product conferring a beneficial property to the host or other advantageous characteristic including, but not limited to, herbicide resistance or tolerance ⁇ e.g., glyphosate resistance or glufosinate resistance), stress tolerance ⁇ e.g., salt tolerance), sterility, improved food content or increased yields ⁇ e.g., a product affecting starch biosynthesis or modification such as starch branching enzymes, starch synthases, ADP-glucose pyrophosphorylase, products involved in fatty acid biosynthesis such as desaturases or hydroxylases and products altering sucrose metabolism such as invertases, sucrose isomerases or sucrose synthases) as well as disease resistance or tolerance ⁇ e.g.
  • resistance to bacterial, viral, nematode, helminth, insect, protozoan or viral pathogens a product conferring insect resistance such as crystal toxin protein of Bacillus thuringiensis; a product conferring viral resistance such as a viral coat or capsid protein; a product conferring fungal resistance such as chitinase, ⁇ -l,3-glucanase or phytoalexins).
  • the apoptosis-inhibiting polynucleotides, the polynucleotides of interest and the modulatory polynucleotides as broadly described above are typically operably connected to a regulatory element (e.g. , a promoter and a 3' non-translated region) that is functional in plant cells to create a nucleic acid construct, designed for genetic transformation of plants.
  • a regulatory element e.g. , a promoter and a 3' non-translated region
  • Promoters contemplated by the present invention may be native to the plant or may be derived from an alternative source, where the promoter is functional in the plant. The selection of a particular promoter depends on the cell type used to express the desired polynucleotide.
  • promoters that are active in plant cells have been described in the literature, illustrative examples of which include the nopaline synthase (NOS) promoter, the octopine synthase (OCS) promoter (which is carried on tumor- inducing plasmids of Agrob ⁇ cterium tumefaciens), the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter and the CaMV 35S promoter, the figwort mosaic virus 35S-promoter, the light-inducible promoter from the small subunit of ribulose-l,5-bis-phosphate carboxylase (ss RUBISCO), the Adh promoter, the sucrose synthase promoter, the R gene complex promoter, the GST-II-27 gene promoter and the chlorophyll a/b binding protein gene promoter, etc.
  • NOS nopaline synthase
  • OCS octopine synthase
  • CaMV cauliflower mosaic virus
  • promoters driving expression of a particular gene have relatively high expression in these specific tissues.
  • promoters examples include the chloroplast glutamine synthetase GS2 promoter from pea, the chloroplast fructose- 1,6- biphosphatase (FBPase) promoter from wheat, the nuclear photosynthetic ST-LS 1 promoter from potato, the serine/threonine kinase (PAL) promoter and the glucoamylase (CHS) promoter from Arabidopsis thaliana.
  • FBPase chloroplast fructose- 1,6- biphosphatase
  • ST-LS 1 from potato
  • PAL serine/threonine kinase
  • CHS glucoamylase
  • RbcS ribulose-l,5-bisphosphate carboxylase
  • the promoter for the cab gene, cab ⁇ from pine
  • the promoter for the Cab-1 gene from wheat
  • the promoter for the CAB-I gene from spinach
  • the promoter for the cab IR gene from rice
  • the promoter for the tobacco Lhcbl *2 gene the Arabidopsis thaliana SUC2 sucrose-H+ symporter and the promoter for the thylakoid membrane proteins from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS).
  • Other promoters for the chlorophyll a/b-binding proteins may also be utilised in the invention, such
  • a number of promoters for genes with tuber-specific or tuber-enhanced expression are known, including the class I patatin promoter, the promoter for the potato tuber ADPGPP genes, both the large and small subunits, the sucrose synthase promoter, the promoter for the major tuber proteins including the 22 kd protein complexes and protease inhibitors, the promoter for the granule-bound starch synthase gene (GBSS) and other class I and II patatins promoters.
  • GBSS granule-bound starch synthase gene
  • promoters can also be used to express a selected polynucleotide sequence in specific tissues, such as seeds or fruits.
  • promoters include the 5' regulatory regions from such genes as napin, phaseolin, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, soybean ⁇ ' subunit of ⁇ -conglycinin (soy 7s), and oleosin.
  • Further examples include the promoter for ⁇ -conglycinin.
  • zeins which are a group of storage proteins found in corn endosperm.
  • Genomic clones for zein genes have been isolated and the promoters from these clones, including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD and genes, could also be used.
  • Other promoters known to function, for example, in corn include the promoters for the following genes: waxy, Brittle, Shrunken 2, Branching enzymes I and II, starch synthases, debranching enzymes, oleosins, glutelins and sucrose synthases.
  • promoters suitable for expression in wheat include those promoters for the ADP glucose pyrosynthase (ADPGPP) subunits, the granule bound and other starch synthase, the branching and debranching enzymes, the embryogenesis-abundant proteins, the gliadins and the glutenins.
  • promoters in rice include those promoters for the ADPGPP subunits, the granule bound and other starch synthase, the branching enzymes, the debranching enzymes, sucrose synthases and the glutelins.
  • promoters for barley include those for the ADPGPP subunits, the granule bound and other starch synthase, the branching enzymes, the debranching enzymes, sucrose synthases, the hordeins, the embryo globulins and the aleurone specific proteins.
  • Root specific promoters may also be used.
  • An example of such a promoter is the promoter for the acid chitinase gene. Expression in root tissue could also be accomplished using the root specific subdomains of the CaMV35S promoter that have been identified.
  • constitutive plant promoters useful for expressing genes in plant cells include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, maize ubiquitin (JJbi-1) promoter, rice actin ⁇ Act) promoter, nopaline synthase promoter, the 1'- or 2'-promoter derived from tDNA of A. tumefaciens and the octopine synthase promoter.
  • CaMV cauliflower mosaic virus
  • JJbi-1 maize ubiquitin
  • rice actin ⁇ Act rice actin ⁇ Act
  • nopaline synthase promoter the 1'- or 2'-promoter derived from tDNA of A. tumefaciens
  • the octopine synthase promoter A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals to name but a few can also be used for expression of foreign genes in plant cells, including promoters regulated by heat
  • heat shock promoters Hsp70
  • light e.g. , pea rbcS- 3A or maize rbcS promoters or chlorophyll a/b-binding protein promoter
  • phytohormones such as abscisic acid or hormones such as estrogen (ERE); wounding (e.g., wunl); anaerobiosis (e.g., Adh); and chemicals such as methyl jasminate, salicylic acid, or safeners (elnl).
  • organ-specific promoters such as endosperm-, embryo-, root-, phloem-, or trichome-specific promoters, for example.
  • the apoptosis-inhibiting polynucleotide and/or the polynucleotide of interest are conditionally expressible, for example through the use of an inducible promoter.
  • transcription or expression under the control of an inducible promoter is "stimulated” or increased in response to an applied stimulus.
  • the nature of the stimulus varies between promoters.
  • Some inducible promoters cause little or undetectable levels of expression of the polynucleotide in the absence of the appropriate stimulus.
  • Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus.
  • promoters there are a number of chemically inducible promoter systems that have been developed for plants.
  • Illustrative examples of such promoters include but are not limited to the ethanol inducible promoter system, Felenbok, (1991, J Biotechnol, 17(1):11-17), the glucocorticoid inducible promoter system, Aoyama and Chua, (1997, Planta, 213(3):370-378) which has already been used to control the expression of a pro- apoptosis gene in Arabidopsis protoplasts and the methoxyfenozide inducible promoter system, Padidam et ah, (2003, Transgenic Res, 12(1): 101 -109).
  • Chemically inducible promoters function in such a way that expression is switched on in the presence of the inducer, the inducer does not have a negative effect on the tissue to be transformed and there is no expression in the absence of the inducer.
  • An alternative to a chemically inducible promoter system is a system wherein the promoter is repressed by a stimulus, and once the stimulus is removed, expression of the operably connected sequence is switched on. Examples of such a system include the Saccharomyces cerevisiae JENl promoter, which is repressed in the presence of certain sugars such as glucose, fructose and mannose (Chambers et ah, 2004, Applied. Enviro. Microbiol., 70(l):8-17).
  • Inducible promoters may also be selected from environmentally inducible promoters including, but not limited to, the light-inducible promoter from the small subunit of ribulose-l,5-bis-phosphate carboxylase (ss RUBISCO); the drought- inducible promoter of maize (Buske? al, 1997, Plant J., 11:1285-1295), the cold, drought, and high salt inducible promoter from potato (Kirch, 1997, Plant MoI. Biol.
  • environmentally inducible promoters including, but not limited to, the light-inducible promoter from the small subunit of ribulose-l,5-bis-phosphate carboxylase (ss RUBISCO); the drought- inducible promoter of maize (Buske? al, 1997, Plant J., 11:1285-1295), the cold, drought, and high salt inducible promoter from potato (Kirch, 1997, Plant MoI. Biol.
  • promoters under developmental control include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, fruit, seeds, or flowers.
  • An exemplary promoter is the anther specific promoter 5126 (U. S. Patent Nos. 5,689,049 and 5,689,051).
  • seed-preferred promoters include, but are not limited to, 27 kD gamma zein promoter and waxy promoter as described in: Boronat, A, et al, (1986, Plant 5"c/.,47:95-102; Reina, M, et al, 1990, Nucleic Acids Res., 18 (21):6426 and; Kloesgen, R. B, et al, 1986, MoI. Gen. Genet., 203:237-244). The disclosures of each of these are incorporated herein by reference in their entirety.
  • the barley or maize Nucl promoter, the maize Cim 1 promoter or the maize LTP2 promoter can be used to preferentially express in the nucleus. See for example WO 00/11177 and U. S. Application number 6,225,529 (2001).
  • Either heterologous or non-heterologous (/. e. , endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention.
  • the constructs of the present invention can comprise a 3' non- translated sequence.
  • a 3' non-translated sequence refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.
  • the polyadenylation signal is characterized by effecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • Polyadenylation signals are commonly recognized by identity with the canonical form 5' AATAAA-3' although variations are not uncommon.
  • the 3' non-translated regulatory DNA sequence suitably includes from about 50 to 1,000 nucleotide base pairs and may contain plant transcriptional and translational termination sequences in addition to a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.
  • suitable 3' non-translated sequences are the 3' transcribed non-translated regions containing a polyadenylation signal from the nopaline synthase (nos) gene of Agrobacterium tumefaciens (Bevan et al, 1983, Nucl. Acid Res., 11:369) and the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens.
  • suitable 3' non-translated sequences may be derived from plant genes such as the 3' end of the protease inhibitor I or II genes from potato or tomato, the soybean storage protein genes and the pea E9 small subunit of the ribulose- 1,5-bisphosphate carboxylase (ss RUBISCO) gene, although other 3' elements known to those of skill in the art can also be employed.
  • 3' non-translated regulatory sequences can be obtained de novo as, for example, described by An (1987, Methods in Enzymology, 153:292).
  • the nucleic acid constructs of the present invention can further include enhancers, either translation or transcription enhancers, as may be required. These enhancer regions are well known to persons skilled in the art, and can include the ATG initiation codon and adjacent sequences. The initiation codon must be in phase with the reading frame of the coding sequence relating to the foreign or endogenous DNA sequence to ensure translation of the entire sequence.
  • the translation control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • Translational initiation regions may be provided from the source of the transcriptional initiation region, or from the foreign or endogenous DNA sequence. The sequence can also be derived from the source of the promoter selected to drive transcription, and can be specifically modified so as to increase translation of the mRNA.
  • transcriptional enhancers include, but are not restricted to, elements from the CaMV 35S promoter and octopine synthase genes as for example described by Last et al. (U.S. Patent No. 5,290,924). It is proposed that the use of an enhancer element such as the ocs element, and particularly multiple copies of the element, will act to increase the level of transcription from adjacent promoters when applied in the context of plant transformation.
  • the omega sequence derived from the coat protein gene of the tobacco mosaic virus may be used to enhance translation of the mRNA transcribed from a polynucleotide according to the invention.
  • leader sequences include those that comprise sequences selected to direct optimum expression of the R polypeptide gene.
  • leader sequences include a preferred consensus sequence which can increase or maintain mRNA stability and prevent inappropriate initiation of translation as for example described by Joshi (1987, Nucl. Acid Res., 15:6643).
  • other leader sequences e.g., the leader sequence of RTBV, have a high degree of secondary structure that is expected to decrease mRNA stability and/or decrease translation of the mRNA.
  • leader sequences that do not have a high degree of secondary structure, (ii) that have a high degree of secondary structure where the secondary structure does not inhibit mRNA stability and/or decrease translation, or (iii) that are derived from genes that are highly expressed in plants, will be most preferred.
  • sucrose synthase intron as, for example, described by Vasil et al. (1989, Plant Physiol. , 91:5175)
  • Adh intron I as, for example, described by Callis et al. (1987, Genes Develop., II
  • TMV omega element as, for example, described by Gallie et al. (1989, The Plant Cell, 1:301)
  • Other such regulatory elements useful in the practice of the invention are known to those of skill in the art.
  • targeting sequences may be employed to target a polypeptide product of the apoptosis-inhibiting polynucleotide or of the polynucleotide of interest or of the modulator polynucleotide to an intracellular compartment within plant cells or to the extracellular environment.
  • a DNA sequence encoding a transit or signal peptide sequence may be operably linked to a sequence encoding a polypeptide product as described above such that, when translated, the transit or signal peptide can transport the polypeptide product to a particular intracellular or extracellular destination, and can then be post-translationally removed.
  • Transit or signal peptides act by facilitating the transport of proteins through intracellular membranes, e.g., endoplasmic reticulum, vacuole, vesicle, plastid, mitochondrial and plasmalemma membranes.
  • the targeting sequence can direct a desired protein to a particular organelle such as a vacuole or a plastid ⁇ e.g., a chloroplast), rather than to the cytosol.
  • the nucleic acid construct can further comprise a plastid transit peptide encoding DNA sequence operably linked between a promoter region or promoter variant according to the invention and the foreign or endogenous DNA sequence.
  • a promoter region or promoter variant for example, reference may be made to Heijne et al. (1989, Eur.
  • Plasmid vectors include additional DNA sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic and eukaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors.
  • Additional DNA sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert DNA sequences or genes encoded in the nucleic acid construct, and sequences that enhance transformation of prokaryotic and eukaryotic cells.
  • the vectors may contains one or more element(s) that permit either stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome of the cell.
  • the vector may be integrated into the host cell genome when introduced into a host cell.
  • the vector may rely on a foreign or endogenous DNA sequence present therein or any other element of the vector for stable integration of the vector into the genome by homologous recombination.
  • the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location in the chromosome.
  • the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination.
  • the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell.
  • the integrational elements may be non-encoding or encoding nucleic acid sequences.
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in a host cell such as a bacterial cell.
  • bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUBl 10, pE194, pTA1060, and pAM ⁇ l permitting replication in Bacillus.
  • the origin of replication may be one having a mutation to make its function temperature-sensitive in a Bacillus cell (see, e.g., Ehrlich, 1978, Proc. Natl.
  • plant expression vectors include, for example, one or more cloned plant sequences under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker.
  • selectable marker will not be required, as the invention comprises a system which does not require selection of transformed plants with the polynucleotide or polypeptide of interest through the use of marker genes.
  • Such plant expression vectors can also contain a promoter regulatory region ⁇ e.g., a regulatory region controlling inducible or constitutive, environmentally, or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • a promoter regulatory region e.g., a regulatory region controlling inducible or constitutive, environmentally, or developmentally-regulated, or cell- or tissue-specific expression
  • a transcription initiation start site e.g., a regulatory region controlling inducible or constitutive, environmentally, or developmentally-regulated, or cell- or tissue-specific expression
  • a transcription initiation start site e.g., a promoterating inducible or constitutive, environmentally, or developmentally-regulated, or cell- or tissue-specific expression
  • RNA processing signal e.g., a transcription termination site, or cell- or tissue-specific expression
  • a transcription termination site e
  • Plant expression vectors optionally include RNA processing signals, e.g., introns, which may be positioned upstream or downstream of a polypeptide- encoding sequence in the transgene.
  • the expression vectors may also include additional regulatory sequences from the 3' non-translated region of plant genes, e.g., a 3' non-translated region to increase mRNA stability of the mRNA, such as the PI- II terminator region of potato or the octopine or nopaline synthase 3' non-translated regions.
  • Vectors suitable for the transfer of multiple genes are also well known in the art and include for example; Ye, et al , (2000, Science, 28:303-305).
  • typical vectors useful for expression of genes in higher plants include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacte ⁇ um tumefaciens described by Rogers et ah, (1987, Methods in Enzymology, 153:253-277).
  • a typical binary vector system may comprise an octopine-type vir helper strain such as LBA4404 that harbors the disarmed Ach5 Ti plasmid and a binary vector such as pBinl9 which is commonly used for plant transformation.
  • the available range of vir helper strains has been expanded with the nopaline-type MP90 and the L, L- succinamopine-type EHAlOl.
  • the bacterial kanamycin resistance gene in EHAlOl was deleted to develop the vir helper strain EHA105, EHAlOl and EHA105.
  • Popular A. tumefaciens vectors in the art include but are not restricted to plasmids pKYLX ⁇ and pKYLX7 of Schardl et ah, (1987, Gene, 61:1-11) and; Berger et ah, (1989, Proc. Natl. Acad. Sci. USA, 86:8402-8406).
  • the vector can be linearized prior to its introduction into plant cells for the purposes of genetically modifying cellular endogenous genomic sequences as linear vectors exhibit significantly higher targeting frequencies than those that are circular (Thomas et ah, 1986, Cell, 44:49). It is, however, possible to successfully utilize vectors for these purposes without linearization.
  • An exemplary high expression vector system that may be used to practice the methods of the present invention is the InPACT system (International Publication No. WO 01/72996, Dale et ah, 2001).
  • a chosen gene sequence e.g., the polynucleotide of interest or the anti-apoptosis polynucleotide or the modulator polynucleotide
  • REP viral-derived replicating protein
  • the system has two fundamental features: the gene to be expressed is split such that exon 1 is 3' of exon 2 and separated by the terminator sequence and the promoter sequence respectively, and the intergenic regions incorporating the stem/loop sequences and the interons are embedded in introns.
  • this "split gene construct" does not express the encoded gene.
  • the sequence is released by replicative release to become an episomal single stranded DNA molecule.
  • This ssDNA molecule is converted into a transcriptionally active double stranded DNA molecule by the host and the gene is transcribed.
  • the intergenic region which is embedded in the intron located between exons 1 and 2 is processed out and the gene product is expressed.
  • this strategy provides an expression platform where there is no expression in the absence of REP, but high expression levels in the presence of REP, as not only will the first copied ssDNA molecule by produced by replicative release, but this molecule itself can be further replicated by REP, providing an in vivo amplification system.
  • the polynucleotide of interest is introduced into plant cells using Agrobacterium-mediated transformation.
  • the apoptosis-inhibiting polynucleotide and the modulator polynucleotide/polypeptide may also be introduced into the plant cells using this method; however, this is not essential and other methods may be used.
  • Agrob ⁇ cterium species such as A. tumef ⁇ ciens and A. rhizogenes are capable of infecting a wide range of plant species, causing Crown Gall diseases.
  • the Agrob ⁇ cterium has natural transformation abilities which can be exploited in plant biotechnology techniques.
  • the introduction of new genes in the T- region by means of recombinant DNA techniques are often carried out in Escherichia coli.
  • the Ti plasmids normally cannot be maintained in E. coli, since it does not replicate in this host. Therefore, in the existing procedures well known in the art, a so-called shuttle vector is used which replicated in E. coli and A. tumefaciens and into which the T-region is introduced.
  • the complete Ti plasmid is necessary in order to transform cells via the method of Agrobacterium-mediated transformation, since the Ti plasmid contains the essential Vir-region on which genes are positioned for selection of T-DNA and transfer into a plant cell.
  • Agrobacterium-mediated transformation plant tissues are typically cut in small pieces, e.g., 10 x 10mm, and soaked for 10 minutes in a fluid containing suspended Agrob ⁇ cterium. Some cells along the cut tissue will be transformed by the bacterium that inserts its DNA into the nucleome of the cell. Placed on selectable rooting and shooting media, the plants will regrow from the transformed cells or tissues. Some plants species can be transformed just by dipping the unopened flowers into suspension of agrobacteria and then planting the seeds in a selective medium.
  • Vacuum infiltration allows the penetration of pathogenic bacteria into the inter cell spaces of plant tissues. Physically, the vacuum generates a negative atmospheric pressure that causes the air spaces between the cells in the plant tissue to decrease. The longer the duration and the lower the pressure of the vacuum, the less air space within the plant tissue. The increase in the pressure allows the infiltration medium, including the infective transformation vector to relocate into the plant tissue.
  • vacuum is applied to a plant part in the presence of Agrobacte ⁇ um for a certain time period. The length of time that a plant part or tissue is exposed to vacuum is critical as prolonged exposure causes hyperhydricity.
  • Vacuum infiltration-facilitated transformation can be performed in planta, in which the plant part to be transformed, e.g. flower, is not excised from the plant, thus eliminating in vitro regeneration of plants. It also offers other several advantages such as the generation of many independently transformed plants from a single plant, a reduction in somaclonal variation by avoiding tissue culture steps, the possibility of testing many constructs in a short time frame as the process is in itself fast, and is potentially useful for transformation of plants recalcitrant to plant tissue culture and regeneration.
  • the floral-dip method is well known in the art, Clough and Bent, (1998, Plant J, 16(6):735-743) and is a very efficient method for generating a number of transgenic plants, not limited to but including for example Arabidopsis thaliana plants. These methods allow plant transformation without the need for tissue culture. A large volume of bacterial culture grown in liquid media is necessary for this transformation method.
  • Floral dipping comprises the method of dipping floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 ⁇ L / L of surfactant Silwet L-77. Sucrose and surfactant are critical to the success of the floral dip method. Plants inoculated with numerous immature floral buds and few siliques present produce transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylaminopurine and pH adjustments are not necessary for transformation, and Agrobacterium can be applied to plants at a range of cell densities. The application step of Agrobacterium dipping may be repeated and may improve transformation rates and the overall yield of transformants by approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raises transformation rates twofold.
  • Agrobacterium transformation protocol involves a combination of wounding by particle bombardment, followed by use of Agrobacterium for DNA or protein delivery Bridney et al. (1992, Plant MoI. Biol, 18:301-313). Further methods include but are not restricted to the split meristem method, the regeneration of plants from leaf explants Horsch et al, (1985, Science, 227:1229-1231).
  • the apoptosis-inhibiting polynucleotide or modulator polynucleotide of the invention are introduced into plant tissues or plant cells by any number of routes, including microinjection, electroporation and particle bombardment acceleration methods, illustrative examples of which include microinjection (Crossway et al, 1986, Biotechniques 4:320-334), electroporation (Riggs et al, 1986, Proc. Natl. Acad ScL USA, 83:5602-5606), and ballistic particle acceleration (see, for example, Sanford et al, U.S. Pat. No. 4,945,050; Tomes et al, U.S. Pat. No.
  • recipient plant cells are employed that are susceptible to transformation and subsequent regeneration into stably transformed, fertile plants.
  • monocot transformation for example, immature embryos, meristematic tissue, gametic tissue, embryogenic suspension cultures or embryogenic callus tissue can be employed as a source of recipient cells which is useful in the practice of the invention.
  • organ and tissue cultures can be employed as a source of recipient cells.
  • tissues, e.g., leaves, seed and roots, of dicots can provide a source of recipient cells useful in the practice of the invention.
  • Cultured susceptible recipient cells are suitably grown on solid supports. Nutrients are provided to the cultures in the form of media and the environmental conditions for the cultures are controlled.
  • both dicotyledonous and monocotyledonous plants that are amenable to transformation can be modified by introducing of the anti-apoptosis and/or modulator constructs of the invention into a recipient plant cell and growing a new plant that harbors the anti-apoptosis and/or modulator constructs.
  • Illustrative transformation methods include electroporation and microprojectile bombardment, to name but a few. Transformation techniques that fall within these and other classes are well known to workers in the art, and the particular choice of a transformation technology will be determined by its efficiency to transform the selected plant species.
  • the anti-apoptosis and/or modulator constructs may be introduced into the plant cells prior to, simultaneous with or after agroinfection-mediated introduction of the polynucleotide of interest.
  • the anti-apoptosis and/or modulator constructs are introduced into plant cells prior to the introduction of the polynucleotide of interest.
  • embryo formation can then be induced from the protoplast suspension, to the stage of ripening and germination as natural embryos.
  • the culture media will generally contain various amino acids and hormones, necessary for growth and regeneration. Examples of hormones utilized include auxins and cytokinins. It is sometimes advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these variables are controlled, regeneration is reproducible. Regeneration also occurs from plant callus, explants, organs or parts. Transformation can be performed in the context of organ or plant part regeneration as, for example, described in Methods in Enzymology, Vol.
  • the mature transgenic plants are propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use.
  • Genetically modified plants derived from plant cells genetically modified through utilization of the expression system of the invention include, but are not limited to, a transgenic TO or RO plant, i.e., the first plant regenerated from transformed plant cells, a genetically modified Tl or Rl plant, i.e., the first generation progeny plant, and progeny plants of further generations derived there from which the first, second and optionally third constructs of the invention in their nucleomes.
  • Parts obtained from the regenerated plant are included in the invention, provided that these parts comprise cells that have been transformed as described. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the first, second and optionally third constructs of the invention in their cellular nucleome sequences.
  • the literature describes numerous techniques for regenerating specific plant species and more are continually becoming known. Those of ordinary skill in the art can refer to the literature for details and select suitable techniques without undue experimentation. Further more detailed descriptions of the Agrobacterium-mediated transformation techniques for use with this invention are detailed below.
  • the present invention may be used for the transformation of any plant species, monocotyledonous and dicotyledonous, including but not limited to corn (Zeamays), Brassica sp. (e.g., B. napus, B. rapa and B.
  • juncea particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecerea/e), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), safflower(Carthamustinctorius), millet (Pennisetumg/aucum, Panicummiliaceum, Eleusinecoracana, Setariaitalica), wheat (Triticumaestivum), soybean (Glycine max), tobacco (Nicotianatabacum), potato (Solanumtuberosum), peanuts (rachis hypogaea), cotton (Gossypiumhirsutum), sweet potato (Ipomoea battus), cassava (Manihotesculenta), coffee (Cofea spp.), coconut (Cocosnucifera), pineapple (Ananascomosus), citrus trees (Citrus spp.), cocoa
  • Vegetables include tomatoes (Lycopersiconesculentum), lettuce (e. g., Lactuca sativa), green beans (Phaseolusvulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genusCucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C.me/o).
  • Ornamentals include azalea (Rhododendron spp.), hydrangea
  • Conifers that may be employed in practicing the present invention include, for example, pines such asloblolly pine (Pinustaeda), slash pine (Pinuselliotil), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinuscontorta), and Monterey pine (Pinusradiata);Douglas- fir (Pseudotsugamenziesil); Western hemlock (Tsugacanadensis); Sitka spruce (Piceaglauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abiesamabilis) and balsam fir (Abiesbalsamea); and cedars such as Western red cedar (Thujaplicata) and Alaska yellow-cedar (Chamaecyparisnootkatensis).
  • pines such asloblolly pine (Pinustaeda), slash pine (Pin
  • Plants of the present invention include crop plants (for example, corn, alfalfa, sunflower, safflower, canola, soybean, casava, cotton, peanut, sorghum, rice, wheat, millet, tobacco, rye, oats, barley, turf grass, etc.).
  • plants of the present invention include corn, soybean, canola, rice, sunflower, wheat and sorghum plants, and in another corn and soybean plants.
  • Plant cells that have been transformed may be grown into plants in accordance with conventional ways, McCornicket al, (1986, Plant Cell Reports, 5:81- 84). Transformed plants may be grown or pollinated with the same transformed strain or different strains and the resulting hybrid having expression of the isolated polynucleotide of interest. Two or more generations may be grown to ensure that the polynucleotide of interest has been inherited if so desired. 8. Transgene analysis
  • a variety of assays may be performed. Such assays include but are not restricted to, for example, "molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting techniques and Polymerase Chain Reaction (PCR) and related DNA amplification techniques.
  • a protein expressed by the heterologous DNA may be analyzed by high performance liquid chromatography or ELISA (e.g., nptll) as is well known in the art.
  • Competent Cells Before Agrobacterium can be transformed with a vector which contains a construct of choice, the cells must be made to be competent.
  • Such methods of preparing competent cells are well known in the art and for example include such methods as follows:
  • the culture is then incubated at 28° C with vigorous agitation. If the culture is started in the late afternoon, it can be harvested the following morning. When the cells have reached log phase (ODsso 0.5-0.8), the culture can be chilled by gently swirling in an ice-water bath. After this stage, the cells must be kept at 4° C for all further steps. The next stage of the procedure involved pelleting the cells by centrifuging at 400Og for 10 minutes in a pre-chilled 4° C rotor. The supernatant is discarded and 5-10 mL of ice-cold H 2 O is added as a wash step (a wide-bore pipette to pipette the cells gently up and down can be used).
  • the final volume must be adjusted to 500 mL with ice-cold H 2 O.
  • the last two steps are repeated twice, for example; i. After the first repeat, the cells are resuspended in a final volume of 250 mL of ice-cold H 2 O; ii. After the second repeat, the cells are resuspended in a final volume of 50 mL of ice-cold H 2 O. Finally, the cells are pelleted as described above, and resuspended in 5 mL oflO% (v/v) ice-cold, sterile glycerol. Competent Agrobacterium cells can then be dispensed in50 ⁇ L aliquots of cells into microcentrifuge tubes and snap-frozen in liquid nitrogen, for storage at-70° C.
  • Competent Agrobacterium cells as described above can be transformed with a polynucleotide of interest using a number of techniques known in the art, non-limiting examples of which include the freeze thaw method and electroporation of the cells.
  • the method of electroporation of cells is provided in further detail below: [00103] Competent cells stored at -70° C can be defrosted on ice at a volume of approximately (50 ⁇ L per transformation). After the cells are defrosted, plasmid DNA (1 ⁇ l of E. coli miniprep or 1-5 ⁇ gof CsCl-purified plasmid DNA) may be added to the cells. Both the cells and the DNA can be mixed together on ice.
  • Electroporation of the cells can be carried out as recommended for E. coli by the manufacturer of the chosen electroporator. For example, when using an electroporator with a 2 mm cuvette, the following conditions can be used: Capacitance: 25 ⁇ F; Voltage: 2.4 kV; Resistance: 200 ⁇ and Pulse length: 5 msec. Immediately after electroporation, 1 mL of LB is added to the cuvette, and the bacterial suspension can be transferred to a 15-mL culture tube, before incubating for 4 hours at 28° C with gentle agitation.
  • the suspended cell can be collect by centrifuging briefly, and spread on LB agar plates containing the appropriate antibiotic or selection. The plates are then incubated for approximately 3-4 days at 28° C, until colonies begin to form, but before satellite colonies are produced. Any colonies that have grown during this time can be streaked on to a new LB agar plate and re-incubated at 28° C until the colonies have grown. The plate can then be kept sealed at 4° C as a stock plate. The colonies that grew should contain the polynucleotide of interest. To verify whether this is the case, small cultures of each independent colony can be grown and mini-preps and PCR undertaken to check for the insertion of the desired sequence. Finally, glycerol stocks of the appropriate clones can be stored at-20° C. Variations of the freeze thaw method are provided in the art. The invention is not restricted to the methods described above.
  • E. coli the polynucleotide can be transferred into Agrobacterium by the freeze-thaw method. Although the transformation frequency by these methods is low (approximately 10 3 transformants per ⁇ g DNA) compared to other methods, the technique is reliable and very rapid. Steps in the procedure are well known in the art and may include: [00105] Primarily, an Agrobacterium strain containing an appropriate helper
  • Ti plasmid in 5 mL of medium is grown overnight at 28° C.
  • the next step involves the addition of 2 mL of the overnight culture to 50 mL of medium in a 250-mL flask.
  • the culture is then shaken vigorously (250 rpm) at 28°C until the culture grows to an OD 600 of 0.5 to 1.0.
  • the cells are chilled on ice. Keeping the cultures chilled, the cell suspension is centrifuged at 3000 g for 5 min at 4°C. The supernatant of this solution is discarded and the cells resuspended in 1 mL of 20 mM CaCl 2 solution (ice-cold). Aliquots of the cells are placed into pre-chilled
  • the second stage of this method comprises adding about 1 ⁇ g of plasmid DNA to the cells before snap freezing them in liquid nitrogen.
  • the cells are further thawed by incubating the test tubes in a 37° C water bath for 5 min.
  • 1 mL of fresh LB medium is added to the tube and incubated at 28° C for 2-4 h with gentle shaking. This period allows the bacteria to express the antibiotic resistance genes if there is a selection step in place.
  • the cells are further centrifuged in their tubes for 30 s in an Eppendorf centrifuge and the supernatant solution is removed. The pelleted cells are resuspended in 0.1 mL LB medium.
  • the cells can be spread on a LB agar plate containing appropriate antibiotic selection, before incubation at 28° C. Transformed colonies containing the polynucleotide of choice should appear in approximately 2-3 days. Variations of the freeze thaw method are provided in the art and the invention is not restricted to the method described above.
  • Sucrose starvation (Riou-Khamlichi et al. , 2000) was used for synchronization of suspension cells. Cells were washed three times with suspension maintenance media lacking sucrose and then resuspended in 100 mL of the same. Flasks were put back on the shaker at 27° C, 90rpm for 24 hours before sucrose was added to the medium to a final concentration of 3%. The cultures were incubated under conditions as above and 4 day old embryogenic cell suspensions (ECS) were used for transformation. To reduce variations between different flasks, ECS were pooled before each experiment and re-aliquoted out into 2 mL tubes and used as starting material for experiments.
  • ECS embryogenic cell suspensions
  • Embryogenic cell suspensions are the most regenerable banana tissue and are frequently used for banana transformation.
  • Agrobacterium-medieAed transformation protocol using two cultivars, Grand Nain (AAA) and Lady Finger (AAB) and A. tumefaciens strains AGLl and LBA4404. They inventors also observed Agrobacterium- induced cell death in banana ECS and discovered that heat-shock could control it but only to a very limited extent. Effect of A. tumefaciens on embryogenic cell suspensions (ECS) was then analyzed in greater detail, with the aim of further improving their transformation efficiency. EXAMPLE 4
  • Binary vectors pPTN254, pPTN255, pPTN261, pPTN396 and pPTN290, used for transformation had the maize polyubiquitin-1 (Ubi-1) promoter controlling the constitutive expression of Bcl-xL (chicken), Bcl-xL G138A (one loss-of- function substitution at codon 138 of Bcl-xL protein), Ced-9 (from C. elegans), Bcl-2 3' UTR (human) and UidA reporter gene encoding ⁇ -glucuronidase (GUS), respectively.
  • Ubi-1 maize polyubiquitin-1
  • the vectors were electroporated into Agrobacterium tumefaciens strain AGLl, Lazo et al, (1991, Bio/Technol, 9:963-967) and selected on yeast-mannitol (YM) medium, Vincent, (1970, A Manual for the practical study of root nodule bacteria. IBP Handbook 15, Blackwell Scientific Publications: London) supplemented with carbenicillin (250 mg/liter), spectinomycin (100 mgL '1 ), and rifampicin (50 mgL '1 ).
  • Agrobacterium culture and banana ECS transformation were carried out as previously described Khanna et al, (2004, supra). ECS were not subjected to heat shock prior to transformation, except in one specified experiment.
  • bound trypan blue was extracted from 50 mg scv of stained ECS in a 500 ⁇ L solution of 50% methanol/ 1% SDS for 1 h at 50° C and quantified using a spectrophotometer by measuring the absorbance at 595 run.
  • TUNEL TdT-mediated dUTP nick-end labeling
  • TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling
  • the inventors applied TUNEL staining and propidium iodide counterstaining to banana ECS and root cells infected with Agrobacterium. Control cells incubated on co-culture media without Agrobacterium infection contained intact nucleus, as visualized by non-specific DNA staining with propidium iodide ( Figures 2a and 8a) and specific labeling of fragmented DNA was rare ( Figure 2b and 8b).
  • TUNEL assays showed a high percentage of positive staining after 48 hours of Agrobacterium co-culture but trypan blue staining and visible browning of cells was not visible until after 4 days indicating that nuclear DNA fragmentation preceded the loss of membrane integrity, suggesting that the observed DNA fragmentation was caused by apoptotic rather than necrotic cell death.
  • Transformed cells were subjected to TUNEL assay on third day of co- culture.
  • a high percentage of TUNEL-positive cells were detected in ECS transformed withpPTN255 (Figure 2h) or pPTN290 ( Figure 2j) indicating that Bcl-xL G138A or vector only control induced cell death comparable to that caused by AGLl alone ( Figure 2d), confirming that transformation process itself had not contributed significantly to the cell death at that stage.
  • the number of cells displaying TUNEL- positive staining dropped significantly in cells transformed with pPTN254 ( Figure 2f), pPTN261 and pPTN396.
  • apoptosis Another typical feature of apoptosis is the cleavage of DNA at specific chromosomal sites by DNA endonucleases, Eastman et al, (1994, Endonucleases associated with apoptosis; In: Apoptosis (Mihich, e and Schimke, R. T. eds) 249-259).
  • the in situ TUNEL assay does not discriminate between random fragmentation and oligonucleosome-sized cleavage of DNA.
  • PCD programmed cell death
  • ECS were selected on kanamycin and regenerated as previously described (Khanna et al. , 2004).
  • Grand Nain ECS transformed with pPTN254 also gave rise to an average of 4630 embryos from four different experiments whereas cells transformed with pPTN255 formed less 100 embryos, as did the control pPTN290 transformed cells. Similarly, approximately 3150 and 820 embryos were formed from ECS transformedCed-9 and Bcl-2 3'UTR respectively. Comparable results were obtained with Lady finger ECS (Table 2). These results suggest that all three anti-apoptosis genes had a dramatic effect on cell death in transformed cells, leading to recovery of almost all transformed cells. Since the Agrobacterium-mediated transformation protocol used was very efficient, T-DNA transfer efficiency was very high.
  • Genomic DNA was isolated from plant tissues using CTAB (cetyltrimethylammonium bromide) buffer supplemented with 1.4% (w/v) 2- mercaptoethanol and standard chloroform extractions.
  • Gene specific primers was used to confirm presence of transgenes and non-T-DNA region specific primers was used to confirm absence of vector DNA backbone or Agrobacterium contamination in the intracellular spaces of plant tissues.
  • PCR reaction contained 8 picomoles of each primer, 200 ⁇ M dNTPs, 50ng genomic DNA and 0.25units of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) with 1 x buffer (2.5 mM MgCl 2 ).
  • the primer sequences used were Bcl-xFl (5'atgagtcagagcaaccgggagctg3') and Bcl-xRl (5'tcatttccgac tgaagagtgagccc3') for Bcl-xL and Bcl-xL (Gl 38A); Ced-9F2 (S'gatggcgactggcgagatgaagS 1 ) and Ced-9R2 (5'gaaaccgccgaacgagattagacc3') for Ced-9; Bcl-F2 (5'acatgcctgccccc aaacaaata3') and Bcl-R2 (5'ggtgatccggccaacaacat3') for Bcl-2 3'UTR and Spec-Fl(5'agtgatattgatttgctggttac3') and SpecRl (5'atgacgggc
  • DNA was isolated from infected roots at various intervals and tested for DNA laddering. For in situ detection of apoptosis using TUNEL, root tips were fixed and assayed as described above.
  • Transgenics and un-transformed control plantlets were rooted in MS4, Khanna et al. , (2004, supra) liquid media and young actively growing roots from plants transformed with anti-apoptotic genes to Agrobacterium were used to determine the cytoprotective effect of these transgenes on the stably transformed root cells.
  • Roots were tested for Agrobacterium-induced cell death by exposing them to AGLl at using a high density inoculum (OD 600 of 1.0). Absence of TUNEL positive nuclei in Bcl-xL transformed root cells (Figure 8f), as compared to untransformed controls ( Figure 8d), Bcl-xL (Gl 38A) transformed plants ( Figure 8h) and pPTN290 transformed plants ( Figure 8j) further confirmed that expression of anti- apoptotic transgenes had a cytoprotective effect on the banana cell that protected the cells from Agrobacterium-induced programmed cell death.
  • the exogenous nucleic acid used in all microprojectile based transformation experiments was either a reporter gene encoding Gfp driven by UBIl promoter or one of the negative regulators of apoptosis Bcl-xL and Bcl-xL G138A (a loss of function mutant) of animal origin (Dickman et ⁇ l., 2001, Proc. N ⁇ tl. Ac ⁇ d. Sci USA, 98: 6957-6962) AtB ⁇ g4 (Doukhanina et ⁇ l., 2006, J Biol.
  • Hsp70h (S) (severe) or Hsp70h (M) (mild) of plant virus origin driven by (CaMV) 35 S promoter For selection of transformed sugarcane cells, all constructs carried the nptll gene (which confers resistance to geneticin) marker gene driven by the 35S promoter and terminated by the (CaMV) 35S poly A region. For bacterial selection all constructs carried a spectinomycin resistance gene.
  • a Hmdlll-SOcI fragment (2817 bp) carrying maize ubiquitin promoter, Gfp coding region and nos terminator digested out from vector pA53 was electrophoresed and purified from agarose gel and cloned into HzrcdIII and S ⁇ cl digested T-DNA region of superbinary vector p ⁇ K(S), that carried an extra virulence gene (virG) in the backbone for enhancing monocot transformation.
  • HmdIII and Spel digested 3145 bp fragment containing ubi promoter, nptll coding region and nos terminator was also cloned into Hwdlll and Spel digested T-DNA region of the superbinary vector as a plant selection marker.
  • the final superbinary vector pUGFPnptll was electroporated into A. tumef ⁇ ciens strain LBA4404 and used for sugarcane transformation.
  • MSD-3 media (Table 3) supplemented with anti-oxidants citric acid and ascorbic acid (150 mg L "1 each) was used.
  • Explants were cultured in tissue culture dishes (90 x 25 mm) with 25 mL agar-solidified medium.
  • explants were inoculated in 250 mL flasks containing 50 mL liquid MSD-3 medium. Liquid cultures were agitated continuously on a shaker at 120 rpm. All cultures were incubated at 25- 28° C in dark, except for the suspension cultures that were maintained under 16 hrs photoperiod provided by cool, white fluorescent tubes. Sub-culturing was done at least once in ten days, or more frequently if media or explants turned brown due to phenolic exudation.
  • A. tumefaciens strains LBA4404 and AGLl were grown in 5 mL of liquid LB (Luria-Bertani broth) medium at 200 rpm agitation with appropriate antibiotics at 28° C for 24 hrs.
  • a 0.5 mL culture was added to 50 mL of liquid YMB medium (Table 3) and grown until the culture reached an OD600 of 1.0.
  • the culture was centrifuged at 5000 x g for 10 min at room temperature. The pellet was resuspended in AIM medium (Table 3), and the bacterial suspension was adjusted to optical density of 0.7 measured at 600 nm wavelength (OD600 0.7) for inoculation of sugarcane cultures.
  • Cells were inoculated with Agrobacterium for 10 min. Treated cells were co-cultivated on co-culture media SCM-I medium (Table 3) for 2 days in the dark at 25° C. Cells were washed after co-cultivation with sterile distilled water and visually assessed for browning at periodic intervals.
  • Liquid cell suspension cultures from Experiment 2 described above but exposed to A. tumefaciens LBA4404 and AGLl at two different inoculum densities (OD660 0.1 and 1.0), as well as the unexposed control cells were fixed in 4% (v/v) formaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.2) 48 hrs after the exposure to Agrobacterium. They were permeabilized using 0.1% sodium citrate containing 1% Triton XlOO.
  • DNA was labelled by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) using an in situ Cell Death Detection kit (ROCHE DIAGNOSTICS, Basel, Switzerland), according to the manufacturer's instructions.
  • Samples were counterstained with 0.5 ⁇ g ml/ 1 propidium iodide and confocal images were obtained using Leica 63 x 1.4NA oil PL APO objective lenses under a Leica TCS 4D confocal laser scanning microscope (LEICA, Heidelberg, Germany), equipped with Argon/Krypton and UV lasers (ex. splitter DD488/586) and Leica TCS-4D software.
  • Plasmid DNA was prepared using a Qiagen Plasmid MaxiPrep Kit according to the manufacturer's instructions. Approximately 4 hrs prior to microprojectile bombardment, sugarcane explants (4, 14, 24 and 34 days after culture initiation) were placed in a circle (about 3 cm in diameter) on solid MSD-3 media containing osmoticum (0.2 M mannitol). Explants were bombarded using a particle inflow gun (Finer et ⁇ l, 1992, Plant Cell Reports, 11: 323-328).
  • Gold particles (Bio- Rad), 1.0 ⁇ m in diameter, were used as microprojectiles, and were prepared for bombardment by mixing 3 ⁇ g of gold particles with 2 ⁇ g of plasmid, 25 ⁇ L of 2.5 M CaCl 2 and 5 ⁇ L of 0.1 M spermidine-free base. All solutions were kept on ice. The gold was kept in suspension for 5 min with occasional vortexing, and then allowed to precipitate for 10 min on ice; 22 ⁇ L of supernatant was subsequently removed. The remaining suspension was vortexed, and 5 ⁇ L aliquots were used for each bombardment.
  • Target tissue was placed 7.5 cm from the point of particle discharge and covered by a 210 mm stainless steel mesh baffle. Helium pressure used for particle launch was 1500 KPa and chamber vacuum was at -84 KPa.
  • Agrobacterium strain LBA4404 carrying superbinary vector pUGfpnptll(S) was grown at 28° C in YMB media, supplemented with Rifampicin 20 mg L '1 , Streptinomycin 200 mg L '1 and Spectinomycin 100 mg L '1 . After three days, the cells were subcultured and grown overnight at 28° C in YMB media. Cells were centrifuged down at 5000 x g for 10 min and resuspended in AIM (Table 3) medium containing 100 ⁇ M acetosyringone to an OD 66O of 0.7.
  • AIM Table 3
  • the bacterial suspension was left at 25° C in the dark, shaking for 4 hrs before using it for co-cultivation.
  • Explants were placed in 5 mL of induced Agrobacterium suspension (with 0.02% Pluronic F-68) and placed in the vacuum (25 mm Hg) for 5 min, to allow the bacteria to infiltrate the explant.
  • Untransformed control was vacuum infiltrated with AIM media without Agrobacterium (but containing acetosyringone and Pluronic F-68).
  • Excess bacteria were then removed and explant dried on a sterile filter paper for 10 s. Control experiments consisted of untransformed explants, microprojectile transformed only and Agrobacterium transformed only explants.

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Abstract

La présente invention porte sur des procédés et des constructions perfectionnés pour transformer de façon stable ou transitoire des cellules végétales par transformation à médiation par Agrobacterium, dans des conditions qui inhibent l'apoptose, sans effectuer une sélection en fonction de la présence d'un gène marqueur.
PCT/AU2008/000478 2007-04-03 2008-04-03 Procédés et constructions perfectionnés pour transformation à médiation par agrobacterium sans marqueur WO2008119136A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2274973A1 (fr) * 2008-03-31 2011-01-19 Japan Tobacco, Inc. Procede au moyen d'agrobacterium de production d'une plante transformee
US20140283202A1 (en) * 2013-03-15 2014-09-18 The Texas A&M University System Compositions, organisms, systems, and methods for altering cold, drought, and salt tolerance in plants
EP2856862A4 (fr) * 2012-05-31 2015-12-30 Kaneka Corp Procédé de transformation de plantes utilisant une hormone inhibant la croissance des plantes
CN110004174A (zh) * 2019-04-10 2019-07-12 东北林业大学 长白落叶松瞬时转化实生苗体系构建方法

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CN110004174A (zh) * 2019-04-10 2019-07-12 东北林业大学 长白落叶松瞬时转化实生苗体系构建方法

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