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WO2009150435A1 - Vecteurs de transformation du plaste permettant l’excision de gènes marqueurs - Google Patents

Vecteurs de transformation du plaste permettant l’excision de gènes marqueurs Download PDF

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Publication number
WO2009150435A1
WO2009150435A1 PCT/GB2009/001482 GB2009001482W WO2009150435A1 WO 2009150435 A1 WO2009150435 A1 WO 2009150435A1 GB 2009001482 W GB2009001482 W GB 2009001482W WO 2009150435 A1 WO2009150435 A1 WO 2009150435A1
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Prior art keywords
plant
promoter
recombinase
acyl
fatty acid
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PCT/GB2009/001482
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English (en)
Inventor
Simon Geir MØLLER
Chua Nam-Hai
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University Of Stavanger
Webber, Philip, Michael
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Application filed by University Of Stavanger, Webber, Philip, Michael filed Critical University Of Stavanger
Publication of WO2009150435A1 publication Critical patent/WO2009150435A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8214—Plastid transformation

Definitions

  • the invention relates to a method for producing a transformed plant cell. More particularly, the method involves the transformation of a plant cell with a Transformation Cassette which is targeted to plant plastids and/or mitochondria and which comprises a selection gene, for example isopentenyl transferase (IPT), and one or more transgenes encoding polypeptides involved in fatty acid biosynthesis, modification or degradation.
  • a selection gene for example isopentenyl transferase (IPT)
  • IPT isopentenyl transferase
  • transgenes encoding polypeptides involved in fatty acid biosynthesis, modification or degradation.
  • expression of a recombinase is induced in the plant cell, which leads to the excision of the selection gene from the plastid and/or mitochondria and the expression of the transgene in the plastid and/or mitochondria.
  • the invention also provides cells and plants comprising the Transformation Cassette.
  • GM genetically modified
  • Plastids are organelles unique to plants. Each plant cell contains approximately 100 plastids each of which contains approximately 100 genomes. This in effect means that when a gene is inserted into the plastid genome each plant cell contains approximately 10,000 copies of that gene compared to plant nuclear transformation where each plant cell would have at best 2-3 copies of the gene.
  • transgenes in plastids has numerous advantages over nuclear gene expression: (i) transgenes are not spread by pollen; (ii) proteins are expressed to high levels (up to 47% of total cellular protein); (iii) "toxic" effects of proteins are reduced due to plastid containment; (iv) the simultaneous expression of several genes allows for biochemical pathway engineering; and (V) gene silencing is eliminated. Plastid genetic engineering has clear fundamental and applied applications in that potentially any protein can be produced to high levels with little environmental risk opening up the possibility of producing edible vaccines, biopharmaceuticals and products of agronomical value.
  • the ability to transform mitochondria in plants has several uses:
  • Mitochondrial transformation has clear advantages over nuclear transformation in that high levels of proteins can most probably be produced and that the transformed mitochondria are maternally inherited (i.e. from the mother) providing genetic control. Mitochondrial transformation will also be useful for assaying mitochondrial functions in terms of agricultural traits.
  • the invention is based on a selection and regeneration system for plastid and/or mitochondria transformation based on the over-expression of a gene such as the isopentenyl transferase (IPT) gene (cytokinin biosynthesis) in plastids and/or mitochondria.
  • IPT isopentenyl transferase
  • This system allows for the direct selection of cells containing transformed plastids and/or mitochondria on media lacking cytokinin due to cytokinin production within plastids and/or mitochondria.
  • the system therefore provides an antibiotics-free selection and regeneration system which, in the case of plastids, will overcome problems with spontaneous spectinomycin mutants and concerns regarding the use of antibiotic resistance genes in GMOs.
  • IPT has previously been used as a selectable marker in plant nuclear transformation (e.g. EP 1 069 855 A), its use in plastid and/or mitochondria transformation has not previously been suggested.
  • plastids and mitochondria are semi-autonomous organelles within plant cells with their own genomes and metabolism.
  • IPT precursors are made in plastids or mitochondria or that any IPT produced is capable of diffusing out of the plastid and/or mitochondria into the cell cytoplasm in order to initiate cytokinin signalling to stimulate shoot regeneration.
  • the criteria for choosing plas ⁇ d-selec ⁇ on and mitochondria-selection genes are distinct from those for choosing genome- selection genes.
  • the plant- hormone biosynthetic gene is preferably removed after selection and initial regeneration has occurred.
  • the transgene in question (expressing the polypeptide of interest) might only be activated after the removal of the plant- hormone biosynthetic gene 3 so that any adverse effects due to transgene expression during selection and regeneration are also eliminated.
  • the invention provides a method for producing a transformed plant cell, the method comprising the step: (i) transforming the plant cell with a genetic construct, wherein the genetic construct comprises first and second homologous recombination elements flanking a Transformation Cassette, wherein the first and second homologous recombination elements are capable of directing the integration of the Transformation Cassette into the genome of at least one plastid and/or mitochondria which is present in the plant cell, wherein the Transformation Cassette comprises:
  • Excision Cassette comprises: (bl) a first site-specific recombination element
  • nucleotide sequences encoding one or more plant- hormone biosynthetic polypeptides and optionally a nucleotide sequence encoding a polypeptide which confers resistance to an antibiotic
  • the method additionally comprises the step:
  • the method additionally comprises the step:
  • the method of the invention is suitable for all plants that can be transformed and regenerated.
  • the plant may be a monocot or dicot.
  • suitable plants are cereals (rice, wheat, barley, oats, sorghum, corn), legumes (alfalfa, lentils, peanut, pea, soybean), oil crops (palm, sunflower, coconut, canola, olive), cash crops (cotton, sugar cane, cassava), vegetable crops (potato, tomato, carrot, sweet potato, sugar-beet, squash, cucumber, lettuce, broccoli, cauliflower, snap bean, cabbage, celery, onion, garlic), fruits/trees and nuts (banana, grape cantaloupe, muskmelon, watermelon, strawberry, orange, apple, mango, avocado, peach, grapefruit, pineapple, maple, almond), beverages (coffee, tea, cocoa), and timber trees (oak, black walnut, sycamore).
  • Other suitable plants include mosses and duckweed.
  • the plant is tobacco or lettuce.
  • the plant cells which are being transformed may be used in any convenient form, for example, as individual cells, groups of cells, in dissociated form or undissociated form, or as part of a plant tissue or plant part.
  • the cells are present in leaves that are removed from intact plants. It is preferable to use actively-growing leaves.
  • Plastid is intended to cover all organelles which are found in the cytoplasm of eukaryotic plants, which contain DNA, which are bounded by a double membrane, and develop from a common type, i.e. a proplastid. Plastids may contain pigments and/or storage materials.
  • plastids examples include chloroplasts, leucoplasts, amyloplasts, etioplasts, chrornoplasts, eJaioplasts and gerontoplasts.
  • the plastid is a green plastid, most preferably a chloroplast.
  • the term "genetic construct” refers to a nucleic acid molecule comprising the specified elements and Cassettes.
  • the genetic construct may, for example, be in the form of a vector or a plasmid. It may also contain other elements which enable its handling and reproduction, such as an origin of replication, selection elements, and multiple cloning sites.
  • the genetic construct will be a double-stranded nucleic acid molecule, preferably a dsDNA molecule.
  • the first and second homologous recombination elements are ones that are capable of directing the integration of the Transformation Cassette into the genome of at least one plastid and/or mitochondria which is present in the plant cell.
  • the first and second homologous recombination elements recombine with corresponding sequences in the genome of the selected plastid or plastids and/or mitochondria, resulting in the insertion of the Transformation Cassette into the genome of the selected plastid or plastids and/or mitochondria.
  • the nucleotide sequences of the homologous recombination elements are selected such that the Transformation Cassette is specifically targeted to one or more selected pJastids and/or mitchondria.
  • the nucleotide sequences of the homologous recombination elements are selected such that no or essentially no Transformation Cassettes become integrated into the nuclear genome of the plant.
  • the nucleotide sequences of the homologous recombination elements are preferably plastid- specific or mitochondria-specific, i.e. corresponding sequences are not present in the nuclear genome and preferably not present in the mitochondrial and/or plastid genome, respectively, of the plant in question. This may be done by avoiding sequences which are present in the nuclear genome of the plant.
  • the Transformation Cassette is specifically targeted to one or more plastids.
  • Transformation Cassette is specifically targeted to mitochondria.
  • the skilled person will readily be able to detect whether a specific sequence is or is not present in the nuclear genome by standard means, for example, by Southern Blotting of the nuclear genome with a labelled sequence probe or by sequence analysis.
  • any sequences can be used from the plastid and/or mitochondrial genome as long as the selected insertion site is not lethal to the cell, i.e. it does not result in the death of the cell.
  • the insertion sites are not in coding regions of plastid and/or mitochondrial genes.
  • the orientation of the sequences of the first and second homologous recombination elements should be the same as the orientation in the plastid and/or mitochondrial genome to allow for efficient homologous recombination.
  • the nucleotide sequences of the first and second homologous recombination elements must be identical or substantially identical to sequences in the genome of the selected plant plastid and/or mitochondria.
  • nucleotide sequences of the first and second homologous recombination elements should preferably not be identical or substantially identical to sequences in the nuclear genome of the selected plant.
  • first and second homologous recombination sequences will independently be 50-2500, 50-2000, 50-1500 or 50-1000 nucleotides each, more preferably about 150, about 1000 or about 1200 nucleotides in length.
  • the distance between the first and second homologous recombination sequences in the plastid and/or mitochondrial genome may be 0-4000 nucleotides or more. Preferably, the distance is about 1-100, 100-500, 500- 1000 or 1000-3000 nucleotides.
  • the total length of the genetic elements which are present between the first and second homologous recombination is preferably less than 4000 nucleotides.
  • the first plastid homologous recombination sequence is nucleotides 104091-105380 of the Nicotiana tabacum (accession no. Z00044) chloroplast genome DNA; and/or preferably, the second plastid homologous recombination sequence is nucleotides 105381-106370 of the Nicotiana tabacum (accession no. Z00044) chloroplast genome DNA.
  • the first pJastid homologous recombination sequence is preferably nucleotides 102925-101857 of the Nicotiana tabacum (accession no. Z00044) choloroplast genome DNA; and/or the second plastid homologous recombination sequence is nucleotides 100933-100130 of the Nicotiana tabacum (accession no. Z00044) choloroplast genome DNA.
  • the first mitochondrial homologous recombination sequence is nucleotides 72001-72501 of the Nicotiana tabacum (GenBank accession no. BA000042) mitochondrial genome DNA; and/or preferably, the second mitochondrial homologous recombination sequence is nucleotides 72502-73000 of the Nicotiana tabacum (GenBank accession no. BA000042) mitochondria] genome DNA.
  • the Transformation Cassette promoter (a) must be one that is operable in the selected plant plastid and/or mitochondria.
  • the promoter is one which is capable of initiating transcription of the transgene once the
  • Excision Cassette has been excised; and of initiating the transcription of the nucleotide sequence encoding a plant-hormone biosynthetic polypeptide, in cases where the Excision Cassette does not contain its own promoter.
  • the promoter might, for example, be one derived from a plant or bacterial gene.
  • the promoter is plant specific.
  • Suitable promoters include PsbA, RbcL, 16S rRNA, T3, T7, ATPase and Prrn promoters.
  • the promoter is a Prrn promoter (e.g. Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • Prrn promoter e.g. Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • the promoter is an inducible promoter. This allows inducible, controlled expression of the selection gene(s).
  • the inducible promoter may be inducible by IPTG, e.g. the PrrnL promoter.
  • Other inducible promoters include those inducible by light, dark, ethanol, drought, metals, pathogens, growth regulators, heat, cold, galactose and other sugars.
  • the promoter is a high-expression level promoter.
  • the Excision Cassette comprises a first site-specific recombination element; optionally, a second promoter; a nucleotide sequence encoding a plant-hormone biosynthetic polypeptide; a terminator sequence; and a second site-specific recombination element.
  • the first and second site-specific recombination elements are sequences of nucleotides which are capable of being recognised and/or bound by die site-specific recombinase which is produced by a recombinase.
  • the site-specific recombination elements must flank the genetic elements in the Excision Cassette, e.g. elements (b2) (if used), (b3), (b4), any other desired elements.
  • the sequences of the first and second recombination elements will be identical or substantially identical to each other; and will be in the same orientation relative to each other (e.g. both 5'-3' or both 3'-5').
  • the site-specific recombination sequences are preferably lox sequences.
  • Site-specific lox recombination sites are 34 bp sequences; these act as binding sites for the Cre recombinase polypeptide. Wild-type lox sequences are preferred (Zuo J, Niu QW, M ⁇ ller
  • the Excision Cassette promoter when present, must be one that is operable in the selected plant plastid and/or mitochondria.
  • the promoter is one which is capable of initiating transcription of the plant-hormone biosynthetic gene.
  • the promoter might be one derived from a plant or bacterial gene.
  • the promoter is plant specific. Examples of suitable promoters include PsbA, RbcL and Prrn promoters.
  • the promoter is a Prrn promoter (e.g. PIastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • the plant-hormone biosynthetic polypeptides act as selection markers, allowing the selection of plant cells which have been transformed with the Transformation Cassette.
  • the plant-hormone biosynthetic polypeptides may be any polypeptides which are involved in the synthesis of a plant cytokinin or auxin or other plant growth regulator, or which regulate the production or metabolism of a plant cytokinin or auxin or other plant growth regulator.
  • nucleotide sequences encoding 1, 2, 3, or 4 plant- hormone biosynthetic polypeptides there are nucleotide sequences encoding 1, 2, 3, or 4 plant- hormone biosynthetic polypeptides.
  • the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides may be present in an operon, with a single optional promoter and terminator element.
  • the plant- hormone biosynthetic polypeptide nucleotide sequences may each have their own promoters and terminator elements.
  • two or more of the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides are present as fusion proteins, optionally with a short linker sequence joining the proteins (e.g. encoding a 1-10 amino acid linker sequence, e.g.
  • nucleotide sequences encoding the plant-hormone biosynthetic polypeptides may be present in an operon and/or as fusion proteins, and others have their own promoters and/or terminators.
  • the or a plant-hormone biosynthetic polypeptide is IPT (isopentenyl transferase) which is an enzyme involved in cytokinin biosynthesis.
  • IPT isopentenyl transferase
  • the IPT nucleotide sequence may be from any suitable source. Due to codon usage, bacterial IPT genes are preferred, because nuclear genes may not be expressed to maximum levels in chloroplasts.
  • the IPT nucleotide sequence is from Agrobacterium tumefaciens or from a plant (e.g. from the plant which is being transformed).
  • the or a plant-hormone biosynthetic polypeptide is iaaH (indoleacetamide hydrolase) and/or iaaM (tryptophan mono-oxygenase), which are enzymes involved in auxin biosynthesis.
  • the nucleotide sequences may be from any source. Due to codon usage, bacterial iaaH and/or iaaM genes are preferred.
  • the iaaH and/or iaaM nucleotide sequences are from Agrobacterium tumefaciens.
  • the transformed plants may be selected on media lacking auxins.
  • auxins include 4-chloro indoleacetic acid, phenyl acetic acid (PAA) and indole-3-butyric acid (IBA).
  • the plant hormone biosynthetic enzymes are iaaH and iaaM, preferably from Agrobacterium tumefaciens.
  • the Excision Cassette terminator prevents the premature expression of the transgene(s) prior to the excision of the Excision Cassette. Any terminator can be used for this provided that it is recognised in the plant cell being transformed.
  • the terminator may be a plant terminator or a bacterial terminator, inter alia. Examples of suitable terminators include those of rrn, psbA, rbcL, T3,
  • a preferred terminator is a TrbcL terminator (e.g. Ribulose-1,5- Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539- 102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • TrbcL terminator e.g. Ribulose-1,5- Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539- 102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA.
  • the promoter and terminator used in the Excision Cassette do not both originate from the same plastid and/or mitochondrial gene.
  • transgene i.e. element (c)
  • element (c) is used to refer to a nucleic acid molecule which is being introduced into the genome of the plastid and/or mitochondria.
  • the transgene may, for example, be a genomic DNA, cDNA or synthetic nucleic acid molecule coding for a peptide or polypeptide; a nucleic acid molecule encoding a mRNA, tRNA or ribozyme; or any other nucleic acid molecule.
  • the transgenes of the invention encode polypeptides involved in fatty acid biosynthesis and/or fatty acid modification and/or fatty acid degradation.
  • Fatty acid biosynthesis in plants mainly occurs in plastids, although mitochondria are also capable to synthesize fatty acids.
  • the active sites for fatty acid biosynthesis are photosynthetic leaf mesophyll cells, seeds, and oil- accumulating fruits.
  • the initial condensation of an acetyl group and a malonyl group to give rise to acetoacetyl-ACP is carried out by the condensing enzyme isoform KAS III.
  • the enzyme specifically uses the substrates acetyl-CoA and malonyl-ACP. There is no evidence for the existence, or physiological significance if exists, of a bacterial version of the initial condensation reaction, which utilizes acetyl-ACP and malonyl-ACP.
  • Acetoacetyl-ACP (a beta-ketoacyl-ACP) then enters the elongation cycle, composed of four reactions.
  • One cycle extends the chain length of an acyl- ACP by two carbon units using malonyl-ACP as the carbon donor.
  • the condensing enzyme isoforms KASI and KASII are responsible for elongation steps from C4 to C 16, and from C 16 to C 18, respectively. Unlike their bacterial counterparts, plants KAS I and KAS II do not accept unsaturated acyl- ACP substrates.
  • the primary products of the fatty acid elongation cycle are 16.O-ACP and 18.0-ACP.
  • the first double bond is then introduced to 18:0-ACP by delta9 18:0-ACP desaturase, which is ubiquitous to all plants. Additional double bonds are inserted to oleate and palmitate after they are incorporated into lipids (glycolipid desaturation, phospholipid desaturation) .
  • Plants also contain very long-chain fatty acids (chain length larger than 18). The additional chain elongation occurs via the very long chain fatty acid biosynthesis pathway (very long chain fatty acid biosynthesis) located in the ER. Free fatty acids are released from acyl-ACPs and are exported out of plastids.
  • transgenes there may be 1, 2, 3, 4, 5, 6 or more such transgenes present in the Transformation Cassette. Preferably, there are 1, 2 or 3 transgenes.
  • nucleotide sequences encoding the polypeptides involved in fatty acid biosynthesis and/or fatty acid modification and/or fatty acid degradation may be present in an operon, with a single optional promoter and terminator element.
  • nucleotide sequences encoding the polypeptides involved in fatty acid biosynthesis and/or fatty acid modification and/or fatty acid degradation may each have their own promoters and terminator elements.
  • a further option is that two or more of the nucleotide sequences encoding the polypeptides involved in fatty acid biosynthesis and/or fatty acid modification and/or fatty acid degradation are present as fusion proteins, optionally with a short linker sequence joining the proteins (e.g. encoding a 1- 10 amino acid linker sequence, e.g. a poly-glycine linker).
  • nucleotide sequences encoding the polypeptides involved in fatty acid biosynthesis and/or fatty acid modification and/or fatty acid degradation may be present in an operon and/or as fusion proteins, and others have their own promoters and/or terminators.
  • the nucleotide sequences encoding the polypeptides involved in fatty acid biosynthesis and/or fatty acid modification and/or degradation have their own promoters
  • different promoters for each of the polypeptide-encoding sequences may be used.
  • different promoters which show different levels of expression in the plastid or mitochondria may be used.
  • the polypeptides encoded by the transgenes may be expressed at different levels.
  • the transgenes may be from any source, for example plants, fish, marine microbes, humans, animals, or mammals.
  • the transgenes are from plants or bacteria.
  • the polypeptide involved in fatty acid biosynthesis and/or fatty acid modification and/or fatty acid degradation is a polypeptide which is found in the fatty acid synthase complex.
  • the fatty acid synthase is a protein complex where the enzymes involved in die fatty acid pathway are held together.
  • the polypeptide involved in fatty acid biosynthesis and/or fatty acid modification and/or fatty acid degradation is a desaturase (preferably a fatty acid desaturase), a hydroxylase (preferably a fatty acid hydroxylase), a dehydrogenase (preferably a fatty acid dehydrogenase), a hydratase (preferably a fatty acid hydratase), a thiolase
  • KAS fatty acid thiolase
  • KAS I KAS I
  • the polypeptide involved in fatty acid degradation is one involved in beta oxidation.
  • the polypeptide might be a dehydrogenase, e.g. one which catalyses the dehydrogenation between the alpha and beta (C2 and C3) carbons in a FAD- linked reaction.
  • the polypeptide might alternatively be an enoyl CoA hydratase, e.g. one which catalyses hydration of a double bond.
  • the polypeptide might alternatively catalyse a second dehydrogenation in a NAD- linked reaction.
  • It might alternatively be a beta-keto acyl CoA thiolase, e.g. one which catalyses the thiolytic cleavage of a thioester.
  • polypeptides involved in fatty acid biosynthesis and/or fatty acid modification and/or fatty acid degradation are given below: EC 6.4.1.2 acetyl-coA carboxylase EC 2.3.1.39 [acyl-carrier protein] s-malonyl transferase EC 1.1.1.1003-oxoacyl-[acyl-carrier protein] reductase EC 2.3.1.41 3-oxoacyI- [acyl-carrier protein] synthase EC 2.3.1.41 3-keto-acyl-ACP synthase I (KASI) EC 3.1.2.14 acyl-ACP thioesterase EC 1.3.1.9 enoyl- [acyl-carrier protein] reductase (NADH) EC 1.14.19.2 acyl-(acyl-carrier-protein) desaturase, stearoyl-ACP desaturase stearoyl-acyl carrier protein desaturase C 12 hydroxylase
  • the transgenes comprise nucleotide sequences encoding KASI + KAS II, KAS I + KAS III, KAS II + KAS III, or KAS I + KAS II + KAS III.
  • the transgenes comprise a nucleotide sequence encoding a stearoyl-acy] carrier protein desaturase.
  • the transgenes comprise nucleotide sequences encoding a fatty acid desaturase and a stearoyl-acyl carrier protein desaturase.
  • the transgenes comprise a nucleotide sequence encoding a C 12 hydroxylase.
  • the transgene sequence may additionally encode a protein purification tag fused to the polypeptide of interest.
  • protein purification tags include the N-terminal influenza haemagglutinin-HA-epitope (HA) and a sequence of six histidine amino acids (HIS6) and the Strep tag.
  • HA N-terminal influenza haemagglutinin-HA-epitope
  • HIS6 histidine amino acids
  • Strep tag six histidine amino acids
  • the first promoter is capable of driving the expression of the transgene, leading to the accumulation of the product of the transgene in the plastid and/or mitochondria.
  • the product of the transgene may be purified or isolated from the plant cell by any suitable means.
  • the transgene is constitutively expressed independently of whether or not the Excision Cassette has been removed.
  • the transgene may be expressed from its own promoter. Such embodiments are useful if the plant does not show any detrimental effects of the transgene expression during regeneration.
  • the Transformation Cassette terminator terminates the expression of the transgene (s). Any terminator can be used for this provided that it is recognised in the plant cell being transformed.
  • the terminator may be a plant terminator or a bacterial terminator, inter ⁇ li ⁇ . Examples of suitable terminators include TrbcL or Tspb A polyA addition sequences.
  • a preferred terminator is the psbA polyA addition sequence.
  • the elements are preferably operably linked in the order (a), (b), (c), (d).
  • the first and second site-specific recombination elements must flank the optional promoter (when present), the nucleotide sequence encoding a plant-hormone biosynthetic polypeptide and the terminator element.
  • the nucleotide sequence encoding a plant-hormone biosynthetic polypeptide and the terminator element will be downstream (i.e. 3') to the promoter of the Transformation Cassette and hence the latter promoter will be capable of driving expression of the plant-hormone biosynthetic polypeptide.
  • the Transformation Cassette comprises:
  • a second terminator element (b5) a second site-specific recombination element, wherein the first and second site-specific recombination elements are capable of being recognised by a recombinase; (c) one or more transgenes encoding polypeptides involved in fatty acid biosynthesis, modification or degradation (d) a first terminator element, operably linked in the order specified above in a 5'-3' direction.
  • the first promoter is capable of driving expression of the nucleotide sequence encoding the plant-hormone biosynthetic polypeptides (for example, as shown in Figures 3-4). After the removal of the Excision Cassette . , the first promoter drives expression of the transgene(s).
  • the Excision Cassette will be in the reverse orientation compared to the first promoter, transgene(s) and first terminator element.
  • the expressed parts of the Excision Cassette will be present in the nucleotide strand which is complementary to that which codes for the first promoter, transgene(s) and first terminator element, and in the reverse direction.
  • the Excision Cassette will comprise a second promoter, capable of driving the expression of the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides.
  • the Transformation Cassette comprises:
  • the second promoter drives expression of the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides (for example, as shown in Figures 5-6).
  • the first promoter drives expression of the transgene(s).
  • Transformation Cassette is not restricted to the parts (a)-(d) specified herein. It may, for example, additionally comprise a 5'-UTR to increase the expression level of the transgene(s) and/or 3'-additional amino acids to increase protein stability.
  • the Transformation Cassette additionally comprises a second selectable marker gene, e.g. an antibiotic resistance gene, preferably a nucleotide sequence encoding spectinomycin adenyltransferase (e.g. the aadA gene). This polypeptide confers resistance to the antibiotic spectinomycin.
  • the nucleotide sequence enoding spectinomycin adenyltransferase may be placed downstream of one of the promoters in the Transformation Cassette.
  • It may, for example, be placed downstream and operably linked to the first promoter; or downstream and operably linked to the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides (e.g. IPT); or upstream and operably linked to the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides (e.g. IPT).
  • the plant-hormone biosynthetic polypeptides e.g. IPT
  • upstream and operably linked to the nucleotide sequences encoding the plant-hormone biosynthetic polypeptides e.g. IPT
  • the Transformation Cassette excludes a second selectable marker gene and/or excludes a nucleotide sequence which confers resistance to an antibiotic.
  • the Transformation Cassette additionally comprises the Lad gene, preferably under control of an appropriate promoter (e.g. T7), in order to allow for inducible expression.
  • an appropriate promoter e.g. T7
  • transformed cells are selected on media lacking the plant-hormone biosynthetic polypeptide.
  • plants are regenerated by adding cytokinin and auxin. Because the transformed plastids and/or mitochondria will preferably produce IPT and therefore cytokinin, plants can be selected and regenerated in the presence of auxin only.
  • the cells for selection will preferably be leaf cells.
  • plants are selected in the presence of cytokinin inhibitors (e.g. cytokinin oxidase or chemical inhibitors) in order to minimise leakage of the cytokinin from the site of biosynthesis to other parts of the plant. This reduced mosaicism in the plant.
  • cytokinin inhibitors e.g. cytokinin oxidase or chemical inhibitors
  • the method additionally comprises the step: (iii) expressing a recombinase in the plant cell, wherein the recombinase is one which recognises the first and second site-specific recombination elements.
  • the recombinase is a site-specific recombinase.
  • a site-specific recombinase is a polypeptide which is capable of binding to site-specific recombination elements and inducing a cross-over event in the nucleic acid molecule in the vicinity of the site-specific recombination elements.
  • the expression of the recombinase leads to the excision of the Excision Cassette from the plastid and/or mitochondrial genome.
  • the recombinase is one which is capable of binding to the first and second site-specific recombination elements which are present in the Excision Cassette, leading to the excision of the Excision Cassette in a standard manner.
  • site-specific recombination elements/site-specific recombinases include Cre-lox, the FLP-FRP system from Saccharomyces cerevisae (O'Gorman S, Fox DT 3 Wahl GM. (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science. 25, 1351-1555.), the GIN/gix system from bacteriophage Mu (Maeser S and Kahmann R. (1991) The Gin recombinase of phage Mu can catalyse site- specific recombination in plant protoplasts. MoI Gen Genet.
  • the nucleotide sequence which codes for the recombinase may comprise an intron, preferably a plant-specific intron. The presence of such an intron will suppress the expression of the recombinase polypeptide in prokaryotes, for example bacteria.
  • the preferred recombination site is lox in combination with the recombinase Cre.
  • the recombinase sequence used is a cDNA sequence encoding a Cre polypeptide.
  • the Excision Cassettes are excised from the plastid and/or mitochondrial genome by the recombinase.
  • the skilled person will understand, however, that some or all of the sequences of one or more recombination elements might remain in the plastid and/or mitochondrial genome.
  • the recombinase may be expressed in the cell by any suitable means.
  • the plant cell is one which already comprises an expressible construct which is integrated into the nuclear genome, wherein the expressible construct comprises a nucleotide sequence encoding a plastid-targeting and/or mitochondrial-targeting transit peptide and a recombinase (operably linked, i.e. in frame).
  • the expressible construct might, for example, have been introduced into the nuclear genome by homologous recombination. In such cases, the recombinase must be under the control of an inducible promoter.
  • an inducible promoter may have been introduced with the construct or the construct may have been integrated adjacent to an endogenous inducible promoter.
  • Plants containing nuclear-located sequences encoding recombinases may be removed from a desired population by crossing, wherein the sequences may be lost due to segregation of this trait.
  • the plant cell is one which already comprises an expressible construct which is integrated into the genome of the desired plastid(s) and/or mitochondria, wherein the expressible construct comprises a nucleotide sequence encoding a recombinase.
  • the expressible construct might, for example, have been introduced into the plastid and/or mitochondrial genome by homologous recombination. In such cases, the recombinase must be under the control of an inducible promoter.
  • an inducible promoter may have been introduced with the construct or the construct may have been integrated adjacent to an endogenous inducible promoter.
  • step (iii) comprises: (i ⁇ ) inducing the expression of a recombinase in the plant cell from an inducible promoter operably linked to a nucleotide sequence encoding a recombinase which is present in the plant cell, wherein the recombinase recognises the first and second site-specific recombination elements.
  • the inducible promoter operably linked to a nucleotide sequence encoding a recombinase is present in the nuclear genome of the plant cell.
  • the inducible promoter operably linked to a nucleotide sequence encoding a recombinase is present in a plastid and/or mitochondrial genome of the plant cell.
  • the plant cell is transformed with a Recombinase Vector which comprises a promoter operably linked to a nucleotide sequence encoding a recombinase, either before step (i), simultaneously with step (i) or after step (i); or before step (ii), simultaneously with step (ii) or after step (ii).
  • the Recombinase Vector is a nucleic acid vector that comprises a promoter element that is capable of driving the expression of a downstream recombinase.
  • the vector is preferably designed such that the recombinase is either expressed only or substantially only in plastids and/or mitochondria or is targeted specifically or substantially specifically to plastids and/or mitochondria.
  • the promoter in the Recombinase Vector must be one that is operable in the plant cell which is to be transformed.
  • the promoter might, for example, be one derived from a plant or bacterial gene.
  • the promoter is plant-specific or plastid-specif ⁇ c and/or mitochondria-specific.
  • the promoter is an inducible promoter such as XVE (Zuo J, Niu QW, Chua NH. (2000) Technical advance: An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24, 265-273.).
  • plant-specific means plant-specific or substantially plant-specific.
  • plastid-specific means specific or substantially specific to plastids.
  • mitochondria-specific means specific or substantially specific to mitochondria.
  • the promoter may or may not be an inducible promoter. If the Recombinase Vector is introduced to the plant cell before or during selection (step (ii)), it is preferable that the promoter is inducible. Examples of inducible promoters which are capable of operating in plants include light inducible promoters, metal inducible promoters, heat-shock promoters and other environmentally-inducible promoters.
  • the promoter is an inducible promoter, for example an XVE promoter (Zuo J, Niu QW, Chua NH. (2000) Technical advance: An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24, 265-273.) or a lac promoter.
  • XVE promoter Zao J, Niu QW, Chua NH. (2000) Technical advance: An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J. 24, 265-273.
  • a lac promoter a lac promoter.
  • the Recombinase Vector comprises a promoter, operably linked to a nucleotide sequence encoding a plastid-targeting and/or mitochondria-targeting transit peptide and a recombinase.
  • a polypeptide product may be produced comprising a plastid-targeting and/or mitochondria-targeting transit peptide operably linked to a recombinase polypeptide.
  • the promoter may or may not be plastid-specific or mitochondria-specific.
  • plastid-targeting transit peptide means a peptide sequence which is capable of targeting the recombinase polypeptide to a plastid in a specific or substantially specific manner. Upon expression, the recombinase polypeptide will be produced and specifically imported into plastids by means of the plastid-targeting peptide.
  • plastid-targeting transit peptides examples include plastid-targeting transit peptides from plastid-targeted proteins.
  • the plastid-targeting transit peptide is one which is capable of targeting the recombinase polypeptide to a chloroplast.
  • the plastid-targeting transit peptide is a plastid- targeting transit peptide from a stromal plastid targeted protein.
  • plastid-targeting transit peptides include: Transit peptide from AtABCl (Simon Geir M ⁇ ller, Tim Kunkel and Nam-Hai Chua. (2001) "A plastidic ABC protein involved in intercompartmental communication of light signaling", Genes and Dev. 15, 90-103.); from AtMinEl (Jodi Maple, Nam-Hai Chua and Simon Geir M ⁇ ller (2002) "The topological specificity factor AtMinE 1 is required for correct plastid division site placement in Arabidopsis", Plant J. 31, 269-277); and from GIANT
  • CHLOROPLAST 1 (Jodi Maple, Makoto T. Fujiwara, Nobutaka Kitahata, Tracey Lawson, Neil Baker, Shigeo Yoshida and Simon Geir M ⁇ ller (2004) "GIANT CHLOROPLAST 1 is essential for correct plastid division in Arabidopsis". Current Biology. 14, 776-781)).
  • mitochondria-targeting peptide means a peptide sequence which is capable of targeting the recombinase polypeptide to a mitochondria in a specific or substantially specific manner. Upon expression, the recombinase polypeptide will be produced and specifically imported into mitochondria by means of the mitochondria-targeting peptide.
  • mitochondria-targeting transit peptides examples include mitochondria-targeting transit peptides from mitochondria-targeted proteins.
  • Specific mitochondrial targeting transit peptides include: 1) 28 amino acid mt targeting peptide of mtSSB Edmondson AC, Song D, Alvarez LA, Wall MK, Almond D, McClellan DA,
  • the Recombinase Vector comprises an XVE promoter, operably linked to a nucleotide sequence encoding a plastid- targeting and/or mitochondrial-targeting transit peptide and CRE recombinase.
  • the Recombinase Vector may also comprise other elements, for example, the nptll gene (kanamyci ⁇ resistance) to allow for selection of transformed cells.
  • the nptll gene kanamyci ⁇ resistance
  • step (iii) comprises: (iii) transforming the plant cell with a Recombinase Vector comprising a promoter, a nucleotide sequence encoding a plastid-targeting and/or mitochondrial-targeting transit peptide and a recombinase, wherein the recombinase is one which recognises the first and second site-specific recombination elements.
  • step (iii) of the invention comprises:
  • a Recombinase Vector comprising a promoter, a nucleotide sequence encoding a plastid-targeting and/or mitochondrial-targeting transit peptide and a recombinase, wherein the promoter is capable of driving the expression of the nucleotide sequence encoding the plastid-targeting and/or mitochondrial-targeting transit peptide and the recombinase in the plant cell, and wherein, upon expression in the plant cell, the recombinase polypeptide is targeted by the transit peptide to the plastid and/or mitchondria.
  • the promoter is an inducible promoter.
  • the genetic construct additionally comprises the Recombinase Vector as defined herein.
  • nucleic acid vectors include direct DNA uptake into protoplasts, PEG-mediated uptake to protoplasts, microparticle bombardment, electroporation, heat-shock, micro-injection of DNA, micro- particle bombardment of tissue explants or cells, vacuum-infiltration of plant tissues, and T-DNA mediated transformation of plant tissues by Agrobacterium, and plant (preferably tobacco) liquid cultures.
  • any such suitable method may be used.
  • the plant cells to be transformed are guard cells . , i.e. stomatal guard cells. Such cells have been shown to be totipotent and therefore regeneration should be more efficient.
  • Guard cells may be used as epidermal strips or as isolated guard cell protoplasts. (Hall et al. 1996. 112 889-892, Plant Physiology; Hall et al. 1996, 14. 1133-1138, Nature Biotechnology).
  • biolistic transformation is preferred. This involves shooting nucleic acid vector-coated gold particles (micro-projectiles) into plastids and/or mitochondria of plant tissues, followed by selection of the transformed plastids and/or mitochondria and plant regeneration.
  • the plant tissue is a plant leaf, although callus, as for rice transformation, may also be used.
  • the method of the invention preferably also comprises the additional step of inducing the expression of the recombinase in the plant cell.
  • this step will take place after selection of the plant cells on media lacking the plant-hormone cytokinin.
  • the expression of the recombinase may be induced by applying an inducing agent which results in the activation of the promoter which is present in the Recombinase Vector or endogenous promoter or expressible construct.
  • the recombinase polypeptide is expressed and it then binds to the first and second site-specific recombination elements in the Excision Cassette, leading to the excision of that Cassette.
  • one of the site-specific recombination elements and some adjacent sequence may be left in the plastid and/or mitochondrial genome).
  • the promoter which is present in the Transformation Cassette will then be able to direct expression of the downstream transgene(s), thus producing the polypeptides (s) of interest.
  • plants are regenerated in the presence of cytokinin (shoot formation) and auxin (root formation).
  • cytokinin shoot formation
  • auxin root formation
  • appropriate cells/tissues e.g. leaf segments
  • auxin only for root regeneration
  • shoot regeneration will occur due to the presence of the IPT gene.
  • the invention also provides a method for making a transgene product, comprising the method for producing a transformed plant cell, as described hereinbefore, and additionally comprising purifying the transgene product from the plastids and/or mitochondria.
  • a particularly preferred embodiment of the invention includes a method for producing a transformed plant cell, the method comprising the step:
  • Transformation Cassette comprises:
  • Excision Cassette comprises: (bl) a first site-specific recombination element
  • b3 one or more nucleotide sequences encoding one or more plant- hormone biosynthetic polypeptides, (b4) a second terminator element, (b5) a second site-specific recombination element, wherein the first and second site-specific recombination elements are capable of being recognised by a recombinase, and wherein the first and second site- specific recombination elements are lox elements and the recombinase is Cre.
  • a further particularly preferred embodiment of the invention includes a method for producing a transformed plant cell, the method comprising the step: (i) transforming the plant cell with a genetic construct, wherein the genetic construct comprises first and second homologous recombination elements flanking a Transformation Cassette, wherein the first and second homologous recombination elements are capable of directing the integration of the Transformation Cassette into the genome of at least one plastid and/or mitochondria which is present in the plant cell, wherein the Transformation Cassette comprises:
  • a first site-specific recombination element (b2) an optional second promoter (b3) one or more nucleotide sequences encoding one or more plant- hormone biosynthetic polypeptides, wherein one or more of the polypeptides is IPT, (b4) a second terminator element, (b5) a second site-specific recombination element, wherein the first and second site-specific recombination elements are capable of being recognised by a recombinase.
  • a yet further particularly preferred embodiment of the invention includes a method for producing a transformed plant cell, the method comprising the step: (i) transforming the plant cell with a genetic construct, wherein the genetic construct comprises first and second homologous recombination elements flanking a Transformation Cassette, wherein the first and second homologous recombination elements are capable of directing the integration of the Transformation Cassette into the genome of at least one plastid and/or mitochondria which is present in the plant cell, wherein the Transformation Cassette comprises:
  • Excision Cassette comprises: (bl) a first site-specific recombination element, (b2) an optional second promoter
  • Yet a further particularly preferred embodiment provides a method for producing a transformed plant cell, the method comprising the steps: (i) transforming the plant cell with a genetic construct, wherein the genetic construct comprises first and second homologous recombination elements flanking a Transformation Cassette, wherein the first and second homologous recombination elements are capable of directing the integration of the Transformation Cassette into the genome of at least one plastid and/or mitochondria which is present in die plant cell, wherein the Transformation Cassette comprises: (a) a first promoter which is operable in said plant cell,
  • a second site-specific recombination element wherein the first and second site-specific recombination elements are capable of being recognised by a recombinase and wherein the first and second site- specific recombination elements are lox elements and the recombinase is Cre, (ii) selecting for transformed plant cells on media which is lacking IPT; and (iii) expressing a Cre recombinase in the plant cell at a level which results in excision of the Excision Cassette from the plastid genome.
  • the invention also provides a Transformation Cassette as herein defined, and a Recombinase Vector as herein defined.
  • the invention further provides a plant cell comprising a Transformation Cassette of the invention, a plant cell comprising a Recombinase Vector of the invention, and a plant cell comprising a Transformation Cassette and a Recombinase Vector of the invention.
  • the invention further provides a transgenic plant comprising a Transformation Cassette of the invention, a transgenic plant comprising a Recombinase Vector of the invention, and a transgenic plant comprising a Transformation Cassette and a Recombinase Vector of the invention.
  • the invention further provides a plant seed comprising a Transformation Cassette of the invention, a plant seed comprising a Recombinase Vector of the invention, and a plant seed comprising a Transformation Cassette and a Recombinase Vector of the invention.
  • the invention further provides a plant plastid comprising a Transformation Cassette of the invention, a plant plastid comprising a Recombinase Vector of the invention, and a plant plastid comprising a Transformation Cassette and a Recombinase Vector of the invention.
  • the invention further provides a plant mitochondria comprising a
  • Transformation Cassette of the invention a plant mitochondria comprising a Recombinase Vector of the invention, and a plant mitochondria comprising a Transformation Cassette and a Recombinase Vector of the invention.
  • the invention provides a plant cell obtainable or obtained using a method of the invention.
  • Figure I Schematic diagram showing the overall principle of the IPT gene excision and YFP gene activation.
  • pPTI001-YFP containing the IPT gene sandwiched between two lox sites which allows for CRE-mediated IPT excision after regeneration.
  • the removal of the IPT gene results in simultaneous transgene (YFP) activation.
  • Figure 2 CRE induced excision of the IPT cassette in pPTIOOl- YFP.
  • Figure 3 Schematic diagram of the (A) pPTIOOl and (B) pPTIOO 1- YFP vectors.
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (mi) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; TrbcL: Ribulose-1,5-Bisphosphate
  • Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); YFP: Yellow fluorescence protein; TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • Figure 4 Schematic diagram of the modified pPTI001-YFP vector containing protein purification tags (pPTIOOl vector series).
  • C pPTIOOl c-YFP, pPTIOOl -YFP containing a C-terminal influenza hemagglutinin-HA-epitope tag (HA3).
  • D pPTIOO Id-YFP, pPTIOOl -YFP containing six C-terminal histidine amino acids (HIS6).
  • FIG. 5 Schematic diagram of the (A) pPTI002 and (B) pPTI002- YFP vectors.
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (mi) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; TrbcL: Ribulose-1 ,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); YFP: Yellow fluorescence protein; TpsbA: psbA polyA addition sequence; HOM2:
  • Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • the construction of the vector series pPTI002 is to ensure that there is no leaky expression from the Prrn promoter to the transgene in question (in this case YFP) prior to CRE-mediated excision.
  • Figure 6 Schematic diagram of the modified pPTI002-YFP vector containing protein purification tags (pPTI002 vector series) .
  • pPTI002a-YFP pPTI002-YFP containing an N-terminal influenza hemaggJutinin-HA-epitope tag (HA).
  • pPTI002b-YFP pPTI002-YFP containing six N-terminal histidine amino acids (HIS 6).
  • C pPTI002c-YFP, pPTI002-YFP containing a C-terminal influenza hemagglutinin-HA-epitope tag (HA).
  • the IPT DNA sequence was modified changing adenine to guanidine at nucleotide position 519 (+1 taken as adenine in the start codon) thereby removing an endogenous EcoRV site.
  • FIG 7 Schematic diagram of pERlO/TPCRE.
  • XVE acts as the inducible promoter that drives TP-CRE (Transit peptide fused to CRE) expression. Selection of this transgene is by Kanamycin resistance conferred by the nptll gene.
  • Figure 8 Selection and regeneration of positive transplastomic plants using the IPT selectable marker.
  • Figure 9 Confirmation of transgene insertion into the tobacco plastid genome. Insertion of pPTI001/YFP into the tobacco chloroplast genome at the homologous recombination sites was confirmed using a primer in the flanking region of the tobacco chloroplast genome and a primer that anneals to TrbcL terminator within the cassette. Lanes shown: WT, wild-type; #1, regenerant 1; #2 regenerant 2; M 3 marker.
  • Figure 10 Induction of YFP expression in tobacco leaf cells.
  • FIG. 11 Plastid transformation vector pPTIOOS :
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA), PpsbA: Plastidic psbA promoter; aadA: spectinomycin adenyltransferase gene; T7: T7 transcription terminator sequence: Prrn: Plastidic ribosomal RNA (rni) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacte ⁇ um tumefaciens; TrbcL; Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homolog
  • Figure 12 Plastid transformation vector pPTI007:
  • HOM3 Homologous recombination sequence (nt 102925-101857, accession Z00044 Nicotiana tabacum chloroplast genome DNA); PpsbA: Plastidic psbA promoter; aadA: spectinomycin adenyltransferase gene; T7: T7 transcription terminator sequence: Prrn: Plastidic ribosomal RNA (rr ⁇ ) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; TrbcL: Ribulose-1 ,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM4: Homologous
  • Plastid transformation vector pPTI008 HOM3: Homologous recombination sequence (nt 102925-101857, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rr ⁇ ) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; aadA: spectinomycin adenyltransferase gene; TrbcL: Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM4: Homologous recombination sequence (nt 100933-100
  • HOM3 Homologous recombination sequence (nt 102925-101857, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (mi) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; aadA: spectinomycin adenyltransferase gene; TrbcL: Ribulose-1 ,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM4: Homologous recombination sequence (nt 100933-10013O 3 accession Z00044 Nicoti ⁇ n ⁇
  • FIG. 15 Plastid transformation vectors pinPTI.
  • Each pPTI vector contains a constitutively expressed L ⁇ cl gene.
  • pPTI003 is shown as an example.
  • Modified Prrn promotors (PrrnL) will be inserted upstream of the IPT.aadA cassette, allowing inducible, controlled expression of the IPT.aadA cassette.
  • B pind2PTI, pPTI containing the L ⁇ cl open reading frame under the control of the T7 promoter sequence and modified PrrnL promoter upstream of the IPT.aadA cassette.
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicoti ⁇ n ⁇ t ⁇ b ⁇ cum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (mi) operon promoter (nt 59034-59303, accession Z00044 Nicoti ⁇ n ⁇ t ⁇ b ⁇ cum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrob ⁇ cterium tumef ⁇ ciens; aadA: spectinomycin adenyltransferase gene; TrbcL: Ribulose-lj,5-Bisphosph ⁇ te Carboxylase/Oxygenase poly A addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (n
  • Each pPTI vector will be modified to contain a modified Prrn-trbcL (Prrn promoter construct, composed of the Prrn promter and rbcL 5' translation control region, to ultimately produce higher levels of expression of the foreign proteins.
  • Prrn promoter construct composed of the Prrn promter and rbcL 5' translation control region, to ultimately produce higher levels of expression of the foreign proteins.
  • pPTI003 is shown as an example.
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn-t: Plastidic ribosomal RNA (rrri) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA) and rbcL 5' translation control region; IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; aadA: spectinomycin adenyltransferase gene; TrbcL: Ribulose-1 , 5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA) ; Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; aadA: spectinomycin adenyltransferase gene; TrbcL: Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2:
  • the IPT.aadA cassette is constructed as an operon. Both of the IPTand aadA genes are preceded by a shine delgardo sequence and a six base pair spacer.
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; aadA: spectinomycin adenyltransferase gene; TrbcL: Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloro
  • FIG. 18 Plastid transformation vector pPTAOOl HOMl: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); iaaH: indoleacetamide hydrolase from Agrobacterium tumefaciens; iaaM: tryptophan monooxygenase from Agrobacterium tumefaciens; TrbcL: Ribulose-1,5-Bisphosphate
  • Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • the iaaH.iaaM cassette is constructed as an operon. Both of the iaaH and iaaM genes are preceded by a shine delgardo sequence and a six base pair spacer.
  • Plastid transformation vector pPTA002 HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); iaaH: indoleacetamide hydrolase from Agrobacterium tumefaciens; iaaM: tryptophan monooxygenase from Agrobacterium tumefaciens; TrbcL: Ribulose-1,5-Bisphosphate
  • Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • the iaaH.iaaM cassette is expressed as a fusion protein.
  • the iaaH a gene is preceded by a shine delgardo sequence and a six base pair spacer and the two open reading frames are separated by an eight glycine linker sequence.
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TrbcL: Ribulose-1,5- Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539- 102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); iaaH: indoleacetamide hydrolase from Agrobacte ⁇ um tumefaciens; iaaM: tryptophan monooxygenase from Agrobacte ⁇ um tumefaciens; Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370,
  • Figure 21 PIastid transformation vector pPTA004
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TrbcL: Ribulose-1,5- Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539- 102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); iaaH: indoleacetamide hydrolase from Agrobacte ⁇ um tumefaciens; iaaM: tryptophan monooxygenase from Agrobacte ⁇ um tumefaciens; Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370,
  • the iaaH. iaaM cassette is expressed as a fusion protein.
  • the iaaH a gene is preceded by a shine delgardo sequence and a six base pair spacer and the two open reading frames are separated by an eight glycine linker sequence.
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); PpsbA: Plastidic psbA promoter; aadA: spectinomycin adenyltransferase gene; T7: T7 transcription terminator sequence: Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); iaaH: indoleacetamide hydrolase from Agrobacte ⁇ um tumefaciens; iaaM: tryptophan monooxygenase from Agrobacte ⁇ um tumefaciens; TrbcL: Ribulose-1,5-Bisphosphate
  • Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • the iaaH.iaaM cassette is constructed as an operon. Both of the iaaH and iaaM genes are preceded by a shine delgardo sequence and a six base pair spacer.
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); PpsbA: Plastidic psbA promoter; aadA: spectinomycin adenyltransferase gene; T7: T7 transcription terminator sequence: Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); iaaH: indoleacetamide hydrolase from Agrobacte ⁇ um tumefaciens; iaaM: tryptophan monooxygenase from Agrobacte ⁇ um tumefaciens; TrbcL: Ribulose-1,5-Bisphosphate
  • Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); TpsbA: psbA polyA addition sequence; HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • the iaaH.iaaM cassette is expressed as a fusion protein.
  • the iaaH a gene is preceded by a shine delgardo sequence and a six base pair spacer and the two open reading frames are separated by an eight glycine linker sequence.
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacte ⁇ um tumefaciens; T7: T7 terminator sequence; KASI: 3- keto-acyl-ACP synthase I gene; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); PpsbA: psbA promoter sequence; KASII: 3-keto-acyl-ACP synthase II gene; PrbcL: rbcL
  • HOM2 Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • Figure 25 Plastid transformation vector pPTFA2
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303;, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacte ⁇ um tumefaciens; T7: T7 terminator sequence; KASI: 3- keto-acyl-ACP synthase I gene; KASII: 3-keto-acyl-ACP synthase II gene; KASIII: 3-keto-acyl-ACP synthase III gene; TrbcL.
  • Ribulose-1,5- Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539- 102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • the KASI.KASII.KASIII cassette is expressed as a fusion protein.
  • the KASI a gene is preceded by a shine delgardo sequence and a six base pair spacer and each of the three open reading frames are separated by an eight glycine linker sequence.
  • FIG. 26 Plastid transformation vector pPTFA3 HOMl: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacte ⁇ um tumefaciens; T7: T7 terminator sequence; KASI: 3- keto-acyl-ACP synthase I gene; KASII: 3-keto-acyl-ACP synthase II gene;
  • KASIII 3-keto-acyl-ACP synthase III gene
  • TrbcL Ribulose-1,5- Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539- 102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA)
  • HOM2 Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • KASI.KASII.KASIII cassette is organised as an operon in which each of the KAS genes are preceded by a shine delgardo sequence and a six base pair spacer.
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacte ⁇ um tumefaciens; T7: T7 terminator sequence; SAD: stearoyl-ACP desaturase gene; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • Figure 28 Plastid transformation vector pPTFAS
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacte ⁇ um tumefaciens; T7: T7 terminator sequence; FAD: fatty acid desaturase gene; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA) .
  • HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacte ⁇ um tumefaciens; T7: T7 terminator sequence; SAD: stearoyl-ACP desaturase gene; FAD: fatty acid desaturase gene; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome
  • HOM2 Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • SAD FAD cassette is expressed as a fusion protein.
  • the SAD gene is preceded by a shine delgardo sequence and a six base pair spacer and the SAD and FAD open reading frames are separated by an eight glycine linker sequence.
  • FIG. 30 Plastid transformation vector pPTFA7 HOMl: Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); Prrn: Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacte ⁇ um tumefaciens; T7: T7 terminator sequence; C12 hydroxylase: C 12 hydroxylase gene; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloro
  • Plastid transformation vector pPTFA8 HOMl Homologous recombination sequence (nt 104091-105380, accession Z00044 Nicotiana tabacum chloroplast genome DNA); PrrnL: Modified plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacte ⁇ um tumefaciens; T7: T7 terminator sequence; KASI: 3-keto-acyl-ACP synthase I gene; KASII: 3-keto-acyI-ACP synthase II gene; KASIII: 3-keto-acyl-ACP synthase III gene; TrbcL:
  • Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase polyA addition sequence (nt 102539-102685, accession Z00044 Nicotiana tabacum chloroplast genome DNA); PpsbA: psbA promoter sequence; Lad: Lad gene from Escherichia coli (accession NP_414879); HOM2: Homologous recombination sequence (nt 105381-106370, accession Z00044 Nicotiana tabacum chloroplast genome DNA).
  • the KASI.KASII.KASIII cassette is expressed as a fusion protein.
  • the KASI a gene is preceded by a shine delgardo sequence and a six base pair spacer and each of the three open reading frames are separated by an eight glycine linker sequence.
  • FIG. 32 Plastid transformation vector pPTFA9 HOMl: Homologous recombination sequence (nt 104091-10538O 3 accession Z00044 Nicotiana tabacum chloroplast genome DNA); PrrnL: Modified plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA); IPT: isopentenyltranferase gene from Agrobacterium tumefaciens; T7: T7 terminator sequence; KASI: 3-keto-acyl-ACP synthase I gene; KASII: 3-keto-acyl-ACP synthase II gene; KASIII: 3-keto-acyl-ACP synthase III gene; TrbcL: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase poJyA addition sequence (nt 102539-102685, accession
  • the plastid transformation vectors pPTIOOl and pPTI002 were constructed as detailed in Figure 3 and Figure 5 using a 1289 bp homologous recombination sequence (104091-105380 nt) and a 989 bp homologous recombination sequence (105381-106370 nt) from the chloroplast genome from Nicotiana tabacum (Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J 3 Yamaguchi-Shinozaki K, Ohto C, Torazawa K, Meng BY, Sugita M, Deno H, Kamogashira T, Yamada K, Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H, Sugiura M.
  • coli DH5 ⁇ cells were produced and transformed with the vector pERlO-TP.CRE, which constitutively expresses the TP. CRE fusion protein, and selected on LB media containing spectinomycin. Subsequently chemically competent E. coli DH5 ⁇ cells containing the pERlO-TP.CRE vector were produced and transformed with pPTI001-YFP. Cells containing both vectors were selected for on LB media containing spectinomycin and chloramphenicol.
  • the expression of high levels of foreign protein in plants can lead to detrimental effects on plant development because of toxic effects.
  • the described system overcomes this by combining insertion of the transgene(s) into the plastid genome where it remains dormant until the IPT selectable marker gene is removed by CRE/lox mediated recombination.
  • the transgene encoding the "plant-toxic" protein is inserted into one of the pPTIOOl or pPTI002 vectors ( Figure 3 and 5) between the TrbcL and the TspbA polyA addition sequences in the pPTIOOl vector series or between the Prrn promoter and the TspbA polyA addition sequence in the pPTI002 vector series and the construct transformed into plastids using the protocol shown in Appendix 1 followed by cytokinin-mediated selection and regeneration. Once regenerated, the IPT gene is removed by CRE-mediated recombination and the toxic transgene is activated leading to minimal adverse effects on initial plant regeneration. Once expressed, the recombinant protein can be purified using one of the affinity tags present in either the pPTIOOl or pPTI002 vector series shown in Figures 4 and 6.
  • the transgene is inserted into one of the pPTIOOl or pPTI002 vectors
  • the present system can be used for the expression of eukaryotic proteins in plastids using IPT marker gene selection and transgene activation. As described in Examples 2 and 3, any gene encoding a eukaryotic protein may be inserted into one or all of the pPTIOOl series or the pPTI002 series of vectors followed by transformation, selection and regeneration as described previously. Following regeneration, the expressed protein may be purified using the affinity tags shown in Figures 4 and 6 and used for downstream applications .
  • a non-exclusive list of possible eukaryotic protein families that will be expressed includes antibodies, enzymes, enzyme inhibitors and design peptides.
  • prokaryotic proteins Due to the endosymbiotic origin of plastids, it is possible to express prokaryotic proteins in plastids. As described in Examples 2 and 3, any gene encoding a prokaryotic protein may be inserted into one or all of the pPTIOO 1 series or the pPTI002 series of vectors followed by transformation, selection and regeneration as described previously. Following regeneration, the expressed protein may be purified using the affinity tags shown in Figures 4 and 6 and used for downstream applications.
  • Example 7 Expression of YFP from pPTIOO 1/YFP in plastids of tobacco pPTI001/YFP vector was bombarded into tobacco leaf cells and regenerants selected on media containing only auxin and on media lacking all hormones. Regenerants were obtained ( Figure 8) and transferred to secondary selection media containing auxin to induce root formation before transfer to soil.
  • the incorporation of the transformation cassette in the tobacco plastid genome was confirmed by PCR using vector-specific primers and primers in the flanking region of the tobacco chloroplast genome ( Figure 9). Leaves from the regenerated plants were infiltrated with pERlO/TP.CRE, a binary vector expressing the TP. CRE fusion, followed by induction. After 72 hours the infiltrated tissue was analysed by fluorescence microscopy revealing cells containing GFP fluorescing chloroplasts ( Figure 10).
  • HOM4 that may increase more efficient homologous recombination and transgene insertion.
  • auxin biosynthesis as a selectable marker for plastid transformation
  • the plastid transformation vectors pPTA00l-pPTA004 are constructed as detailed in Figures 18-21 using a 1289 bp homologous recombination sequence (104091-105380 nt) and a 989 bp homologous recombination sequence (105381-106370 nt) from the chloroplast genome from Nicotiana tabacum (Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohto C, Torazawa K, Meng BY, Sugita M, Deno H, Kamogashira T, Yamada K 3 Kusuda J, Takaiwa F 3 Kato A 5 Tohdoh N, Shimada H, Sugiura M.
  • the expression of high levels of foreign protein in plants can lead to detrimental effects on plant development because of toxic effects.
  • the described system overcomes this by combining insertion of the transgene(s) into the plastid genome where it remains dormant until the auxin selectable marker genes are removed by CKE ⁇ ox mediated recombination.
  • the transgene encoding the "plant-toxic" protein is inserted into one of the pPTA001-pPTA004 vectors ( Figures 18-21) between the Prrn promoter and the TpsbA polyA addition sequences and the construct transformed into plastids using the protocol shown in Appendix 3 followed by auxin-mediated selection and regeneration. Once regenerated, the auxin genes are removed by CRE-mediated recombination and the toxic transgene is activated leading to minimal adverse effects on initial plant regeneration. Once expressed, the transplastomic plants are used conferring a desired trait or the recombinant protein is purified.
  • Plastids play an integral role during plant development and it may therefore be of interest to express proteins (plant or non-plant) inside plastids that would have a positive effect on plant growth, development and/or confer modified characteristics to the plant as whole. Due to the high expression levels of transgenes in plastids coupled to the fact that the iaaH and iaaM as selectable marker genes does not lead to the generation of spontaneous ribosomal mutants (as with the common spectinomycin selectable marker gene), the present invention represents an ideal system for this application.
  • the transgene encoding the plastidic protein is inserted into one of the pPTA001-pPTA004 vectors ( Figures 18-21) between the Prrn promoter and the TrbcL polyA addition sequences and the construct transformed into plastids using the protocol shown in Appendix 3 followed by auxin-mediated selection and regeneration. Once regenerated, the iaaH and iaaM genes are removed by CRE-mediated recombination and the transgene is activated leading to minimal adverse effects on initial plant regeneration.
  • the present system can be used for the expression of eukaryotic proteins in plastids using iaaH and iaaM marker gene selection and transgene activation.
  • any gene encoding a eukaryotic protein may be inserted into one or all of pPTA00l-pPTA004 series of vectors followed by transformation, selection and regeneration as described previously. Following regeneration, the expressed protein may be purified and used for downstream applications.
  • a non-exclusive list of possible eukaryotic protein families that will be expressed includes antibodies, enzymes, enzyme inhibitors and design peptides.
  • prokaryotic proteins Due to the endosymbiotic origin of plastids, it is possible to express prokaryotic proteins in plastids. As described in Examples 10 and 11, any gene encoding a prokaryotic protein may be inserted into one or all of ppTA001-pTA004 vectors followed by transformation, selection and regeneration as described previously. Following regeneration, the expressed protein may be purified and used for downstream applications.
  • Transgenes encoding one or more fatty acid biosynthetic enzymes are inserted into plastid transformation vectors as shown in Figures 24-26 and 31-32.
  • the vectors are transformed into plastids using the protocol described in Appendix 1, followed by cytokinin-mediated selection and regeneration. Once regenerated, the IPT gene is removed by CRE-mediated recombination.
  • Transgenes encoding one or more fatty acid modifying enzymes are inserted into plastid transformation vectors as shown in Figures 27-30.
  • the vectors are transformed into plastids using the protocol described in Appendix 1, followed by cytokinin-mediated selection and regeneration. Once regenerated, the IPT gene is removed by CRE-mediated recombination.
  • PROTOCOL FOR CHLOROPLAST AND/OR MITOCHONDRIAL TRANSFORMATION USING CYTOKININ SELECTION TIME COURSE The standard procedures produce transformed plants in 3-5 months.
  • Vacuum release rate Attenuate the release so it approximates the speed of vacuum inflow.
  • the key to successful bombardment is usually in the spread of particles on the macrocarrier.
  • the ethanol/gold/DNA mixture should quickly spread out over the centre of the macrocarrier.
  • the resulting spread should be a very fine dusting of particles, evenly spread and containing few chunks. Chunk causes increased cell death.
  • Tobacco plants are micropropagated using sterile technique in magenta boxes containing MS media.
  • Rooted shoots are transferred to soil approx. 3-5 weeks after isolation. Plants are allowed to grow in standard tobacco conditions (16:8 photoperiod at 25°C).
  • Transformation frequency for an average experiment is anywhere from 1.5 stably transformed plants to 0.3 stably transformed plants per bombardment.
  • MFB media for bombardement
  • MTS media for transgenic selection
  • This time includes second selection time
  • Vacuum release rate Attenuate the release so it approximates the speed of vaccum inflow.
  • the key to successful bombardment is usually in the spread of particles on the macrocarrier.
  • the ethanol/gold/DNA mixture should quickly spread out over the centre of the macrocarrier.
  • the resulting spread should be a very fine dusting of particles, evenly spread and containing few chunks. And chunk causes cell death.
  • Tobacco plants are micropropagated using sterile technique in magenta boxes containing MS media • Expanded leaves are excised and placed abaxial surface up on a
  • Green shoots can be collected from the bleached explants in 3-8 weeks. • Leaves from these shoots are cut up (2mm square) and subcultured in the same selective media for approx. 4 weeks.
  • Plants are allowed to grow in standard tobacco conditions (16:8 photoperiod at 25°C).
  • Transformation frequency for an average experiment is anywhere from 1.5 stably transformed plants to 0.3 stably transformed plants per bombardment.
  • MFB media for bombardment
  • MTS media for transgenic selection
  • This time includes second selection time
  • Level of macrocarrier lauch assembly 1 (from top) • Level of Petri dish holder: 4 (from top)
  • Vacuum release rate Attenuate the release so it approximates the speed of vaccum inflow.
  • the key to successful bombardment is usually in the spread of particles on the macrocarrier.
  • the ethanol/gold/DNA mixture should quickly spread out over the centre of the macrocarrier.
  • the resulting spread should be a very fine dusting of particles, evenly spread and containing few chunks. And chunk causes cell death.
  • TISSUE CULTURE Tobacco plants are micropropagated using sterile technique in magenta boxes containing MS media • Expanded leaves are excised and placed abaxial surface up on a Whatman filter paper laying on top of RMOP media. The leaves are allowed to desiccate slightly (1-2 hours) prior to bombardment on the filter paper. Each bombardment can treat 1-3 leaves covering approx. 1/3 of the 9 cm Petri dish. • Post bombardment, the plates are sealed and the leaves are left at
  • Rooted shoots are transferred to soil appox. 3-5 weeks after isolation. Plants are allowed to grow in standard tobacco conditions (16:8 photoperiod at 25 0 C).
  • Transformation frequency for an average experiment is anywhere from 1.5 stably transformed plants to 0.3 stably transformed plants per bombardment.
  • MSS MS Salts and Vitamins (IX) Magenta Box 30 g/1 Sucrose 6 g/1 Phytagar pH 5.8
  • MTS media for transgenic selection
  • This time includes second selection time
  • Vacuum release rate Attenuate the release so it approximates the speed of vaccum inflow.
  • the key to successful bombardment is usually in the spread of particles on the macrocarrier.
  • the ethanol/gold/DNA mixture should quickly spread out over the centre of the macrocarrier.
  • the resulting spread should be a very fine dusting of particles, evenly spread and containing few chunks. And chunk causes cell death.
  • Expanded leaves are excised and placed abaxial surface up on a Whatman filter paper laying on top of RMOP media.
  • the leaves are allowed to desiccate slightly (1-2 hours) prior to bombardment on the filter paper.
  • Each bombardment can treat 1-3 leaves covering approx. 1/3 of the 9 cm Petri dish.
  • Post bombardmentj the plates are sealed and the leaves are left at 12:12 photoperiod at 24 0 C for 2 days.
  • Green shoots can be collected from the bleached explants in 3-8 weeks.
  • Rooted shoots are transferred to soil appox. 3-5 weeks after isolation. Plants are allowed to grow in standard tobacco conditions (16:8 photoperiod at 25°C) .
  • Transformation frequency for an average experiment is anywhere from 1.5 stably transformed plants to 0.3 stably transformed plants per bombardment.
  • MSS MS Salts and Vitamins (IX) Magenta Box 30 g/1 Sucrose 6 g/1 Phytagar pH 5.8
  • MFB media for bombardment
  • MTS media for transgenic selection
  • This time includes second selection time

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Abstract

L’invention concerne un procédé de fabrication d’une cellule de plante transformée. Plus particulièrement, le procédé comprend la transformation d’une cellule de plante avec une cassette de transformation qui cible les plastes et/ou les mitochondries des plantes et qui comprend un gène de sélection, par exemple isopentényle transférase (IPT), et un ou plusieurs transgènes codant pour des polypeptides impliqués dans la biosynthèse, la modification ou la dégradation d’acides gras. Après la sélection des plastes et/ou mitochondries transformés, l’expression d’une recombinase est induite dans la cellule de plante, ce qui conduit à l’excision du gène de sélection du plaste et/ou de la mitochondrie et à l’expression du transgène dans le plaste et/ou la mitochondrie. L’invention concerne également des cellules et des plantes comprenant la cassette de transformation.
PCT/GB2009/001482 2008-06-13 2009-06-12 Vecteurs de transformation du plaste permettant l’excision de gènes marqueurs WO2009150435A1 (fr)

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WO2015158919A1 (fr) * 2014-04-18 2015-10-22 Møller Simon Geir Procédé de transformation de plastide de maïs
WO2017022587A1 (fr) * 2015-08-06 2017-02-09 花王株式会社 Procédé de production de lipides
CN109943657A (zh) * 2019-04-04 2019-06-28 中国计量大学 鉴定核桃乳的dna条形码基因及核桃真伪性的分子鉴定方法

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WO2015158919A1 (fr) * 2014-04-18 2015-10-22 Møller Simon Geir Procédé de transformation de plastide de maïs
WO2017022587A1 (fr) * 2015-08-06 2017-02-09 花王株式会社 Procédé de production de lipides
CN109943657A (zh) * 2019-04-04 2019-06-28 中国计量大学 鉴定核桃乳的dna条形码基因及核桃真伪性的分子鉴定方法

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