WO2024065009A1 - Procédés de manipulation de plantes - Google Patents

Procédés de manipulation de plantes Download PDF

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Publication number
WO2024065009A1
WO2024065009A1 PCT/AU2023/050942 AU2023050942W WO2024065009A1 WO 2024065009 A1 WO2024065009 A1 WO 2024065009A1 AU 2023050942 W AU2023050942 W AU 2023050942W WO 2024065009 A1 WO2024065009 A1 WO 2024065009A1
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Prior art keywords
plant
genetically modified
nuclease
safflower
seed
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PCT/AU2023/050942
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English (en)
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Daniel ISENEGGER
Matthew James Hayden
German Carlos Spangenberg
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Agriculture Victoria Services Pty Ltd
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Priority claimed from AU2022902820A external-priority patent/AU2022902820A0/en
Application filed by Agriculture Victoria Services Pty Ltd filed Critical Agriculture Victoria Services Pty Ltd
Publication of WO2024065009A1 publication Critical patent/WO2024065009A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/14—Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce
    • A01H6/1416—Carthamus tinctorius [safflower]

Definitions

  • the present invention relates to methods for manipulating plants, more particularly methods for manipulating target genes in safflower plants.
  • Alternate methods to grafting include ex vitro rooting, which have been established for a range of plant species produced by micropropagation (Benmahioul et al. 2012, Ranaweeraa et al. 2013, Murphy and Adelberg 2021). This approach relies on conditions that stimulate the formation of adventitious roots in deflasked micro-shoots and has the potential to reduce the time in tissue culture, as well as to increase the recovery of fertile plants from tissue culture.
  • TALEN Transcription Activator-Like Effector Nuclease
  • ZFN Zinc Finger Nuclease
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • a challenge in generating transgenic or gene edited safflower is associated with generating roots on regenerated shoots in the transgenic or gene edited safflower plants.
  • grafting methods may be used to overcome this problem, in safflower plants, this method is technically challenging and not always successful. Without roots, it is not possible to recover seed from regenerated plants so the transgenic or gene editing outcome is not passed on through inheritance. This makes it difficult to apply accelerated breeding techniques to safflower plants.
  • the present invention provides a method for generating a genetically modified safflower plant, said method including: a. selecting a nuclease having cleavage efficacy at a target gene in the safflower plant genome and introducing a gene encoding the nuclease into safflower plant material; and/or b. introducing a transgene into safflower plant material; and c. establishing a genetically modified microshoot from the safflower plant material; and d.
  • the genetically modified microshoot to ex vitro rooting by: i) transferring the genetically modified microshoot to a growing material; ii) retaining the genetically modified microshoot in a light- and temperature- controlled growth chamber to establish a TO genetically modified safflower plant.
  • the present invention provides a method for manipulating a target gene in a safflower plant, said method including: a. selecting a nuclease having cleavage efficacy at the target gene in the safflower plant genome and introducing a gene encoding the nuclease into safflower plant material; and/or b. introducing a transgene into safflower plant material; and c. establishing a genetically modified microshoot from the safflower plant material; and d.
  • the genetically modified microshoot to ex vitro rooting by: i) transferring the genetically modified microshoot to a growing material; ii) retaining the genetically modified microshoot in a light- and temperature- controlled growth chamber to establish a TO genetically modified safflower plant with altered expression of the target gene.
  • the methods are for accelerating precision breeding in the plant.
  • a method of precision breeding including: a) obtaining a genetically modified plant according to the method of above; and b) inter-crossing the genetically modified plant to establish offspring having a combination of traits, wherein at least one trait corresponds to the target gene or transgene.
  • the safflower plant is also known as Carthamus tinctorium.
  • precision breeding as used herein is meant targeted genome alterations such as nucleotide changes, additions, or deletions in a plant that introduce a novel trait into the plant.
  • trait as used herein is meant a genetically determined characteristic, such as, for example, tolerance to one or more biotic (e.g., pest resistance) or abiotic (e.g., drought) stresses, or plant yield indices, such as fruit or seed size, extent of the fruiting season, and the like.
  • biotic e.g., pest resistance
  • abiotic e.g., drought
  • a novel trait is meant that the trait corresponding to the target gene is different in the genetically modified plant or its offspring, as compared with a wild type plant.
  • the trait may be improved tolerance to one or more biotic (e.g. pest resistance) or abiotic (e.g. drought) stresses, or improvements in plant yield, such as increased fruit or seed size, extended fruiting season, and the like.
  • biotic efficacy as used in the context of a nuclease, is meant a nuclease which cleaves a site in a plant genome corresponding to a target gene at a significantly higher frequency than that of a control nuclease.
  • target gene as used herein is meant a region of a nucleic acid which encodes a trait of interest. As such, a target gene may include a fragment of DNA or RNA.
  • the genetically modified plant is fertile.
  • the offspring are fertile.
  • ‘fertile’ as used in the context of a plant is meant a plant capable of producing viable offspring through pollination including, for example self-pollination or cross-pollination.
  • a genetically modified plant as used herein is meant a plant having a modified genome.
  • a genetically modified plant may include a plant into which one or more exogenous gene(s) have been introduced (e.g. a plant which has undergone transformation).
  • a genetically modified plant may also include a plant in which one or more endogenous gene(s) have been modified or silenced (e.g. a plant which has undergone gene editing).
  • Techniques for introducing exogenous genes into plants or editing genes in plants are known to those skilled in the art.
  • inter-crossing as used herein is meant the fertilization of one plant following pollination by another plant to produce an offspring with genes from both plants.
  • the method may include the steps of: a. selecting a nuclease having cleavage efficacy at a target gene in the safflower plant genome and introducing a gene encoding the nuclease into safflower plant material to produce genetically modified plant material, wherein expression of the target gene in the plant is altered relative to a wild type plant; or b. introducing a transgene into safflower plant to generate a genetically modified safflower plant material, wherein expression of the target gene in the plant is altered relative to a wild type plant.
  • expression of the target gene in the plant may be silenced.
  • a transgene may be introduced into the safflower plant that provides a trait not present in the safflower plant before transformation.
  • the step (a) or (b) of generating a genetically modified safflower plant includes: a. generating an axenic in vitro seedling from a seed of the plant; b. transforming the seedling with a genetic construct.
  • the methods include the further step of generating a genetically modified plant from the microshoot.
  • the genetically modified plant is fertile.
  • the step of transforming the seedling is an Agro bacterium-mediated transformation.
  • the Agrobacterium sp. includes a binary T-DNA vector.
  • the binary T-DNA vector includes a recombinant transgene cassette and a transformation selectable marker gene.
  • the recombinant transgene cassette includes a transgene and/or a gene encoding a nuclease. More preferably, the nuclease is an engineered nuclease selected from the group consisting of TALEN, ZFN, and CRISPR.
  • the step of transforming the seedling includes: a. excising a cotyledon explant from the seedling; and b. subjecting the cotyledon to Agrobacterium-mediated transformation.
  • the step (b) of subjecting the cotyledon to Agrobacterium-mediated transformation involves inoculating and co-culturing the cotyledon with an Agrobacterium sp., preferably Agrobacterium tumefaciens, more preferably Agrobacterium tumefaciens strain EHA105.
  • Agrobacterium sp. preferably Agrobacterium tumefaciens, more preferably Agrobacterium tumefaciens strain EHA105.
  • microshoot as used herein is meant a small shoot initiated during the establishment and multiplication stages of micropropagation.
  • micropropagation as used herein is meant a method of plant propagation using a small piece of plant tissue.
  • the step of ex vitro rooting includes: a. transferring the transformed microshoot to a growing material; b. retaining the transformed microshoot in a light- and temperature-controlled growth chamber to establish a TO plant; and c. optionally supporting the growth of the TO plant such that TO plant flowers and produces T 1 seeds.
  • growing material as used herein is meant a nutrient rich material that may support a microshoot during its early stages of growth and development.
  • a growing material may include, for example, hydrated sphagnum peat moss or coco pellets.
  • the transformed microshoot may be retained in a light- and temperature- controlled growth chamber.
  • the light may be controlled with a 6 to 14 hr photoperiod, especially an 8 to 14 hr photoperiod, more especially a 10 to 14 hr photoperiod, for example, an approximately 12 hr photoperiod.
  • the temperature may be controlled in the range of 16 to 28°C, especially 18 to 26°C, more especially 20 to 24°C, for example at approximately 22°C.
  • supporting the growth of the TO plant may include transplanting TO plants, preferably robust TO plants, to conventional potting media once a root system is established.
  • the TO plant may be retained in a greenhouse.
  • the temperature may be maintained at 16 to 28°C, especially 18 to 26°C, more especially 20 to 24°C, for example at approximately 22°C to 24°C.
  • the photoperiod may be set as a 6 to 16 hr photoperiod, especially an 8 to 16 hr photoperiod, more especially a 12 to 16 hr photoperiod, for example, an approximately 14 hr.
  • axenic as used in the context of a seedling is meant a seedling having a substantially identical genome to that of the parent plant.
  • part of a plant refers to a plant seed, stem, callus, leaf, root, shoot or the like.
  • the protoplast may be isolated from a leaf.
  • the nuclease may be an engineered nuclease, for example an engineered nuclease selected from the group consisting of: Transcription Activator- Like Effector Nuclease (TALEN), Zinc Finger Nuclease (ZFN), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).
  • TALEN Transcription Activator- Like Effector Nuclease
  • ZFN Zinc Finger Nuclease
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the nuclease is ZFN.
  • the protoplast is free of other plant materials.
  • the protoplast is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure, even more preferably at least approximately 99% pure.
  • genetic construct refers to an artificially assembled or isolated nucleic acid molecule which includes the target transgene or a gene encoding a nuclease.
  • the genetic construct is a recombinant nucleic acid molecule.
  • a construct may include the target transgene or the gene encoding a nuclease, a marker gene which in some cases can also be the target gene, and appropriate regulatory sequences. It should be appreciated that the inclusion of marker genes and regulatory sequences in a construct is optional, for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used.
  • construct includes vectors but should not be seen as being limited thereto.
  • the genetic construct is a vector.
  • vector By a ‘vector’ is meant a genetic construct used to transfer genetic material to a target cell.
  • the term vector encompasses both cloning and expression vectors. Vectors are often recombinant molecules containing nucleic acid molecules from several sources.
  • the vector may be of any suitable type and may be viral or non-viral.
  • the vector may be an expression vector.
  • Such vectors include chromosomal, non-chromosomal, and synthetic nucleic acid sequences, e.g., derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens', derivatives of the Ri plasmid from Agrobacterium rhizogenes', phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA.
  • any other vector may be used as long as it is replicable or integrative or viable in the target cell.
  • the vector may include a regulatory element such as a promoter, a nucleic acid or nucleic acid fragment (i.e., a target transgene or a gene encoding a nuclease) according to the present invention, and a terminator; said regulatory element, nucleic acid or nucleic acid fragment and terminator being operatively linked.
  • a regulatory element such as a promoter, a nucleic acid or nucleic acid fragment (i.e., a target transgene or a gene encoding a nuclease) according to the present invention, and a terminator; said regulatory element, nucleic acid or nucleic acid fragment and terminator being operatively linked.
  • promoter is meant a nucleic acid sequence sufficient to direct transcription of an operatively linked nucleic acid sequence.
  • operatively linked is meant that the nucleic acid(s) and a regulatory sequence, such as a promoter, are linked in such a way as to permit expression of said nucleic acid under appropriate conditions, for example when appropriate molecules such as transcriptional activator proteins are bound to the regulatory sequence.
  • a regulatory sequence such as a promoter
  • an operatively linked promoter is upstream of the associated nucleic acid.
  • upstream is meant in the 3’->5’ direction along the nucleic acid.
  • the promoter and terminator may be of any suitable type and may be endogenous to the target cell or may be exogenous provided that they are functional in the target cell.
  • the promoter used in the constructs and methods of the present invention may be a constitutive, tissue specific, or inducible promoter.
  • the promoter may be a constitutive cauliflower mosaic virus (CaMV35S) promoter for expression in many plant tissues, an inducible ‘photosynthetic promoter’ (e.g. ribulose 1 ,5-bisphosphate), capable of mediating expression of a gene in photosynthetic tissue in plants under light conditions, or a tissue specific promoter such as a seed specific promoter, for example from a Brassica napus napin gene.
  • CaMV35S constitutive cauliflower mosaic virus
  • an inducible ‘photosynthetic promoter’ e.g. ribulose 1 ,5-bisphosphate
  • a tissue specific promoter such as a seed specific promoter, for example from a Brassica napus napin gene.
  • the terminator may be from the same gene as the promoter sequence or a different gene, such as Arabidopsis thaliana Ubiquitin-10 (AtUBI-10).
  • the genetic construct in addition to the promoter, the transgene and/or the gene encoding a nuclease and the terminator, may include further elements necessary for expression of the nucleic acid, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase ⁇ nptll) gene, the hygromycin phosphotransferase (hpf) gene, the phosphinothricin acetyltransferase bar or pat) gene], and reporter genes (such as betaglucuronidase (GUS) gene (gus t)].
  • the genetic construct may also contain a ribosome binding site for translation initiation.
  • the genetic construct may also include appropriate sequences for amplifying expression.
  • the selecting a nuclease step of step (a) includes: a. generating an axenic in vitro seedling from a seed of the plant; b. isolating a protoplast from a part of the seedling; c. transforming the protoplast with a genetic construct, wherein the genetic construct includes a gene encoding the nuclease; d. quantifying cleavage efficacy of the nuclease from the frequency of insertion deletions (INDELs) at an expected cleavage site for the target gene in the plant.
  • INDELs frequency of insertion deletions
  • the cleavage efficacy of the nuclease of step (a) is quantified.
  • the cleavage efficacy of the nuclease may be quantified nuclease from the frequency of insertion deletions (INDELs) at an expected cleavage site for the target gene in the plant.
  • INDELs frequency of insertion deletions
  • the cleavage efficacy is ascertained prior to use in the method of the invention.
  • the nuclease is selected based on the cleavage efficacy.
  • the step of quantifying cleavage efficacy may include amplifying a nucleotide fragment corresponding to the target gene to contain paired end sequences and, optionally, a sequence barcode index.
  • protoplast as used herein is meant a plant cell which is lacking a cell wall.
  • the term includes, for example, plant cells in which the cell wall has been substantially removed, for example enzymatically degraded or digested.
  • transforming the protoplast is meant transferring nucleic acid into the protoplast. Methods of transforming protoplasts are known to those skilled in the art, such using polyethylene glycol or electroporation. In a preferred embodiment, the transformation may be a polyethylene glycol mediated transformation.
  • the present invention provides a method for generating offspring of a genetically modified safflower plant, said method including inter-crossing (for example by controlled crossing) the genetically modified plant with another safflower plant.
  • a method of precision breeding including: a) obtaining a genetically modified plant according to the method described above; and b) inter-crossing the genetically modified plant to establish offspring having a combination of traits, wherein at least one trait corresponds to the target gene or transgene.
  • the offspring may be characterized by improved agronomic performance with respect to a parent plant.
  • agronomic performance as used herein is meant the interaction of a plant with its environment.
  • improvements in agronomic performance include, for example, reduced water and/or fertilizer requirements, increased crop yield, and/or improved nutritional value in a crop.
  • the other plant may be of any suitable type.
  • the other plant may be a similar genetically modified plant, a different genetically modified plant, or a native plant.
  • the other plant is of the genus Carthamus. More preferably the other plant is a safflower plant or Carthamus tinctorium.
  • the offspring are fertile.
  • the step of inter-crossing the genetically modified plant to establish offspring having a combination of traits, wherein at least one trait corresponds to the target gene includes controlled crossing of the genetically modified plant with another plant.
  • the controlled crossing of the genetically modified plant may include one or more of: a. accelerating the seed-to-seed cycle of the plant, and b. selectively emasculating the plant.
  • the controlled crossing further includes selectively pollinating the emasculated plant.
  • seed-to-seed cycle as used herein is meant the time between sowing a T1 generation seed and harvesting a T2 generation seed.
  • the step of accelerating the seed-to- seed cycle may be achieved by growing the plants, preferably in a greenhouse, at a desired temperature and/or with a desired photoperiod.
  • the temperature may be between approximately 16 to 28°C, especially 18 to 26°C, more especially 20 to 24°C, for example at approximately 22°C to 24°C.
  • the photoperiod may be set as a 6 to 16 hr photoperiod, especially an 8 to 16 hr photoperiod, more especially a 12 to 16 hr photoperiod, for example, an approximately 14 hr.
  • emasculating in the context of a plant is meant removal of a pollen-producing part of the plant such that self-pollination is substantially prohibited.
  • selectively emasculating the plant may include applying an effective amount of a growth regulator, preferably gibberellic acid, to the plant, preferably to a recipient flower head, for example a capitula.
  • a growth regulator preferably gibberellic acid
  • an effective amount as used herein is meant an amount sufficient to result in substantial, including entire, emasculation of the plant. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration, and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration.
  • the present invention provides safflower plant cells, including protoplasts, plant seeds, plants and plant parts, produced by the methods of the present invention.
  • the methods provided herein may collectively provide a platform to rapidly develop safflower plants, such as safflower plant varieties, with tailored traits including novel traits derived from transgenesis and gene editing approaches.
  • the methods of the present invention may be used individually or in combination.
  • Figure 1 shows a representative binary vector for Agrobacterium-mediated transformation containing zinc finger nuclease expression cassettes targeting an endogenous gene in the safflower genome and the hygromycin phosphotransferase (hpt) gene as a selectable marker for transformation.
  • the zinc finger nuclease expression cassettes are regulated by the Arabidopsis thaliana Ubiquitin-10 (AtUBI-10) promoter and terminator, while hpt gene is regulated by the Cassava vein mosaic virus (CsVMV) promoter and Cauliflower Mosaic Virus derived 35S terminator.
  • AtUBI-10 Arabidopsis thaliana Ubiquitin-10
  • CsVMV Cassava vein mosaic virus
  • Figure 2 shows Agrobacterium-mediated transformation of safflower using ex vitro rooting.
  • Figure 3 shows representative vector maps with left (pAVSSAF049) and right (pAVSSAF050) zinc finger nuclease expression cassettes that target an endogenous gene in safflower.
  • ZFA zinc-finger array
  • Ath Arabidopsis thaliana'
  • Cti Carthamus tinctorius'
  • Zma Zea mays
  • Fokl_XGC extra-GC Fokl nuclease domain
  • ELD and KKR are amino acid sequence variants that are obligate heterodimers with each other
  • Ubi10 is the ubiquitin 10
  • -p indicates a promoter
  • -t indicates a terminator
  • Bia is the ampicillin resistance marker gene
  • attL# sites are Gateway recombination sequences for cassette transfer to an alternative vector backbone.
  • Figure 4 shows a schematic safflower seed-to-seed lifecycle using rapid generational advance.
  • Figure 5 shows chemical emasculation of safflower for efficient controlled crossing. A) nonemasculated flower with pollen grains, and B) emasculated flower without polled grains.
  • methods for manipulating safflower plants for example accelerating precision breeding in safflower plants may be achieved by transgenesis with ex vitro rooting, transient protoplast assays for nuclease efficacy testing, and accelerated generational advance and chemical emasculation. As shown, these methods collectively provide a platform to rapidly develop safflower plant varieties with tailored traits including novel traits derived from transgenesis and gene editing approaches.
  • the following example demonstrates a method for generating fertile safflower plants via transgenesis.
  • the approach combines methods for Agrobacterium-mediated transformation with the recovery of events by ex vitro rooting.
  • a binary T-DNA vector is constructed using standard molecular biology recombination techniques to contain a recombinant transgene cassette encoding a gene of interest, which could include, for example, an engineered nuclease.
  • engineered nuclease may include a Transcription Activator- Like Effector Nuclease (TALEN) (Zhang et al. 2013), a Zinc Finger Nuclease (ZFN) (Shukla et al. 2009), or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) (Mali et al. 2013) to facilitate targeted genome editing at an endogenous gene within the safflower genome.
  • TALEN Transcription Activator- Like Effector Nuclease
  • ZFN Zinc Finger Nuclease
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • T-DNA vector Also included in the T-DNA vector is a transformation selectable marker such as the hygromycin phosphotransferase (hpt) gene.
  • hpt hygromycin phosphotransferase
  • the binary vector is DNA sequence characterised to confirm the gene sequences are in-frame and intact prior to being transferred to a disarmed Agrobacterium strain EHA105 via electroporation.
  • a representative binary T-DNA vector is shown in Figure 1.
  • Seeds (10 g) from commercial safflower cultivar S317 are surface sterilised in a 250 mL container containing freshly prepared 100 mL 0.01 % w/v silver nitrate and 0.01 % v/v TWEEN® 20 surfactant for 6 minutes with gentle agitation followed by four rinses in sterile distilled water. The seeds are transferred to sterile filter paper to remove excess moisture and heat treated in an incubator at 50°C for 3 hours.
  • Sterilised seeds are placed on seed germination medium (0.5x MS basal salts, 10 g/L sucrose, 8 g/L agar, pH 5.7) and incubated at 22°C with a 16 hour photoperiod and a photon flux density of 60 pmol/m 2 /sec from fluorescent lights for three to five weeks to produce axenic in vitro safflower seedlings.
  • seed germination medium 0.5x MS basal salts, 10 g/L sucrose, 8 g/L agar, pH 5.7
  • Transgenic TO shoots are generated using the Agrobacterium-mediated transformation method of Belide et al. (2011) with minor modifications.
  • cotyledon explants from in vitro seedlings are excised, inoculated, and co-cultured with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pDPI001849.
  • Callus initiation and shoot regeneration on selective media containing hygromycin is stimulated in MS media supplemented with plant growth regulators such as 1 -naphthalene-acetic-acid (NAA) and 6-benzyl-amino-purine (BAP) as reported by Belide et al. (2011).
  • NAA 1 -naphthalene-acetic-acid
  • BAP 6-benzyl-amino-purine
  • Fertile TO plants are established in the greenhouse by deflasking and ex vitro rooting. This is achieved by transferring well-developed microshoots to hydrated sphagnum peat moss or coco pellets (such as JIFFY-7® pellets) and enclosing them in a covered conventional seedling tray to retain humidity for 3-4 weeks in a growth chamber programmed at 22°C with a 12 hour photoperiod and a photon flux density of 150-200 pmol/m 2 /sec from fluorescent lamps. This allows for both root formation and acclimatisation. TO plants that survive and form roots are transplanted to conventional potting media and grown at 22-24°C under greenhouse conditions supplemented with conventional high pressure sodium lamps to provide a 14 hour photoperiod. Under these conditions, the TO plants flower and produce T 1 seed that can be harvested within 4-5 months. The transformation process is shown in Figure 2.
  • Leaf tissue harvested from putatively transgenic fertile TO plants is subjected to molecular analysis to confirm the presence and copy number of the transgene.
  • primers and fluorescent probes for quantitative PCR could be designed to detect the presence of T- DNA elements from the hygromycin selectable marker and Arabidopsis thaliana Ubiquitin-10 (AtUBI-10) promoter. These primers and probes are used in combination with primers and probes designed to detect an endogenous gene of known copy number in the safflower genome.
  • the detection of both elements within a TO event would confirm the presence of both transgene cassettes and the probable likelihood of a complete T-DNA integration prior to further DNA sequence validation. Comparison of the fluorescent signals generated by the probes detecting the transgenes and endogenous gene would enable the copy number of the transgenes to be determined. Molecular analysis is performed first on the putative TO event and then on selfed T 1 plants derived from the fertile TO plant grown in the glasshouse. The latter analysis is used to confirm the Mendelian inheritance of the transgenes.
  • Polyethylene glycol (PEG) mediated transfection of safflower leaf mesophyll protoplasts is used to assess the efficacy of engineered nucleases.
  • the preparation and transfection of leaf mesophyll protoplasts from seedlings of safflower is performed as follows:
  • Leaves are excised from three- to five-week-old axenic in vitro seedlings germinated from surface-sterilised seeds as described above (see Example 1 , ‘Plant transformation’).
  • Using a scalpel blade about 1 g of leaves are sliced to 2-3 mm transverse strips and transferred to a PETRITM dish containing 30 mL of enzyme digestion solution (1% w/v cellulase 'Onozuka' R10 (Yakult, Tokyo, Japan), 0.25% w/v macerozyme 'Onozuka' R10 (Yakult, Tokyo, Japan), 0.4 M mannitol, 10 mM CaCh, 20 mM KOI, 0.1% w/v BSA, 20 mM MES, pH 5.7).
  • the enzymatic digestion is incubated in the dark at 24°C overnight (15-18 hours). After incubation, the protoplasts are released by gentle agitation on an orbital shaker at 40 rpm for 30 minutes. The resulting protoplast suspension are split into 15 mL aliguots and gently pipetted through a sterile 100 pm nylon mesh filter placed in a 50 mL collection tube. Each protoplast suspension aliguot is washed with 30 mL of prechilled modified W5 wash buffer (154 mM NaCI, 125 mM CaCh, 5 mM KCI, 5 mM glucose, 2 mM MES, pH 5.7).
  • prechilled modified W5 wash buffer 154 mM NaCI, 125 mM CaCh, 5 mM KCI, 5 mM glucose, 2 mM MES, pH 5.7.
  • the collection tubes containing the protoplast suspension are centrifuged (70 g, 5 min) and the protoplast pellets are gently resuspended in 25 mL W5 wash buffer. Centrifugation and washes are conducted three times. The protoplasts are suspended in 25 mL W5 wash buffer pellets and incubated on ice for 30 min. The yield and viability of the mesophyll protoplasts is estimated using a COUNTESS II FL AUTOMATIC CELL COUNTER instrument (Thermofisher Scientific) according to the manufacturer’s instructions.
  • the protoplasts Prior to transfection, the protoplasts are centrifuged (70 g, 5 min) and resuspended in 0.2 mL MMG solution (0.4 M mannitol, 15 mM MgCl2, 4 mM MES, pH 5.7) to a final concentration of 2-5 x 10 6 cells/mL in 12 mL round bottom tubes.
  • MMG solution 0.4 M mannitol, 15 mM MgCl2, 4 mM MES, pH 5.7
  • the protoplasts are transfected using the method described by Yoo et al. (2007) with modifications.
  • the resuspended protoplasts are mixed with 15 pL of 1 pg/pl of plasmid DNA (treatment sample) or water (control reactions) at room temperature.
  • the plasmid DNA consists of the paired vectors for expressing a zinc finger nuclease (see Figure 3) and a vector for constitutively expressing green fluorescent protein (35S-p::GFP::nos-t).
  • the replicate reactions for each engineered nuclease design are pooled and used for molecular analysis to assess the cleavage efficacy at the targeted gene in the safflower genome.
  • next generation Illumina DNA sequencing could be used to assess the cleavage efficacy at the targeted gene as follows:
  • genomic DNA is extracted from the pooled protoplasts reactions using SPRI paramagnetic bead technology (Agencourt DNAdvance genomic DNA Isolation Kit, Beckman Coulter) with a modified manufacturer’s protocol.
  • the genomic DNA is quantified using a Nanodrop 2000 spectrophotometer (Thermofisher Scientific) and diluted to 10 ng/pL.
  • PCR primers designed to amplify fragments spanning the cleavage sites of each engineered nuclease designs for each target gene are used to assess the efficacy of each engineered nuclease.
  • the forward and reverse primers for each target gene contain at their 5’ ends the Illumina SP1 and SP2 sequences, respectively, which are required to sequence PCR amplicons on the Illumina DNA sequencing instrument.
  • the resultant products are purified using Ampure Magnetic beads (Beckman- Coulter) with a DNA-to-bead ratio of 0.8:1.
  • an additional round of PCR is performed to introduce the Illumina P5 and P7 sequences onto the amplified DNA fragments, as well as a sequence barcode index that could be used to unequivocally attribute sequence reads to the sample from which they originated.
  • primers that are in part complementary to the SP1 and SP2 sequences added in the first round of amplification, and which also contained the sample index and P5 and P7 sequences.
  • the resultant products are purified using Ampure magnetic beads (Beckman-Coulter) with a DNA-to-bead ratio of 0.7:1.
  • the purified PCR fragments are titrated using a PCR-based library quantification kit (KAPA) according to the manufacturer’s instructions and sequenced on an Illumina MiSeq instrument (Illumina) to generate paired end sequence reads according to the manufacturer’s instructions.
  • KAPA PCR-based library quantification kit
  • Bioinformatic analysis is used to quantitate the frequency of insertion-deletions (INDELs) at the expected cleavage sites for each of the engineered nuclease designs in the targeted genes.
  • INDELs insertion-deletions
  • Such INDELs are known to be indicators of in planta zinc finger nuclease activity resulting from non-homologous end joining (NHEJ) DNA repair.
  • NHEJ non-homologous end joining
  • the transient protoplast assay is used to determine the efficacy of zinc finger nuclease designs targeting endogenous genes in the safflower genome.
  • General greenhouse nursery conditions consisting of 22-24°C temperature supplemented with conventional high pressure sodium lamps to provide a 14 hour photoperiod were shown to reduce the seed-to-seed cycle of safflower to about 4 months duration, enabling up to three full lifecycles to be completed per annum (see Figure 4). Under these accelerated growth conditions, between 20 and 100 seeds can be harvested from individual plants across a range of safflower varieties and breeding lines.
  • the prospective plants are inspected, and the recipient capitula are bagged immediately when the first florets emerge from the opening capitulum, typically 3-4 weeks from entry into reproductive phase and from the first GA3 application.
  • Highest priority is directed to the primary capitula as these are typically the largest and have the best prospect for inter-crossing success and consequently high hybrid seed yield.
  • Pollination is performed at 2-3 days after bagging, when multiple florets have emerged from the top of the recipient capitulum and when suitable pollen donor capitula is available.
  • Donor capitula are selected with freshly dehisced pollen, bract extensions are trimmed with scissors and then gently brushed against the stigmas of fresh florets.
  • One donor capitulum can be used to pollinate 2 or 3 recipient capitula. Harvesting of seed from mature capitula is ready when they are fully dried and yellow-brown in colour, about 4-6 weeks post crossing. Once dry, the capitula are removed by cutting and threshed.
  • the chemical emasculation method was found to be effective for achieving male sterility across a range of safflower varieties and breeding lines, with all resulting seed confirmed by molecular analysis to derived from outcrossing.

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Abstract

La présente invention concerne des procédés de manipulation de plantes, plus particulièrement des procédés de génération de plantes génétiquement modifiées, notamment des plantes de carthame.
PCT/AU2023/050942 2022-09-29 2023-09-28 Procédés de manipulation de plantes WO2024065009A1 (fr)

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Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
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