WO2024065009A1 - Methods of plant manipulation - Google Patents

Abstract

The present invention relates to methods for manipulating plants, more particularly methods for generating genetically modified plants, especially safflower plants.

Description

METHODS OF PLANT MANIPULATION

Field of the Invention

The present invention relates to methods for manipulating plants, more particularly methods for manipulating target genes in safflower plants.

Background of the Invention

Methods for the stable transgenesis of plants have been reported using Agrobacterium- mediated transformation (Ying et al. 1992, Belide et al. 2011 , Dhumale et al. 2016). This approach relies on an in vitro regeneration system initiated by a competent explant such as cotyledons or young seedling leaves that are susceptible to infection by disarmed Agrobacterium tumefaciens. Using conventional techniques, regenerated shoots, such as safflower shoots, are recalcitrant to in vitro adventitious rooting and prone to low survival during deflasking, which hampers the recovery of fertile transgenic plants under greenhouse conditions (Belide et al. 2011). To address this limitation, methods for grafting transgenic shoots onto non-transgenic seedlings have been established with moderate success, achieving survival rates of about 50% (Belide et al. 2011).

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.

Most current gene editing techniques are reliant on the ability of an engineered nuclease such as, for example, Transcription Activator-Like Effector Nuclease (TALEN), Zinc Finger Nuclease (ZFN), or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to create a double stranded DNA break at a pre-determined position in the plant genome. Hence, the efficacy of the engineered nuclease is a key determinant of the efficiency of gene editing.

Breeding for new plant varieties having tailored traits, including novel traits derived from transgenesis and gene editing approaches, requires the ability to combine these traits while simultaneously selecting for a range of other breeding targets for superior agronomic performance. As speed for breeding progress is limited by the length of the seed-to-seed cycle, methods to reduce the lifecycle of a given plant are also important. In addition, such gene editing is difficult in some plants, such as safflower.

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. Although 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.

There exists a need to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.

Summary of the Invention

In a first aspect, 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. subjecting 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.

In another aspect, 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. subjecting 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.

In one embodiment, the methods are for accelerating precision breeding in the plant.

In another aspect of the invention, there is provided 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.

By ‘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.

By ‘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.

By 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. For example, 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. By ‘cleavage 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. By ‘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.

Preferably the genetically modified plant is fertile. Preferably the offspring are fertile. By ‘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.

By ‘genetically modified plant’ as used herein is meant a plant having a modified genome. For example, 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.

By ‘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.

In one embodiment of this aspect of the invention, 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.

For example, in some embodiments, expression of the target gene in the plant may be silenced. In other embodiments, a transgene may be introduced into the safflower plant that provides a trait not present in the safflower plant before transformation. In a preferred embodiment, 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.

In preferred embodiments, the methods include the further step of generating a genetically modified plant from the microshoot.

Preferably the genetically modified plant is fertile.

Methods of transforming seedlings are known to those skilled in the art. In a preferred embodiment, the step of transforming the seedling is an Agro bacterium-mediated transformation. In another embodiment, the Agrobacterium sp. includes a binary T-DNA vector. In a further embodiment, the binary T-DNA vector includes a recombinant transgene cassette and a transformation selectable marker gene. Preferably, 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.

In yet another embodiment of this aspect of the invention, the step of transforming the seedling includes: a. excising a cotyledon explant from the seedling; and b. subjecting the cotyledon to Agrobacterium-mediated transformation.

Preferably 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.

By ‘microshoot’ as used herein is meant a small shoot initiated during the establishment and multiplication stages of micropropagation. By ‘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.

By ‘growing material’ as used herein is meant a nutrient rich material that may support a microshoot during its early stages of growth and development. As such, a growing material may include, for example, hydrated sphagnum peat moss or coco pellets. In a preferred embodiment, the transformed microshoot may be retained in a light- and temperature- controlled growth chamber. For example, 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. For example, 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.

In another preferred embodiment, 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. Preferably, the TO plant may be retained in a greenhouse. More preferably, 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. More preferably, 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.

By ‘axenic’ as used in the context of a seedling is meant a seedling having a substantially identical genome to that of the parent plant.

The expression ‘part of a plant’ as used herein refers to a plant seed, stem, callus, leaf, root, shoot or the like. Preferably, the protoplast may be isolated from a leaf.

In a preferred embodiment of the methods of the invention, 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). Preferably, the nuclease is ZFN. By ‘isolating’ as used in the context of a protoplast is meant that the protoplast is removed from its original environment (e.g., a plant or plant material). Preferably, an isolated protoplast is substantially purified. By ‘substantially purified’ as used in the context of a protoplast is meant that the protoplast is free of other plant materials. Preferably, 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.

The term ‘genetic construct’ as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the target transgene or a gene encoding a nuclease. Preferably the genetic construct is a recombinant nucleic acid molecule. In general, 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. The term construct includes vectors but should not be seen as being limited thereto.

In a preferred embodiment of this aspect of the invention, the genetic construct is a 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. However, any other vector may be used as long as it is replicable or integrative or viable in the target cell.

In a preferred embodiment, 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.

By a ‘promoter’ is meant a nucleic acid sequence sufficient to direct transcription of an operatively linked nucleic acid sequence.

By ‘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. Preferably an operatively linked promoter is upstream of the associated nucleic acid.

By ‘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. For example, 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.

A variety of terminators which may be employed in the genetic constructs of the present invention are also well known to those skilled in the art. 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.

Those skilled in the art will appreciate that the various components of the genetic construct are operably linked so as to result in expression of said nucleic acid. Techniques for operably linking the components of the genetic construct of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites.

In some embodiments, 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.

In some embodiments, the cleavage efficacy of the nuclease of step (a) is quantified. For example, 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. In other embodiments, the cleavage efficacy is ascertained prior to use in the method of the invention. In preferred embodiments, the nuclease is selected based on the cleavage efficacy.

In another embodiment, 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.

By ‘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. By ‘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.

In another aspect, 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.

In this aspect, there is provided 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.

In particular embodiments, the offspring may be characterized by improved agronomic performance with respect to a parent plant. By ‘agronomic performance’ as used herein is meant the interaction of a plant with its environment. As such, 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. For example, the other plant may be a similar genetically modified plant, a different genetically modified plant, or a native plant. In preferred embodiments the other plant is of the genus Carthamus. More preferably the other plant is a safflower plant or Carthamus tinctorium.

Preferably the offspring are fertile.

Likewise, in a preferred embodiment, 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.

In preferred embodiments of the methods, 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.

Preferably, the controlled crossing further includes selectively pollinating the emasculated plant.

By ‘seed-to-seed cycle’ as used herein is meant the time between sowing a T1 generation seed and harvesting a T2 generation seed.

In a preferred embodiment of this aspect of the invention, 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. For example, 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. More preferably, 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.

By ‘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.

In a preferred embodiment, 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.

By ‘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.

In a further aspect, 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.

In this specification, the term ‘comprises’ and its variants are not intended to exclude the presence of other integers, components or steps.

In this specification, reference to any prior art in the specification is not and should not be taken as an acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably expected to be combined by a person skilled in the art.

The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

Brief Description of the Drawings/Figures

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.

Figure 2 shows Agrobacterium-mediated transformation of safflower using ex vitro rooting. A) In vitro seedlings established for cotyledon explants for inoculation with Agrobacterium', B) Shoot regeneration from cotyledon explant; C) Selection of putative transgenic shoots resistant to hygromycin; D) Transgenic shoots expressing the fluorescent reporter gene dsRED; E) Transgenic leaf with dsRED expression under brightfield and fluorescence; F) Ex vitro rooted regenerants; G) Ex vitro rooted regenerants shown with adventitious roots growing out through Jiffy pellet; H) Flowering safflower plant recovered from 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.

Detailed Description of the Embodiments

In the following examples, it is demonstrated that 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.

Example 1 - Transgenesis with ex vitro Rooting

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.

Production of Binary T-DNA Vectors

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. Such 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. Also included in the T-DNA vector is a transformation selectable marker such as the hygromycin phosphotransferase (hpt) gene. 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.

Plant Transformation

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/m²/sec from fluorescent lights for three to five weeks to produce axenic in vitro safflower seedlings.

Transgenic TO shoots are generated using the Agrobacterium-mediated transformation method of Belide et al. (2011) with minor modifications. In brief, 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).

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/m²/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.

Initial experiments performed to optimise the ex vitro rooting step using non-transgenic microshoots from safflower cultivar S317 found that fertile plants could be recovered at a frequency ranging from 30 to 70%, with an average of 56%. Initial experiments performed to assess the efficiency of transformation showed that transgenic TO events expressing the fluorescent reporter gene dsRED could be recovered at a frequency of about a 5%. Hence, the overall efficiency of the method for transformation of safflower using ex vitro rooting is expected to be about 2.5%; i.e., 5% of the average frequency or 0.05x56%.

Molecular Analysis and Characterisation of Transgenic Plants

Leaf tissue harvested from putatively transgenic fertile TO plants is subjected to molecular analysis to confirm the presence and copy number of the transgene. For example, to determine the copy number of the transgenes in the T-DNA vector shown in Figure 1 , 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.

Example 2 - Transient Protoplast Assays for Engineered Nuclease Efficacy Testing

Here, a method to quantify in planta the efficacy of engineered nucleases in safflower using a transient protoplast assay is described. Assessing Zinc Finger Nuclease Efficacy using Transient Protoplast Assays

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 PETRI™ 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). 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. 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 10⁶ cells/mL in 12 mL round bottom tubes.

The protoplasts are transfected using the method described by Yoo et al. (2007) with modifications. In brief, the resuspended protoplasts are mixed with 15 pL of 1 pg/pl of plasmid DNA (treatment sample) or water (control reactions) at room temperature. For example, in the case of testing the efficacy of a zinc finger nuclease, 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). An egual volume of a freshly prepared PEG solution (40% w/v PEG MW 4000, 0.1 M CaCI2, 0.2 M mannitol) is added and the mixture is incubated at room temperature for 5 min. After incubation, 3 mL of W5 solution is added slowly, followed by gentle mixing and pelleting of the protoplasts by centrifugation (100 g, 1 min). The protoplasts are washed twice using 25 mL W5 wash buffer. The protoplasts are gently resuspended in 1 mL of W5 and incubated for 2 days at 22°C with a 16 hour photoperiod and a photon flux density of 60 pmol/m2/sec from fluorescent lamps. Three protoplast transfection reactions are performed for each zinc finger nuclease paired design. Each reaction is assessed for protoplast viability and the presence of GFP transfected protoplast cells using a fluorescence microscope. An estimate of transfection efficiency is determined using a Countless II FL Automatic cell counter instrument (Thermofisher Scientific) according to manufacturer’s instructions.

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. For example, next generation Illumina DNA sequencing could be used to assess the cleavage efficacy at the targeted gene as follows:

In brief, 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. Following PCR amplification from genomic DNA, the resultant products are purified using Ampure Magnetic beads (Beckman- Coulter) with a DNA-to-bead ratio of 0.8:1. To enable the amplification products to be sequenced using Illumina short read technology, 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. This is achieved using 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. Following amplification, 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. 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. Such INDELs are known to be indicators of in planta zinc finger nuclease activity resulting from non-homologous end joining (NHEJ) DNA repair. The efficacy of each engineered nuclease is calculated as the ratio of INDELs observed at the expected cleavage site in the treatment and control samples.

The transient protoplast assay is used to determine the efficacy of zinc finger nuclease designs targeting endogenous genes in the safflower genome.

Example 3 - Accelerated Generational Advance and Chemical Emasculation

In this example, a novel approach for accelerated precision breeding in safflower that both simplifies inter-crossing and reduces the length of the seed-to-seed cycle is demonstrated. The approach is based on rapid generational advance and chemical emasculation to facilitate controlled crossing.

Rapid Generational Advance

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.

Chemical Emasculation to Facilitate Controlled Crossing

The application by spray of the plant growth regulator GA3 to safflower plants growing under field conditions was reported to effectively reduce the viability of pollen at the pre-meiotic interphase stage such that the progeny derived are mostly from cross-pollination (Baydar and Gdkmen 2003). An adaptation of this method is used for chemical emasculation of safflower plants growing under controlled environment conditions such as a glasshouse. Pollen recipient plants are inspected every 2-3 days and selected for emasculation when primordial secondary inflorescences become visible and are less than 0.5 cm in diameter. A solution of GA3 (100 mg/L) is hand sprayed to cover the capitula but avoiding run-off. The capitula are allowed to air dry, and the GA3 spray application is repeated. A total of three GA3 applications at weekly intervals are performed to achieve male sterility (see Figure 5).

For the selection of recipient capitula and pollination plants, 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.

Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

References

Baydar H, Gdkmen OY (2003) Hybrid seed production in safflower (Carthamus tinctorius) following the induction of male sterility by gibberellic acid. Plant Breeding 122, 459.

Belide S, Hac L, Singh SP, Green AG, Wood CC (2011) Agrobacterium-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting. BMC Plant Methods 7, 12.

Benmahioul B, Dorion N, Kaid-Harche M, Daguin F (2012) Micropropagation and ex vitro rooting of pistachio (Pistacia vera L.). Plant Cell Tissue and Organ Culture 108, 353.

Dhumale DR, Shingote PR, Dudhare MS, Jadhav PV, Kale PB (2016) Parameters influencing Agrobacterium-mediated transformation system in safflower genotypes AKS-207 and PKV Pink. 3 Biotech 3, 181.

Mali P, Yang L, Esvelt KM, Aach J, Guell M, Dicarlo JE, Norville JE, Church GM (2013) RNA- guided human genome engineering via cas9. Science 339, 6121.

Murphy R, Adelberg J (2021) Physical factors increased quantity and quality of micropropagated shoots of Cannabis sativa L. in a repeated harvest system with ex vitro rooting. In Vitro Cellular & Developmental Biology - Plant 57, 923.

Ranaweeraa KK, Gunasekarab MTK, Eeswarac JP (2013) Ex vitro rooting: A low cost micropropagation technique for Tea (Camellia sinensis (L.) O. Kuntz) hybrids. Scientia Horticulturae 155, 8.

Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, Rock JM, Wu Y-Y, Katibah GE, Zhifang G, McCaskill D, Simpson MA, Blakeslee B, Greenwait SA, Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, llrnov FD (2009) Precise genome modification in the crop species Zea mays using zinc- finger nucleases. Nature 459, 437.

Ying M, Dyer WE, Bergman JW (1992). Agrobacterium tumefaciens- mediated transformation of safflower (Carthamus tinctorius L.) Cv. ‘Centennial’. Plant Cell Reports 11 , 581. Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols 2, 1565.

Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ, Voytas DF (2013) Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiology 161 , 20-27.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1 . A method of 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. subjecting 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.

2. The method according to claim 1 , further including supporting the growth of the TO genetically modified safflower plant such that the TO genetically modified safflower plant flowers and produces T 1 seeds.

3. The method according to either one of claim 1 or claim 2, wherein the gene encoding the nuclease or the transgene is introduced into the safflower plant material by: a. generating an axenic in vitro seedling from a seed of the plant; and b. transforming the seedling with a genetic construct, wherein the genetic construct includes the transgene or the gene encoding the nuclease.

4. The method according to claim 3, wherein the genetic construct is a vector.

5. The method according to any one of claims 1 to 4, wherein the nuclease is 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).

6. The method according to claim 5 wherein the engineered nuclease is ZFN.

7. The method according to either one of claim 3 or claim 4, wherein the step of transforming the seedling is an Agrobacterium-mediated transformation that includes: a. excising a cotyledon explant from the seedling; and b. inoculating and co-culturing the cotyledon with an Agrobacterium sp.

8. The method of any one of claims 1 to 7, wherein the growing material is hydrated growing material.

9. The method according to any one of claims 1 to 8, wherein the method includes step (a) or step (b).

10. The method according to any one of claims 1 to 9, wherein the genetically modified plant is a fertile genetically modified plant.

11. The method according to any one of claims 1 to 10, wherein the gene encoding the nuclease is selected by: a. generating an axenic in vitro seedling from a seed of the plant; b. isolating a protoplast from a part of the seedling; and c. transforming the protoplast with a genetic construct; wherein the genetic construct includes a gene encoding the nuclease or the transgene; and 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.

12. A method of accelerated precision breeding including: a) obtaining a genetically modified plant according to the method of any one of claims 1 to 11 ; 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.

13. The method of claim 12, wherein the offspring are characterized by improved agronomic performance with respect to a parent plant.

14. The method according to either one of claim 12 or claim 13, wherein the step of intercrossing the genetically modified plant is characterized by controlled crossing of the genetically modified plant with another plant.

15. The method according to claim 14, wherein controlled crossing of the genetically modified plant includes accelerating the seed-to-seed cycle of the plant and/or selectively emasculating the plant.

16. the method according to claim 15, wherein accelerating the seed-to-seed cycle includes growing the plant in a greenhouse having a temperature between approximately 22°C and

24°C and a photoperiod of approximately 14 hr.

17. The method according to claim 15, wherein selectively emasculating the plant includes applying an effective amount of a growth regulator to a recipient flower head or capitula.

18. The method according to claim 17, wherein the growth regulator is gibberellic acid.

19. The method according to either one of claim 17 or claim 18, wherein the controlled crossing further includes selectively pollinating the emasculated plant.

20. A safflower protoplast, plant cell, plant seed, plant part, or plant, produced by a method according to any one of claims 1 to 19.