Patent Application
20170183672
The present invention relates to processes for the transformation of plant tissues with a genetic construct which comprises a transgene and a selection gene. The selection gene preferably encodes an auxin biosynthetic polypeptide, thus allowing for selection of transformed plants on media lacking plant auxins. The invention particularly relates to processes wherein the selection step is carried out under a light/dark cycle.
The use of genetically modified (GM) food crops in agriculture is rapidly increasing with an approximate £14 billion world market in 2005. The production of transgenic plants is, however, a long process which may take a number of weeks. Any steps which speed up this process will therefore be particularly advantageous.
Currently, the standard method used for nuclear transformation of plants such as maize is the method developed by Iowa State University (Frame B et al., In Vitro Cell. Dev. Biol-Plant 36:21-29). In the Iowa State protocol, a plasmid containing a selectable marker and a screenable marker or gene of interest are introduced into immature zygotic embryos or type II callus by particle bombardment. The bombarded tissue (either embryos or callus) is then selected for stable transformation events by transferring the tissue onto selection media which contains the synthetic plant auxin 2,4-D (typically 2 mg/L 2,4-D).
The prior art methods therefore rely on the use of the synthetic plant auxin 2,4-D in the selection media to regenerate the transformed plants and the plants must be kept in the dark for a prolonged period during the initial regeneration phase. This method therefore results in a time-consuming and lengthy process wherein transformed calli emerge after a minimum of 6-8 weeks post-bombardment.
There is therefore a commercial need for methods of regenerating transformed plants, embryos and callus that is quicker and therefore cheaper than the methods currently used.
To circumvent this problem, the invention is based on a selection and regeneration system for plastid transformation based on the use of a hormone-based auxin selection system which allows for the initial selection of putative transformed plant cells, including maize cells, in the dark before transferring to the light.
The Applicant has discovered a new method of transforming plant embryos and callus using selection media that does not contain the synthetic plant auxin 2,4-D and wherein the plant embryos/callus undergo a light/dark cycle during the regeneration phase.
The Applicant's method allows the production of transformed callus at about 3 weeks post-bombardment of embryos or about 4.5 weeks post-bombardment of callus. Transformed embryos and callus can therefore be produced within a much shorter time frame than the currently used methods. The method of the invention is thus quicker for regenerating whole transformed plants, transformed plant embryos and transformed callus.
In one embodiment, the invention provides a process for producing a transformed plant tissue, the process comprising the steps:
Preferably, the selection gene encodes an auxin biosynthetic polypeptide.
In some preferred embodiments, the process comprises:
In other preferred embodiments, the process comprises:
In yet other preferred embodiments, the process comprises:
Preferably, the transforming is carried out using a biolistic transformation method.
In a preferred embodiment, the invention provides a process for producing somatic plant embryos, the process comprising the steps:
Preferably, the plant is maize.
In a further preferred embodiment, the invention provides a process for producing a transformed plant, the process comprising the steps:
Preferably, the plant is maize.
The method of the invention is suitable for all plants that can be transformed and regenerated, and for which auxin is essential for plant regeneration.
The plant may be a monocot or dicot.
Examples of 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. Preferably, the plant is tobacco or lettuce.
In some embodiments, the plant is rice, soybean, canola, cotton, potato, tomato, carrot, lettuce, cauliflower, cabbage and tobacco.
In other embodiments, the plant is carrot, rice lettuce, cabbage, potato, tomato, oilseed rape, maize, wheat, oats, rye, sugar beet, cotton, sorghum or sugarcane.
Preferably, the plant is maize.
Plant embryos are parts of seeds which contain precursor tissues that eventually develop into leaves, stems and roots, as well as one or more cotyledons.
The plant tissues 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 part. Preferably, the tissues are present in leaves that are removed from intact plants. It is preferable to use actively-growing leaves.
In some embodiments, the plant tissue is a plant embryo or plant callus.
In a preferred embodiment, the genetic construct is targeted to plastids within the plant tissue.
For example, homologous recombination elements may be used which are capable of directing the integration of the genetic construct, or a part thereof, into the genome of at least one plastid which is present in the plant tissue. The homologous recombination elements may, for example, flank the transgene and/or selection gene.
The term “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.
Examples of plastids include chloroplasts, leucoplasts, amyloplasts, etioplasts, chromoplasts, elaioplasts and gerontoplasts. Preferably, the plastid is a green plastid, most preferably a chloroplast.
The genetic construct comprises a transgene and a selection gene.
As used herein, the term “genetic construct” refers to a nucleic acid molecule comprising the specified elements. 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, additional selection elements, and multiple cloning sites. Generally, the genetic construct will be a double-stranded nucleic acid molecule, preferably a dsDNA molecule.
In the context of the present invention, the term “transgene” is used to refer to a nucleic acid molecule which is being introduced into the genome of the plant. 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.
Examples of transgenes include those coding for antibodies, antibiotics, herbicides, vaccine antigens, enzymes, enzyme inhibitors and design peptides.
Single or multiple antigens may be produced from viridae, bacteria, fungi or other pathogens. The antigens may be expressed as single units or as multiple units of several antigens, e.g. for broad-spectrum vaccine development.
Enzymes may be produced for use in cosmetics (e.g. superoxide dismutase, peroxidase, etc.). Enzymes may also be produced for use in detergent compositions.
The invention particularly relates to the production of proteins/enzymes with specific activities, for example, immunostimulants to boost immune responses, such as interferons; and growth factors, e.g. transforming growth factor-beta (TGF-beta), bone morphogenic protein (BMP), neurotrophins (NGF, BDNF, NT3), fibroblast growth factor (FGF), proteolytic enzymes (papain, bromelain), and food supplement enzymes (protease, lipase, amylase, cellulase).
The invention also relates to the production or overexpression of proteins/enzymes in plant tissues that make the plants more resistant to biotic and abiotic stress, such as salts and metals. Examples of this include the generation of transplastomic plants that chelate iron (Fe) for mopping up excess metal in agriculturally important areas for future planting.
The invention further relates to the use of transgenes encoding polypeptides which modify fatty acid biosynthesis in plastids.
One or more transgenes may be inserted in the genetic construct. Preferably, the transgene sequences are contiguous.
The transgene sequence may additionally encode a protein purification tag fused to the polypeptide of interest. Examples of 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. Each of the transgene products may have a different affinity tag.
The selection gene is preferably one which encodes one or more plant auxin biosynthetic polypeptides. The expression of this transgene results in the production of auxin within the plant.
The auxin biosynthetic polypeptides may be any polypeptides which are involved in the synthesis of a plant auxin or other plant growth regulator, or which regulate the production or metabolism of a plant auxin or other plant growth regulator.
Preferably, there are nucleotide sequences encoding 1, 2, 3, or 4 auxin biosynthetic polypeptides. The nucleotide sequences encoding the auxin biosynthetic polypeptides may be present in an operon, with a single optional promoter and terminator element. Alternatively, the auxin biosynthetic polypeptides nucleotide sequences may each have their own promoters and terminator elements. A further option is that two or more of the nucleotide sequences encoding the auxin 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. a poly-glycine linker). In other embodiments, some of the nucleotide sequences encoding the auxin biosynthetic polypeptides 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 auxin biosynthetic polypeptides may be from any suitable source. Due to codon usage, bacterial genes are preferred, because nuclear genes may not be expressed to maximum levels in chloroplasts.
Preferably, the nucleotide sequence encoding the auxin biosynthetic polypeptides is from Agrobacterium tumefaciens or from a plant (e.g. from the plant which is being transformed).
In some preferred embodiments, the or a auxin biosynthetic polypeptides 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. Preferably, the iaaH and/or iaaM nucleotide sequences are from Agrobacterium tumefaciens.
In other embodiments, the auxin biosynthetic polypeptides are selected for the group consisting of AMI1, TAA1, TAR1, TIR2, YUC, AAO1, CYP79B2 and TDC.
The transgene and/or a selection gene may be flanked by homologous recombination elements that are capable of directing the integration of the transgene and/or a selection gene into the genome of the plant tissue.
Upon transformation of the genetic construct into the plant cells the plant tissue, the first and second homologous recombination elements recombine with corresponding sequences in the genome of the selected cells, resulting in the insertion of the transgene and/or a selection gene into the genome of the cells.
The homologous recombination elements may target the transgene and/or a selection gene to the plant nuclear genome, mitochondrial genome or plastid genome, preferably to the plastid genome.
The nucleotide sequences of the homologous recombination elements are selected such that the transgene and/or a selection gene is specifically targeted to one or more selected genomes. In particular, the nucleotide sequences of the homologous recombination elements may be selected such that no or essentially no transgenes and/or a selection genes become integrated into the nuclear genome of the plant or into the mitochondrial genome of the plant. In other words, the nucleotide sequences of the homologous recombination elements may be preferably plastid-specific, i.e. corresponding sequences might not present in the nuclear genome and preferably not present in the mitochondrial genome of the plant in question. This may be done by avoiding sequences which are present in the nuclear genome of the plant and optionally in the mitochondrial genome. 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.
Apart from the above, any sequences can be used from the 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. Preferably, the insertion sites are not in coding regions of genes.
The orientation of the sequences of the first and second homologous recombination elements should be the same as the orientation in the plant cell genome to allow for efficient homologous recombination.
In order to target the transgene and/or a selection gene to the plastid genome, 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.
In the context of the present invention, the term “substantially identical” means that the nucleotide sequences of the first and second homologous recombination sequences are independently more than 95%, preferably more than 98% or more than 99% and particularly preferably 100% identical to sequences which are present in the genome to be transformed. Percentage sequence identities may be determined using the Clustal method of alignment with default parameters, e.g. KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
Similarly, the 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, if targeting to the nuclear genome is to be avoided. In this context, the term “substantially identical” means that the nucleotide sequences of the first and second homologous recombination sequences are independently less than 50%, more preferably less than 70% or less than 90% identical to sequences which are present in the nuclear genome of the plant to be transformed. Percentage sequence identities may be determined using the Clustal method of alignment with default parameters, e.g. KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
Preferably, the lengths of 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 plant 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.
Preferably, the first homologous recombination sequence is nucleotides 68231-69454 of the Zea mays (accession no. NC_001666.2) chloroplast genome DNA; and/or preferably, the second homologous recombination sequence is nucleotides 69455-71184 of the Zea mays (accession no. NC_001666.2) chloroplast genome DNA.
In yet other embodiments, the first homologous recombination sequence is preferably nucleotides 123821-124699 of the Zea mays (accession no. NC_001666.2) choloroplast genome DNA; and/or the second homologous recombination sequence is nucleotides 124764-125784 of the Zea mays (accession no. NC_001666.2) choloroplast genome DNA.
Other preferred pairs of first and second homologous recombination sequences in Zea mays accession no. NC_001666.2 include the following:
(a) 94859-94930 and 95161-96651;
(b) 131265-133501 and 130495-130965;
(c) 13073-13146 and 12509-12579;
(d) 127807-127878 and 126086-127576;
(e) 89236-91472 and 91772-92242;
and
(f) 98038-98916 and 96953-97973.
Prior to the transformation step, the process optionally comprises:
The plant tissue (e.g. immature embryos) may be placed on callusing medium.
Callusing medium can be used to initiate plant cell differentiation. This helps to facilitate the transformation step.
The Callusing Medium may contain an auxin (e.g. 2,4-D).
One example of Callusing Medium is N6E (see Appendix 1).
The step of initiating cell differentiation of the plant tissue is preferably carried out in the dark.
The step of initiating cell differentiation of the plant tissue is preferably carried out for 2 days to 8 weeks.
In embodiments of the invention wherein the plant tissue is immature embryos, the step of initiating cell differentiation of the plant tissue is preferably carried out for 1-4 days, preferably 2-3 days.
In embodiments of the invention wherein the plant tissue is callus, the step of initiating cell differentiation of the plant tissue is preferably carried out for 5-9 weeks, preferably 6-8 weeks.
The step of initiating cell differentiation of the plant tissue is preferably carried out at 21-32° C., preferably at about 28° C.
The plant tissue may also be pre-cultured in Osmotic Medium prior to the transformation step. Osmotic medium is used to reduce turgor pressure in the plant cells.
An osmotic agent may be used (e.g. sorbitol and/or mannitol) to increase gene expression by reducing turgor pressure in cells. This increases the chance of cell survival by avoiding leakage following the shock wave created during bombardment (Rosillo, G., J. Acuña, A. Gaitan & M. Peña De. (2003). “Optimized DNA delivery into Coffea arabica suspension culture cells by particle bombardment”. Plant Cell Tiss. Org. Cult. 74: 45-49). In addition, it is thought that a high concentration of osmotic agents may also induce changes in cell membranes, leading to increased cell tolerance to biolistic delivery impact (Ingram, H. M., J. B. Power, K. C. Lowe & M. R. Davey. (1999). “Optimization of procedures for microprojectile bombardment of microspore-derived embryo in wheat”. Plant Cell Tiss. Org. Cult. 57: 207-210).
One example of Osmotic Medium is N6OSM (see Appendix 1).
The pre-cultured in Osmotic Medium step is preferably carried out in the dark or under reduced light conditions.
For plant embryos, the pre-cultured in Osmotic Medium step is preferably carried out for 2-6 hours, preferably about 4 hours.
For plant calli, the pre-cultured in Osmotic Medium step is preferably carried out for 4-26 hours, preferably about 24 hours.
The person skilled in the art will be aware of numerous methods for transforming plant cells with nucleic acid vectors. These 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 maize) liquid cultures.
The transformation method may target the plant nucleus or plastids.
Preferably, the plastids within the plant tissue are transformed. Any such suitable method may be used.
For targeting the genetic construct to plastids, biolistic transformation is preferred. This involves shooting nucleic acid vector-coated gold particles (micro-projectiles) into plastids of plant tissues, followed by selection of the transformed plastids and plant regeneration. Preferably, the plant tissue is immature embryos or callus.
In some embodiments of the invention, 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).
In some embodiments of the invention, the transformation step is followed by a recovery interval.
Preferably, the recovery interval is 12-60 hours, more preferably, 24-48 hours.
If immature embryos are being transformed, the recovery interval is preferably about 48 hours.
If callus is being transformed, the recovery interval is preferably about 24 hours.
The recovery step is preferably carried out in the dark or under low-light conditions.
Preferably, the plant tissue is maintained on Osmotic Medium after the transformation step.
In some embodiments of the invention, the plant tissue is placed on Callusing Medium prior to the selection step for 4-10 days, preferably for about 7 days.
Preferably, this step is carried out in the dark.
Embodiments of the invention which involve biolistic transformation, the particle bombardment uses helium under high pressure to deliver DNA coated gold micro-particles to target cells. This results in damage to target tissue inflicted by the high-pressure helium. The recovery period (continuing callus formation) post bombardment is thought to allow plant tissue time to recover from this damage and may result in a higher transformation efficiency.
In the selection step, transformed plant tissue is selected for on media which is lacking plant auxin using a light/dark cycle. This is significantly different from standard transformation protocols which require an auxin (e.g. 2,4-D) to initiate shoot development.
In the process of the invention, transformed plants express an auxin biosynthetic polypeptide. Hence the transformed plants of the invention do not need to be selected for on a medium which contains an auxin.
As used herein, the term “lacking auxin” is intended to mean that the selection medium does not contain sufficient auxin to enable the production of shoots and/or the regeneration of the plant. Hence the selection media may still contain trace amounts of auxin.
In some embodiments of the invention, the selection step is carried out in the absence of antibiotics.
In other embodiments of the invention, the selection step is carried out in the absence of spectinomycin.
In other embodiments of the invention, the selection step is carried out in the absence of bialaphos.
In particular, the selection medium is lacking any of the following:
In embodiments of the invention where the plant tissue are immature embryos, the plant tissue is preferably transferred to selection medium without auxin about 6-8 days, preferably about 7 days, after transformation.
Preferably, the immature embryos undergo a three-stage selection process:
Preferably, the first selection step is carried out in the dark or under reduced light conditions.
Preferably, the first selection step is carried out at 21-32° C., more preferably at about 28° C.
Preferably, the first selection step is carried out for 6-8, more preferably about 7 days.
Preferably, a second selection step takes place straight after the first selection step or within 1-2 days of the first selection step.
In the second selection step, the plant tissues are placed under continuous light for 2-4 days, preferably for about 3 days.
Preferably, a third selection step takes place straight after the second selection step or within 1-2 days of the second selection step.
In the third selection step, the plant tissues are placed under a light/dark cycle.
Preferably, out of a 24 hour cycle, the light is on for 14-18 hours, more preferably on for about 16 hours.
Preferably, out of a 24 hour cycle, the dark is for 6-10 hours, more preferably for about 8 hours.
Preferably, the third selection step is carried out for 4-8 days, preferably about 6 days.
In the case of transformed embryos, the light/dark cycle selection step is preferably completed less than 4 weeks, more preferably less than 3 weeks and most preferably less than 2 weeks after transformation.
In the case of transformed embryos, green calli are preferably produced less than 4 weeks, more preferably less than 3 weeks and most preferably less than 2 weeks after transformation.
In embodiments of the invention where the plant tissue are calli, the plant tissue is preferably transferred to selection medium without auxin about 12-26 hours, preferably about 24 hours, after transformation.
Preferably, the calli undergo a two-stage selection process:
Preferably, the first selection step is carried out in the dark or under reduced light conditions.
Preferably, the first selection step is carried out at 21-32° C., more preferably at about 28° C.
Preferably, the first selection step is carried out for 6-8 days, more preferably about 7 days.
The first selection step may also be carried out for 3-5 weeks, preferably for about 4 weeks.
Preferably, a second selection step takes place after the first selection step.
In the second selection step, the plant calli are placed under a light/dark cycle. Preferably, out of a 24 hour cycle, the light is on for 14-18 hours, more preferably on for about 16 hours.
Preferably, out of a 24 hour cycle, the dark is for 6-10 hours, more preferably for about 8 hours.
Preferably, the second selection step is carried out for 4-8 days, preferably about 6 days.
In the case of transformed calli, the light/dark cycle selection step is preferably completed less than 6 weeks, more preferably less than 5 weeks and most preferably less than 4 weeks after transformation.
In the case of transformed calli, green calli are preferably produced less than 6 weeks, more preferably less than 5 weeks and most preferably less than 4 weeks after transformation.
Preferably, all of the selection steps are carried out at 21-32° C., more preferably at about 28° C.
In some embodiments, the process comprises
The regeneration step is primarily used to initiate shoot formation.
Shoots are usually then transferred to root-inducing medium for root formation.
The regenerating step is preferably carried out for 2-15 weeks.
In embodiments of the invention where the plant tissue are immature embryos, the regenerating step is preferably 3-5 weeks, more preferably about 4 weeks.
In embodiments of the invention where the plant tissue are calli, the regenerating step is preferably 6-12 weeks, more preferably about 8 weeks.
In embodiments of the invention where the plant tissue are immature embryos, the regeneration step preferably starts with 2-4 days of continuous light, more preferably about 3 days continuous light.
The regenerating step is preferably carried out under a light/dark cycle.
Preferably, out of a 24 hour cycle, the light is on for 14-18 hours, more preferably on for about 16 hours.
Preferably, out of a 24 hour cycle, the dark is for 6-10 hours, more preferably for about 8 hours.
The light/dark cycle is preferably carried out for 2-8 days, more preferably for about 6 days.
The regenerating step is preferably carried out at 22-30° C., more preferably at about 25° C.
After the regeneration step, the transformed embryos are preferably maintained under a 16 hour light/8 hour dark cycle indefinitely. The temperature is preferably maintained at about 25° C.
The genetic construct may further comprise one or more promoters. It may also comprise one or more terminators.
The transgene and the selection gene may have the same or different promoters and the same or different terminators.
The promoter must be one that is operable in the selected plant cell or plastid. The promoter is one which is capable of initiating transcription of the transgene. It may also be necessary for it to be capable of initiating the transcription of the nucleotide sequence encoding an auxin biosynthetic polypeptide, in cases where the gene encoding the auxin biosynthetic polypeptide does not contain its own promoter. The promoter might, for example, be one derived from a plant or bacterial gene. Preferably, the promoter is plant specific.
Examples of suitable promoters include PpsbA, CIpP, RbcL and Prrn promoters.
Preferably, the promoter is a Prrn promoter (e.g. Plastidic ribosomal RNA (rrn) operon promoter (nt 59034-59303, accession Z00044 Nicotiana tabacum chloroplast genome DNA) or a Prrn promoter (nt 95161-96651, accession no. NC_001666.2 Zea mays chloroplast genome DNA).
In some embodiments, the promoter is an inducible promoter. This allows inducible, controlled expression of the selection gene(s). For example, 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. Alternatively, the promoter is a high-expression level promoter.
The terminator may be a plant terminator or a bacterial terminator, inter alia.
Examples of suitable terminators include those of rrn, psbA, rbcL and T7.
The 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).
In yet other embodiments, the genetic construct includes a second selectable marker gene and/or a nucleotide sequence which confers resistance to an antibiotic.
The selection gene may form part of an Excision Cassette, wherein the Excision Cassette is excised from the plant genome after selection.
Such excision may involve the use of site-specific recombination elements/site-specific recombinases.
Examples of site-specific recombination elements/site-specific recombinases include Cre-lox, the FLP-FRP system from Saccharomyces cerevisae (O'Gorman S, Fox D T, Wahl G M. (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. Mol Gen Genet. 230, 170-176.) or the R/RS system from Zygosaccharomyces rouxii (Onouchi H, Yokoi K, Machida C, Matsuzaki H, Oshima Y, Matsuoka K, Nakamura K, Machida Y. (1991) Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells. Nucleic Acids Res. 19, 6373-6378.).
The preferred recombination site is lox in combination with the recombinase Cre. Preferably, the recombinase sequence used is a cDNA sequence encoding a Cre polypeptide.
Once the Excision Cassette has been excised, an appropriate promoter should then be capable of driving the expression of the transgene, leading to the accumulation of the product of the transgene in the plant cells. The product of the transgene may be purified or isolated from the plant cell by any suitable means.
In a preferred embodiment, the invention provides a process for producing somatic plant embryos, the process comprising the steps:
Preferably, the plant is maize.
Preferably, the above steps are carried out in order without significant intervening steps, or without a gap of 12-24 hours between any of the steps.
In a further preferred embodiment, the invention provides a process for producing a transformed plant, the process comprising the steps:
Preferably, the plant is maize.
Preferably, the above steps are carried out in order without significant intervening steps, or without a gap of 12-24 hours between any of the steps.
The invention also provides a process for making a transgene product, comprising the process for producing a transformed plant embryos, as described hereinbefore, and additionally comprising purifying the transgene product from the regenerated plants.
The invention also provides a transgene product obtained or obtainable by a process of the invention.
Additionally, the invention provides a transformed plant embryo or transformed plant obtainable or obtained using a process of the invention.
The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.
The plastid transformation vector pAD001 was constructed as detailed in
For proof-of-principle purposes, the iaaM-iaaH transgene cassette was cloned in between the homologous recombination sequences to generate pAD001 (
The pAD001 construct was transformed into plastids using the protocol shown in Appendix 1 followed by auxin mediated selection and regeneration (
Abiotic stresses such as drought, salinity and temperature can be very detrimental to plants because of their sessile existence and can result in severe reduction in crop yields worldwide. The described system allows for the introduction and selection of transgenes, which can confer tolerance to abiotic stresses, in the maize plastid genome.
Transgenes e.g. the betaine aldehyde dehydrogenase gene, which confers tolerance to salinity and trehalose phosphate synthase, which confers drought tolerance, can be inserted into the pAD001 vector (
Biotic stresses such as bacterial, viral and fungal pathogens in addition to weeds and pests affect crop yields yearly and can result in significant financial losses to both farmers and industry alike.
As described in Example 2, biotic stress resistant transgenes e.g. B. thuringiensis (Bt) cry1A(c), may be inserted into the pAD001 vector followed by transformation, selection and regeneration as described previously.
A major limitation that both farmers and scientists face in crop production worldwide is the loss of up to 30-60% crop yield each year due to a combination of both biotic and abiotic stresses (Dhlamini. Z., Spillane. C., Moss. J P., Ruane. J., Urquia. N., Sonnino. A., (2005). Status of research and applications of crop biotechnologies in developing countries: Preliminary assessment, Roma, Food and Agriculture Organization of the United Nations [ISBN 92-5-105290-5]). As described in Examples 2 and 3, a combination of transgenes conferring both abiotic and biotic stress resistance may be inserted into the pAD001 vector, followed by transformation, selection and regeneration as previously described.
An increase in auxin due to the integration of the iaaM-iaaH transgene cassette may alter the growth characteristics of transformed plant species. To avoid this issue, the system described in Example 1 can be combined with a system for eviction such as a RIRS system from Zygosaccharomyces rouxii, Flp/frt from Saccharomyces cerevisiae, and Gin/gix from bacteriophage Mu removing the iaaM-iaaH transgene cassette and thus eliminating the problem.
The generation of whole transformed plants may be achieved by the following protocol. Transformation vectors containing the iaaM-iaaH gene cassette as a selectable marker are constructed and then bombarding into maize tissue as described above. Following the selection and confirmation of putative transformed calli, as described above, shoot regeneration could then be achieved using a cocktail of plant growth regulators (e.g. cytokinins etc.) to promote organogenesis. Alternatively, an antibiotic resistant gene may be incorporated in addition to the auxin genes (iaaM-iaaH) and the use of a two step selection system, first utilizing the iaaM-iaaH gene cassette for initial selection of transformants in the dark and secondly utilizing the antibiotic resistance gene once the calli are moved into the light.
The standard procedures produce transformed plants in 3-5 months.
4 g/L N6 salts (Chu et al., 1975)
1 ml/L (1000×) N6 vitamin stock
2 mg/L 2,4-D
100 mg/L myo-inositol
2.76 g/L proline
30 g/L sucrose
100 mg/L casein hydrolysate
2.5 g/L agar,
20 pH 5.8 and autoclave
Silver nitrate (25 μM) added after autoclaving.
4 g/L N6 salts
1 ml/L N6 vitamin stock
2 mg/L 2,4-D
100 mg/L myo-inositol
0.69 g/L proline
30 g/L sucrose
100 mg/L casein hydrolysate
36.4 g/L sorbitol
36.4 g/L mannitol (Vain et al, 1993)
2.5 g/L agar
pH 5.8 and autoclave.
Silver nitrate (25 μM) added after autoclaving.
4 g/L N6 salts
1 ml/L N6 vitamin stock
100 mg/L myo-inositol
30 g/L sucrose
2.5 g/L agar
pH 5.8 and autoclave.
Silver nitrate (25 μM) added after autoclaving.
A standard protocol is used as for nuclear transformation.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/058449 | 4/17/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61981386 | Apr 2014 | US |
