WO2001059086A2 - Methods and constructs for agrobacterium-mediated plant transformation - Google Patents
Methods and constructs for agrobacterium-mediated plant transformation Download PDFInfo
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- WO2001059086A2 WO2001059086A2 PCT/CA2001/000136 CA0100136W WO0159086A2 WO 2001059086 A2 WO2001059086 A2 WO 2001059086A2 CA 0100136 W CA0100136 W CA 0100136W WO 0159086 A2 WO0159086 A2 WO 0159086A2
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- 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
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- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/743—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Agrobacterium; Rhizobium; Bradyrhizobium
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- 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
- C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
Definitions
- the present invention relates to novel methods for the transformation of plant species and methods to produce transgenic plant species free of selectable marker genes.
- the present invention also relates to novel Agrobacterium vectors and uses thereof.
- Transformed plant cells are generated by the use of a variety of methods to insert foreign DNA into plant cells. These methods further employ the use of genetic constructs which contain genes encoding resistance to chemical selection agents such as antibiotics (e.g. kanamycin or hygromycin) or herbicides. Plant cells expressing selectable markers are preferentially obtained by culturing a population of plant cells subjected to a transformation process in the presence of media which contains, in addition to the selective agent, specific combinations of phytohormones and nutrients to allow cells to grow and regenerate to intact plants.
- antibiotics e.g. kanamycin or hygromycin
- the process of producing a transformed cell requires a means to physically insert foreign DNA as well as a means to select for those cells which have incorporated the DNA in an expressible and stable form.
- regeneration of cells containing the foreign DNA leads to the recovery of whole plants, usually following shoot-, embryo- or organo-genesis .
- the regenerated plant tissue is induced, by a number of different means, typically by the alteration of tissue culture conditions, more typically by manipulation of phytohormones, to form a whole plant capable of forming seed and / or pollen, which can transfer the foreign DNA to subsequent generations.
- a major limitation is efficient selection and regeneration of plant cells following transfer of DNA, as opposed to the actual transfer of DNA into the cell.
- practical limitations on the use of standard transformation methods generally relate to the efficiency of selection and identification of transformed plant cells, not necessarily the transfer of DNA into the plant cell.
- Another step in the process that is often problematic is the regeneration of transformed cells into whole plants in the presence of a selective agent.
- Brassica species are of special interest. Transformation of members of the Cruciferae family by Agrojacterium and other methods has been reported. A significant body of scientific work has accumulated that describes various methodologies, techniques and studies related to the generation of transgenic Brassica plants that contain foreign DNA of commercial interest and utility. A significant effort to achieve this was made through the early 1980' s and still is being made. The technology has been advanced to the point where transformation is now routine for certain narrow genotypes of a species within the Brassicacea , however, it remains difficult within other species in the same family. In addition there is a strong influence of genotype on transformation success within those Brassicas that have been successfully transformed and technology that is more widely applicable to even the "recalcitrant" genotypes would be of great value to the industry.
- transformation frequencies that have been achieved for Brassica napus are sometimes variable and very low (Fry et al . r Plant Cell Reports 6:321-325, 1987; Mehra-Palta et al . , In Proc 8th Int . Rapeseed Congress, Saskatoon, Saskatchewan, 1991; Swanson and Erickson, Theor. Appl . Genet . 7_8:831-835, 1989).
- Variable and often low transformation frequencies have also been observed with other Brassica species, such as B . oleracea (Christie and Earle, In Proc 5th Crucifer Genetics Workshop, Davis, pp 46-47, 1989; Metz et al .
- the many Brassica species, varieties and cultivars represent a very diverse group with radically different morphologies and physiological characteristics.
- the vegetable Brassicas represent a crop with significant economic value. This value could be enhanced by the addition of certain novel traits such as disease and insect resistance, male sterility systems for hybrid seed production, certain quality traits, and the like. Accordingly efficient transformation systems for broccoli, cabbage, cauliflower, kale and other Brassica vegetables would be valuable.
- Many Brassica vegetable species of commercial interest do not respond well or at all to the methods previously described. A transformation method is required which is useful for substantially all of the Brassica species and especially those previously recalcitrant to transformation.
- methods that permit the efficient transformation of a wide range of crop species and individual varieties, are essentially genotype independent and capable of being used to impart a variety of novel traits will be of significant benefit to the industry.
- methods that avoid, as a primary step in the process, the use of a toxic selective agent which kills non-transformed cells may provide opportunities to achieve the efficient regeneration of plants in species where this has been found to be difficult or in those plant varieties where this is currently not possible.
- Agrobacterium is a free-living Gram- negative soil bacterium. Virulent strains of this bacterium are able to infect plant tissue and induce the production of a neoplastic growth commonly referred to as a crown gall. Virulent strains of Agrobacterium contain a large plasmid DNA known as a Ti-plasmid that contains genes required for plasmid transfer and replication as well as a region of DNA that is called T-DNA. The T-DNA region is bordered by T-DNA border sequences that are crucial to the DNA transfer process.
- T-DNA border sequences are recognized by the vir genes encoded on the Ti-plasmid and the vir genes are responsible for the DNA transfer process.
- the native Ti-Plasmid contains the vir genes as well as the T-DNA region and the T-DNA borders required for efficient DNA transfer to a recipient plant cell.
- the entire complement of these genetic elements, in conjunction with genes encoded on the bacterial chromosome allow the efficient transfer of the T-DNA region into plant cells with subsequent stable integration and expression of the various genes encoded by the T-DNA.
- the T-DNA transferred and integrated into the plant cell nucleus contains a number of genes that encode enzymes for the production of unusual amino acids (opines) and genes that encode enzymes capable of producing plant hormones and genes responsible for the modulation of plant development.
- opioid unusual amino acids
- genes that encode enzymes capable of producing plant hormones and genes responsible for the modulation of plant development there are a relatively large number of genes within the T-DNA that are transferred to the plant cell, the function of all of genes has not been completely elucidated in the Agrobacterium genus.
- the primary effect of the genes in the T-DNA are at the level of plant cell growth and development.
- the plant cells infected with the T-DNA undergo an uncontrolled proliferation due to the activity of the T-DNA genes.
- These genes referred to as "oncogenes" because of the phenotype they confer, permit hormone-independent growth of transformed cells in culture.
- the process of gall or tumor formation is very efficient, essentially 100% of inoculated Agrobacterium susceptible plant tissue will form a tumor.
- the natural Agrobacterium DNA transfer process is exceedingly efficient.
- the physiological processes of crown gall formation are also very efficient in conferring a transformed phenotype.
- most wild-type Agrobacterium have broad host range and are capable of transferring large segments of DNA, typically T-DNA transferred to plant cells contains up to 25 kilobases of DNA (e.g. nopaline strains) or more (e.g. octopine strains) .
- T-DNA transferred to plant cells contains up to 25 kilobases of DNA (e.g. nopaline strains) or more (e.g. octopine strains) .
- the naturally occurring DNA transfer system of Agrobacterium provides an efficient means to transfer DNA into a plant cell and the subsequent formation and identification of transformed tissue.
- these have been based on the engineering of the Ti-plasmid to no longer contain the genes responsible for altered morphology ("oncogenes") and replacing these genes with a recombinant gene encoding a trait of interest.
- oncogenes genes responsible for altered morphology
- Linked to this trait of interest is a gene encoding a selectable marker such that cells that receive the DNA can be selected.
- vir genes are left intact, however some methods include the alteration of the vir genes in one form or another.
- Agrobacterium based plant transformation systems binary and co-integrate methods.
- binary transformation system is described by Schilperoort et al in U.S. Pat. No. 4,940,838.
- co-integrate system is found in Fraley et al . , Biotechnology, 3:629-635, 1985.
- Both the co-integrate and binary systems are based on replacing the normally oncogenic complement of Agrobacterium genes with engineered, non- oncogenic DNA, typically DNA that comprises a selectable marker and a gene encoding a novel trait.
- the T-DNA border repeats are maintained in both systems and the natural DNA transfer process is used to transfer the portion of DNA located between the T-DNA borders into the plant cell.
- these transformation methods avoid the problems associated with recovery of morphologically normal plant cells when using methods wherein the oncogenic region of the Ti-plasmid is inserted into a plant cell. Therefore, these methods rely on the presence of a selectable marker to recovery transformed cells since the selection based on the activity of the oncogenes can no longer be used.
- oncogenes can be included in a transformation vector for plants and the activity of these oncogenes used as a method to screen for transformed cells.
- Ebunuma et al. (Proc. Natl. Acad. Sci. USA 94:2117-2121, 1997, US 5,965,791) describe a plant transformation vector that comprises the isopentyl transferase gene from Agrobacterium (typically referred to as oncogene 4) as a means to identify transformed plant cells.
- oncogene the activity of the oncogene causes the production of cytokinin, a plant hormone that causes shoot production.
- a " shooty" mutant is produced (a morphologically abnormal shoot) and the plant cells are selected on the basis of abnormal shoot formation as well as selection for resistance to kanamycin or herbicides.
- the oncogene is bordered by transposition elements that permit the eventual loss of the oncogene from the transformed plant cell.
- the arrangement of DNA in the vector is such that the gene of interest remains in the transformed plant cell.
- the oncogene 2 can be used for discrimination of transformed plant cells, and recovery of transgenic plants following regeneration and selection of transgenic cells on appropriate media or under appropriate selective conditions.
- Keller et al. (International Publication No. WO 00/37060) demonstrates that the isolated oncogene 2 can be used for discrimination of transgenic plant cells following transformation and culturing on selective media.
- Abnormal plantlets are produced in the presence of the oncogene 2 enzyme and an appropriate substrate for the enzyme.
- the selection of a transformed plant relies on the formation of a morphologically abnormal structure.
- T-DNA transfer process and subsequent formation of crown galls or plant tumors with wild- type T-DNA is a highly efficient process.
- the process is usually very efficient even on plant varieties and species that are recalcitrant to usual tissue-culture protocols for the introduction of DNA by Agrobacterium and recovery of transgenic plants (for example various Brassica species or cotton) .
- transgenic plants for example various Brassica species or cotton
- crown galls will form easily on decapitated plants or wounded stems, even on plant genotypes recalcitrant to transformation using typical dis-armed vectors.
- Crown gall formation is also easily scored and true galls (i.e., those carrying the entire T-DNA region) are not capable of forming any differentiated plant tissue.
- crown galls do not form differentiated cells, even morphologically abnormal differentiated cells unless one or more of the encoded oncogenes undergoes a mutation or loss of function. Crown galls can also be easily cultured by simple growth on hormone free minimal media. Crown galls can also be made bacteria free by culturing in antibiotic solution for a period of time. Thus it is a trivial process to obtain bacteria-free, transformed plant cells from many different plant species comprising intact oncogenic T-DNA.
- a method for plant transformation that could take advantage of this efficient process, along with the easy means of identifying the transformed cells (by hormone- independent growth in culture) can provide many advantages including efficiency of transformation and selection of transformed plant cells.
- the art teaches that when the naturally occurring oncogenic region is used, recovery of morphologically normal plant cells is not possible.
- methods for recovering morphologically normal plant cells following the formation of crown galls are not available and hence the efficiency and wide host range of the natural T-DNA transfer process cannot be used as a means to produce morphologically normal transformed plants from a wide range of plant species.
- the present invention provides a novel method to produce and recover transformed plants that are morphologically normal and free of selectable markers.
- the invention utilizes the efficient and convenient process of T-DNA transfer as found in wild-type Agrobacterium to produce plant cells capable of growth under-hormone free conditions, leading to the formation of cells that can be conveniently identified by visual characteristics and under simple culture conditions.
- the plant cells are then selected from non-transformed cells and induced to regenerate into whole plants.
- the methods of the invention can avoid the antibiotic or herbicide selection of plant cells and offers an efficient means to recover transformed plants, particularly with those plant species that are difficult to transform by standard Agrobacterium and selection techniques.
- the broad method of the present invention utilizes transformation of plant cells with Agrobacterium oncogenes to produce a population of cells that contain oncogenes and are able to grow under conditions insufficent for the growth of untransfor ed cells, such as conditions lacking plant growth hormones. This results in the selection of transformed cells.
- the cells are transformed with the full complement of oncogenes found in wildtype Agrobacterium T-DNA. Although the transformed cells can grow under conditions insufficient for the growth of untransformed cells, the presence of the oncogenes usually prevents the transformed cells from regenerating to form morphologically normal plants.
- the transformed cells are then further modified to eliminate oncogene function, thus restoring the ability to regenerate and grow normally, so that a morphologically normal plant can be recovered.
- the invention provides a method for preparing a transformed plant, comprising:
- the construct comprises, in the 5' to 3' direction, a first recombinase recognition site, at least one Agroba cterium oncogene, and a second recombinase recognition site.
- the negating step then comprises excising the Agroba cterium oncogene (s) or a portion thereof from the construct with a recombinase which recognizes the first and second recombinase recognition sites.
- the recombinase may be encoded by a recombinase coding sequence contained in the original construct containing the oncogene, or it may be introduced into the transformed plant cells in a second construct.
- the recombinase coding sequence may be under the control of an inducible promoter, and it may contain a plant intron.
- either the construct containing the oncogene or the second construct includes a novel trait coding sequence which, when expressed in a plant, confers a novel trait on the plant.
- the novel trait coding sequence is contained in the construct containing the Agrobacterium oncogene or oncogenes, the ability of the transformed cells to grow under conditions under which untransformed cells cannot grow (e.g. in the absence of plant growth hormones), results in selection of the cells transformed with the novel trait coding sequence when the cells are grown without plant growth hormones, or with levels of hormones insufficient for the growth of untransformed cells. The use of an additional selectable marker is therefore not required.
- the construct carrying the oncogene is a modified Agrobacterium T-DNA region comprising, in the 5' to 3' direction, a right T-DNA border sequence, a first recombinase recognition site, at least one oncogene, a second recombinase recognition site, and a left T- DNA border sequence.
- the construct is introduced into the plant cells by Agrobacterium-mediated transformation.
- the recombinase recognition sites are replaced with transposase recognition sites, and a transposase enzyme which recognizes the transposase recognition sites is used to excise the one or more oncogenes or a portion thereof.
- the negating step comprises contacting the construct containing the Agrobacterium oncogene with a DNA binding protein to inhibit the expression of the product encoded by the oncogene.
- the construct containing the Agrobacterium oncogene may include a DNA binding protein coding sequence which encodes the DNA binding protein, or the DNA binding protein coding sequence may be contained in a second construct introduced into the plant cells in a second transformation event.
- the negating step comprises contacting the Agrobacterium oncogene or a transcript thereof with an antisense polynucleotide which inhibits transcription of the oncogene or inhibits translation of the transcript thereof.
- Agrobacterium oncogenes will be apparent to those of skill in the art, including the use of co-suppression or ribozyme techniques .
- the methods of the invention are suitable for use with monocot or dicot plants, preferably dicot plants.
- Preferred plants include members of the family Malvaceae, Linaceae, Composi tae, Solanacae, Fabaceae, Euphorbiaceae, Oleaceae, or Brassicaceae .
- the plant is a member of the genus Brassica , such as, without limitation, broccoli, cabbage, cauliflower, kale, Chinese kale, collard, kohlrabi, Chinese cabbage, pak choi, or turnip.
- the invention also provides plants, plant seeds, plant embryos, plant cells, or plant tissues, prepared according to the methods of the invention.
- the invention also provides various vectors, constructs, and modified Ti-plasmids useful for practicing the methods of the invention.
- the invention provides a DNA vector comprising, in the 5' - 3' direction, a DNA sequence homologous to a left portion of the right border region of the T-DNA region of a Ti-plasmid, a recombinase recognition site, and a region of DNA homologous to a right portion of the right border region of the T-DNA region of a Ti-plasmid.
- a DNA vector comprising, in the 5' - 3' direction, a DNA sequence homologous to a left portion of the left border region of the T-DNA region of a Ti-plasmid, a recombinase recognition site, and a region of DNA homologous to a right portion of the left border region of the T-DNA region of a Ti-plasmid, is provided.
- the DNA vector may further comprise an antibiotic resistance marker useful for selection in Agrobacterium .
- a modified Ti-plasmid comprising a recombinase recognition site 3' to the right T-DNA border is provided.
- the invention provides a modified Ti-plasmid comprising a recombinase recognition site 5' to the left T-DNA border.
- the invention provides a modified Ti-plasmid comprising a recombinase recognition site 3 r to the right T-DNA border and a recombinase recognition site 5' to the left T-DNA border.
- the modified Ti-plasmid may further comprise a recombinase coding sequence expressible in plant cells, and/or a scorable marker coding sequence expressible in plant cells.
- vectors and plasmids include: modified Ti-plasmid pTi-C58 CIMB; modified Ti-plasmid pTi-C58 TIMB; modified Ti-plasmid pTi-C58 RBC-1; DNA vector pRBC-1; DNA vector pLBC-1; and DNA vector pLBC-2, as described herein.
- the invention further provides a construct for transformation of plant cells comprising, in the 5' to 3' direction, a first recombinase recognition site, at least one oncogene, and a second recombinase recognition site.
- the construct may further comprise a recombinase coding sequence which encodes a recombinase.
- the construct further comprises a novel trait coding sequence located either 5' to the first recombinase recognition site or 3' to the second recombinase recognition site, the expression of the novel coding sequence in a plant conferring a novel trait on the plant.
- the recombinase coding sequence may be under the control of an inducible promoter, and may include a plant intron.
- a particularly preferred construct is a modified Agrobacterium T-DNA region comprising, in the 5' to 3' direction, a right T-DNA border sequence, a first recombinase recognition site, at least one oncogene, a second recombinase recognition site, and a left T-DNA border sequence.
- transposase recognition sites may be substituted for the recombinase recognition sites, and a transposase coding sequence substituted for the recombinase coding sequence.
- the invention also provides a construct comprising at least one oncogene and a DNA binding protein coding sequence which encodes a DNA binding protein.
- the construct further comprises a novel trait coding sequence the expression of which in a plant conferring a novel trait on the plant.
- the invention also provides plants, plant seeds, plant embryos, plant cells, or plant tissues, comprising a construct or vector according to the invention.
- the invention further provides an Agrobacterium cell, comprising a construct, vector, or a Ti-plasmid according to the invention.
- the invention also provides a method for modifying a
- Ti-plasmid to contain recombinase or transposase recognition sites comprising:
- step (b) introducing a second DNA vector into the Agrobacterium cells from step (a) under conditions sufficient for a homologous recombination event to occur between the second DNA vector and the modified Ti-plasmid, the second DNA vector comprising, in the 5' - 3 ' direction, a DNA sequence homologous to a left portion of the left border region of the T-DNA region of the modified Ti-plasmid, a recombinase or transposase recognition site, and a region of DNA homologous to a right portion of the left border region of the T-DNA region of the modified Ti-plasmid, to obtain Agrobacterium cells comprising a further-modified Ti-plasmid, the further-modified Ti-plasmid comprising a recombinase or transposase recognition site within the T-DNA region of and in proximity to the left T- DNA border sequence of the further-modified Ti-plasmid and a recombinase or transposase recognition site within
- the Ti-plasmid further comprises within the T-DNA region a recombinase or transpose enzyme coding sequence under the control of an inducible promoter.
- the Ti- plasmid may further comprise within the T-DNA region a scorable marker gene expressible in plant cells.
- Figure 1 illustrates a general approach to using the methods of the invention to recover transformed plant cells and whole plants.
- Figure 2 illustrates the derivation of a modified Ti- plasmid comprising recombinase recognition sites.
- Figure 3 illustrates the use of a repression strategy that may be employed to eliminate oncogene function in plant cells transformed with oncogenes.
- Figure 4 illustrates the derivation of a modified Ti- plasmid comprising a recombinase recognition site at the Right T-DNA border. The use of an ampicillin marker gene to first select for the homologous recombination event is shown.
- Figure 5 illustrates the use of a recombinase enzyme to excise an ampicillin marker gene to derive a modified Ti- plasmid that comprises a recombinase recognition site at the Right T-DNA border.
- Figure 6 illustrates the derivation of a modified Ti- plasmid comprising a recombinase recognition site, recombinase gene and ampicillin marker at the Left T-DNA border.
- Figure 7 illustrates the use of the ampicillin marker gene to select a modified Ti-plasmid that comprises recombinase sites at the Left and Right T-DNA borders as well as a bacterial marker and plant expressible recombinase gene.
- Figure 8 illustrates the result of the expression of the recombinase gene in plant cells transformed with the modified Ti-plasmid. The oncogenic region is lost and the plant DNA contains only the gene encoding the novel trait.
- FIGS 9A and 9B illustrate the regions of homology used for the construction of modified Ti-plasmids.
- the regions of the nopaline Ti-plasmid used are shown.
- ROH refers to region of homology
- the "-R L , ⁇ R R , -L L and -L R " refer to the sub- regions (i.e. portions) of homology (right (9B) and left (9A) regions) used in the construction of vectors for homologous recombination in Agrobacterium .
- Figures 10A-10C provide restriction maps and a representation of the modified FLP recombinase DNA sequences used to construct the modified Ti-plasmids.
- Figures 11A-11B provide restriction maps and a representation of the source (11A, pBinl9) of the modified NOS promoter DNA sequence (PCR product, 11B) used to construct the modified Ti-plasmids.
- Figure 12 provides a restriction map and a representation of the GUS:NPTII fusion DNA sequences used to construct the modified Ti-plasmids.
- Figure 13 provides a restriction map and a representation of the cold inducible promoter DNA sequence used to construct the modified Ti-plasmids.
- Figure 14 provides a restriction map and a representation of the CaMV 35S DNA sequences used to construct the modified Ti-plasmids.
- Figure 15 provides a restriction map and a representation of the constitutive "EntCUP2" promoter DNA sequence used to construct the modified Ti-plasmids.
- Figures 16A-16B provide restriction maps and a representation of the tet repressor/operator DNA sequences used to construct the modified Ti-plasmids.
- Figure 17 provides a restriction map and a representation of the ampicillin bacterial marker DNA sequences used to construct the modified Ti-plasmids.
- Figures 18A-18B provide restriction maps and a representation of the FRT recombinase recognition DNA sequences used to construct the modified Ti-plasmids.
- Figure 19 provides a simplified restriction map and a representation of the components used to derive the homologous recombination plasmid pRBC-1 for insertion of a recombinase sequence into the Right border region of the T-DNA of the C58 nopaline Ti-plasmid.
- Figure 20 provides a simplified restriction map and a representation of the components used to derive the homologous recombination plasmid pLBC-1 for insertion of a recombinase sequence into the Left border region of the T-DNA of the C58 nopaline Ti-plasmid.
- Figure 21 provides restriction maps and a representation of the modified pGEM7Zf (+) vector used to construct the modified Ti-plasmids.
- Figure 22 provides restriction maps and depicts construction of the pUM21 vector used to construct the modified Ti-plasmids .
- Figure 23 illustrates the steps used to construct one of the intermediate vectors used for the modification of the left border region of the T-DNA of the C58 Ti-plasmid and construction of the intermediate vector pAmpTerFloP.
- Figure 24 illustrates the steps used to construct one of the intermediate vectors (pROH (L L ) NosPrFRT) used for the modification of the left border region of the T-DNA of the C58 Ti-plasmid.
- Figure 25 illustrates the steps used to construct one of the intermediate vectors used for the modification of the left border region of the T-DNA of the C58 Ti-plasmid.
- Figure 26 illustrates the steps used to construct one of the intermediate vectors used for the modification of the left border region of the T-DNA of the C58 Ti-plasmid (final derivation of pLBC-1) .
- Figure 27 depicts a detailed restriction map of the homologous recombination plasmid pLBC-1 designed for the insertion of a recombinase sequence into the Left border region of the T-DNA of the C58 nopaline Ti-plasmid.
- the plasmid contains, two flanking regions of homology and located between those regions is a recombinase recognition site (FRT) as well as a cold-inducible promoter (CiPr) controlling the expression of a FLP recombinase gene modified to contain a plant intron (FLoP) with a nos terminator (NosTer) linked to an ampicillin resistance gene for selection of the homologous recombination event .
- FRT recombinase recognition site
- CiPr cold-inducible promoter
- Figure 28 illustrates the steps used to construct one of the intermediate vectors (pAmpFRT(R)) used for the modification of the right border region of the T-DNA of the C58 Ti-plasmid.
- Figure 29 illustrates the steps used to construct one of the intermediate vectors used for the modification of the right border region of the T-DNA of the C58 Ti-plasmid (construction of a 35S polyadenylation signal) .
- Figure 30 illustrates the steps used to construct one of the intermediate vectors (pGUS-pA) used for the modification of the right border region of the T-DNA of the C58 Ti-plasmid.
- Figure 31 illustrates the steps used to construct one of the intermediate vectors (pGUSNPTIIpA) used for the modification of the right border region of the T-DNA of the C58 Ti-plasmid.
- Figure 32 illustrates the steps used to construct one of the intermediate vectors (pAMPFRT (R) Gus) used for the modification of the right border region of the T-DNA of the C58 Ti-plasmid.
- Figure 33 illustrates the steps used to construct one of the intermediate vectors used for the modification of the right border region of the T-DNA of the C58 Ti-plasmid (final derivation of pRBC-1) .
- Figure 34 depicts a detailed restriction map of the homologous recombination plasmid pRBC-1 designed for the insertion of a recombinase sequence into the Right border region of the T-DNA of the C58 nopaline Ti-plasmid.
- the plasmid contains, two flanking regions of homology and located between those regions are two recombinase recognition site (FRT> flanking an ampicillin resistance gene for selection of the homologous recombination event as well as a "pro oterless" GUS- NPTII fusion gene that can be activated upon recombination within the modified Ti-plasmid.
- Figure 35 depicts a simplified restriction map and representation of the components used to derive the homologous recombination plasmid pLBC-2 for insertion of a recombinase sequence into the Left border region of the T-DNA of the C58 nopaline Ti-plasmid.
- This plasmid comprises the tet repressor/operator system for the control of recombinase expression.
- Figure 36 illustrates the steps used to construct the plasmid encoding the tet repressor system, pTetRepSys .
- Figure 37 illustrates construction of the vector pLBC-2. Details of the steps employed to produce a homologous recombination vector for insertion of DNA into the Left Border region of the T-DNA of the C58 Ti-plasmid.
- Figure 38 depicts a detailed restriction map of the homologous recombination plasmid pLBC-2 designed for the insertion of a recombinase sequence into the Left border region of the T-DNA of the C58 nopaline Ti-plasmid.
- the plasmid contains, two flanking regions of homology and located between those regions is a recombinase recognition site (FRT) as well as the tet repressor / operator system controlling the expression of a FLP recombinase gene modified to contain a plant intron (FloP) with a nos terminator (NosTer) linked to an ampicillin resistance gene for selection of the homologous recombination event.
- FRT recombinase recognition site
- NosTer nos terminator linked to an ampicillin resistance gene for selection of the homologous recombination event.
- Figure 39 illustrates the steps used to introduce the vectors pRBC-1, pLBC-1 and pLBC-2 into the C58 Agrobacterium and recovery of the modified Ti-plasmid.
- a “coding sequence” or “coding region” is the part of a gene that codes for the amino acid sequence of a protein, or for a functional RNA such as a tRNA or rRNA.
- a “complement” or “complementary sequence” is a sequence of nucleotides which forms a hydrogen-bonded duplex with another sequence of nucleotides according to Watson-Crick base-pairing rules.
- the complementary base sequence for 5'-AAGGCT-3' is 3 ' -TTCCGA-5 ' .
- “Expression” refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into a protein.
- Polynucleotides are "functionally equivalent” if they perform substantially the same biological function.
- Polynucleotides are "heterologous" to one another if they do not naturally occur together in the same arrangement in the same organism.
- a polynucleotide is heterologous to an organism if it does not naturally occur in its particular form and arrangement in that organism.
- Polynucleotides or polypeptides have "homologous” or “identical” sequences if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described herein. Sequence comparisons between two or more polynucleotides or polypeptides are generally performed by comparing portions of the two sequences over a comparison window to identify and compare local regions of sequence similarity.
- the comparison window is generally from about 20 to about 200 contiguous nucleotides or contiguous amino acid residues.
- the "percentage of sequence identity” or “percentage of sequence homology" for polynucleotides and polypeptides, such as 50, 60, 70, 80, 90, 95, 98, 99 or 100 percent sequence identity may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
- the percentage is calculated by: (a) determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions; (b) dividing the number of matched positions by the total number of positions in the window of comparison; and, (c) multiplying the result by 100 to yield the percentage of sequence identity.
- Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms, or by inspection.
- Readily available sequence comparison and multiple sequence alignment algorithms are, respectively, the Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. 1990. J. Mol. Biol. 215:403; Altschul, S.F. et al . 1997. Nucleic Acids Res. 25: 3389-3402) and ClustalW programs.
- BLAST is available on the Internet at http://www.ncbi.nlm.nih.gov and a version of ClustalW is available at http://www2.ebi.ac.uk.
- stringent conditions are selected to be about 5 ° C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
- Tm is the temperature (under defined ionic strength and pH at which 50% of a complementary target sequence hybridizes to a perfectly matched probe.
- stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C for long probes (e.g. greater than 50 nucleotides) .
- Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide .
- Exemplary low stringency conditions include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 2X SSC at 50°C.
- Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0. IX SSC at 60°C. Hybridization procedures are well-known in the art and are described in Ausubel et al . , (Ausubel et al . , Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994).
- isolated refers to material that is: (1) substantially or essentially free from components which normally accompany or interact with it as found in its naturally occurring environment; or (2) if in its natural environment, the material has been non-naturally altered to a composition and/or placed at a locus in the cell not native to a material found in that environment.
- the isolated material optionally comprises material not found with the material in its natural environment.
- the alteration to yield synthetic material can be performed on the material within or removed from its natural state.
- a naturally occurring nucleic acid becomes an isolated nucleic acid if it is altered, or if it is transcribed from DNA which is altered, by non- natural, synthetic methods performed within the cell from which it originates.
- a naturally occurring nucleic acid becomes isolated if it is introduced by non-naturally occurring means to a locus of the genome not native to that nucleic acid.
- Two DNA sequences are "operably linked” if the nature of the linkage does not interfere with the ability of the sequences to effect their normal functions relative to each other.
- a promoter region would be operably linked to a coding sequence if the promoter were capable of effecting transcription of that coding sequence.
- a "polynucleotide” is a sequence of two or more deoxyribonucleotides (in DNA) or ribonucleotides (in RNA) .
- a "construct” is a nucleic acid molecule that is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature .
- a "polypeptide” is a sequence of two or more amino acids .
- a “promoter” is a cis-acting DNA sequence, generally located upstream of the initiation site of a gene, to which RNA polymerase may bind and initiate correct transcription.
- a "recombinant" polynucleotide for instance a recombinant DNA molecule, is a novel nucleic acid sequence formed in vitro through the ligation of two or more nonhomologous DNA molecules (for example a recombinant plasmid containing one or more inserts of foreign DNA cloned into its cloning site or its polylinker) .
- a "recombinase recognition site” is a sequence of nucleotides that is recognized by and acted upon by a site- specific recombinase enzyme.
- Transformation means the directed modification of the genome of a cell by the external application of recombinant DNA from another cell of different genotype, leading to its uptake and integration into the subject cell's genome.
- transgenic organism such as a transgenic plant
- a “transgenic plant” encompasses all descendants, hybrids, and crosses thereof, whether reproduced sexually or asexually, and which continue to harbour the foreign DNA.
- a "vector” may be any of a number of nucleic acid sequences into which a desired sequence may be inserted by restriction and ligation.
- a vector typically carries its own origin of replication, one or more unique recognition sites for restriction endonucleases which can be used for the insertion of foreign DNA, and usually selectable markers such as genes coding for antibiotic resistance, and often recognition sequences (e.g. promoter) for the expression of the inserted DNA.
- Common vectors include plasmids, phage, phasmids, and cosmids .
- transposase recognition site is a sequence of nucleotides, which, in nature, is ' found at the 5' and 3' ends of a transposable element, and which are acted upon by a transposase enzyme during transposition of the transposable element .
- all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.
- oncogenic function is permanently inhibited by the use of repression strategies that delete or genetically interrupt the oncogenic region itself.
- the oncogenes themselves are engineered to be responsive to novel repression strategies.
- selected oncogenes are used rather than the entire complement of genes normally transferred to plant cells via wild-type Agrobacterium .
- Agrobacterium generally transforms many different crop species at high efficiency. Numerous Agrobacterium species and strains have- been described, the transfer of T-DNA and subsequent tumor formation process is generally conserved and dependent on the function of the introduced oncogenes of which there are many contained within the T-DNA region. It is also known that many Agrobacterium strains, such as nopaline or succinamopine can transform (i.e. form tumors) on many different plant species and virtually any genotype of Brassica species with high efficiency. The present method utilizes this efficiency as means to first derive a population of transformed cells from any species or genotype capable of being infected by Agrobacterium .
- These cells preferably contain the full complement of oncogenes and are not capable of regeneration to morphologically normal plants.
- the oncogenes are introduced to the plant cell by Agrojacterium-mediated transformation in which the Agrobacterium used is an armed strain capable of conferring hormone independent growth on appropriate media.
- the Agrobacterium strain is modified to include a Ti-plasmid that has been altered by the addition of recombinase recognition sites flanking the oncogenic region and a recombinase gene, capable of expression in a plant cell, under the control of an inducible promoter.
- induction of the recombinase gene leads to the expression of the recombinase in a transformed plant cell, causing the loss of the oncogenic region and allowing the plant cells to regenerate into whole plants.
- a preferred method further comprises the introduction of a novel DNA sequence within the Ti-plasmid that is outside of the region excised by the recombinase but within the T-DNA border repeats of the Ti-plasmid. In this fashion, loss of the oncogenic region from the inserted DNA produces a plant cell wherein the novel DNA sequence is stably inserted into the plant genome.
- This method also provides certain advantages in relation to selection.
- the initial transformation event relies on the hormone independent growth of the transformed cells due to the expression of the oncogenes. These cells can be selected for by simply plating on hormone free media. This process has been used for many years to score Agrobacterium infectivity. Only transformed cells (cells with oncogenes) grow on hormone free media. Thus it is a simple matter to obtain a population of cells, all of which are transformed.
- these populations of cells are further manipulated by a transient expression event (which activates a DNA construct capable of causing loss of the oncogenic region) that generates cells capable of regeneration, usually directly from a population of cells originally containing oncogenic region, typically without the needs for added hormones or prolonged and complicated tissue culture procedures.
- a transient expression event which activates a DNA construct capable of causing loss of the oncogenic region
- selection can provide higher regeneration efficiencies since the need to balance the toxic effects of the antibiotic and the death of non-transformed cells (with subsequent negative effects on transformed cells) is eliminated.
- This particular step has been difficult for many plant species and in particular Brassica genotypes. Indeed, selectable markers based on killing non-transformed cells can be efficient, but also require significant manipulation in culture and time in order to be used.
- This new method provides many advantages in developing transgenic lines since less tissue culture experimentation with new genotypes is required and prolonged regeneration protocols are minimized.
- the attributes of this system include:
- various methods may be employed to (1) produce plant cells that initially comprise oncogenic activity convenient for the selection of transformed plant cells, and are (2) subsequently modified to eliminate the activity of the oncogenic region of the T-DNA to permit regeneration to morphologically normal plants.
- the initial step formation of tumor cells can be carried out in a number of different ways. These include direct inoculation of whole plants, inoculation of sterile plant tissue or plant explants, or inoculation of decapitated plantlets. Tumor cells are simply cultured on minimal media, or in some cases tumor cells are allowed to grow directly on the inoculated plant.
- This transformation and regeneration procedure is in contrast to toxic selection techniques where plant cells are first subjected to the transformation process and then subjected to the presence of a phytotoxic agent and introduced genes are linked to a selectable marker that detoxifies the agent. Regeneration is therefore dependent on the selection of transformed cells within a population of non-transformed cells.
- the present invention allows the formation of a population of cells that are substantially all transformed, and regeneration takes place without the selective pressure of a phytotoxic agent .
- a first aspect of the present invention contemplates the use of naturally occurring oncogenes, found in wild-type T- DNA of Agrobacterium and the naturally occurring highly efficient plant transformation process of the Agrobacterial genus to first produce transformed plant cells, which because of the hormone independent growth of said cells as a result of the expression of the oncogenes, permit the easy identification of transformed cells.
- novel strategies for the elimination of the oncogenic region, or elimination of oncogene function are employed to produce cells transformed and capable of regeneration to morphologically normal plants.
- the Ti-plasmid is modified to comprise recombinase recognition sites and a recombinase gene that can be expressed in the plant cell under appropriate conditions.
- the oncogenic region, or portions thereof are excised, eliminating the tumor phenotype and allowing for the regeneration of whole plant cells from the initially transformed plant cells.
- Inclusion of a DNA sequence in the Ti-plasmid encoding a novel trait permits the recovery of whole plants comprising a novel trait inserted into the plant genome.
- the cells transformed with the oncogenic region of the T-DNA are then subjected to a second transformation event with a DNA construct that inhibits the activity of the oncogenes such that morphologically normal plants can be conveniently induced to form from the population of cells initially expressing the oncogenes .
- the use of direct selection on a population of transformed cells is avoided.
- the method allows for the recovery of transformed morphologically normal plants from transformed oncogene containing cells since inhibition of the oncogenes allows cells to regenerate into morphologically normal plants.
- selective agents that kill non-transformed cells and relying on a conversion from a morphologically abnormal state to a morphologically normal state, recovery of whole plants occurs in a convenient and efficient fashion.
- a site-specific recombinase is contemplated as a means to eliminate oncogene activity.
- the use of recombinases can provide a direct means to eliminate oncogene function in transformed cells.
- the resultant cells following exposure to the recombinase, contain only the transgene. The cells are then capable of regeneration to morphologically normal plants.
- the T-DNA can be modified (for example site-specific mutagenesis or homologous recombination as a means to introduce new DNA sequences) to provide sites capable of recognition by site specific recombinases.
- These DNA sequences which are typically short (less than 40 base pairs, often less) can be introduced as flanking regions within the T- DNA borders (i.e. within the portion of DNA transferred to plant cells), oncogenes being contained between two of these sequences. Introduction of a recombinase activity would cause the specific excision of the oncogenes, leaving the T-DNA borders (and any associated novel transgene) in place.
- the recombinase activity could be introduced by stable transformation, transient expression of DNA, such as by biolistics or even viral infection of said cells with recombinant virus containing the recombinase gene, or by introduction of the recombinase enzyme through various means such as electroporation.
- the recombinase gene is included in a Ti-plasmid, but is under the control of an inducible plant promoter such that expression of the recombinase (and loss of the oncogenic region) can be accomplished by placing the cells containing the oncogenic region under appropriate induction conditions.
- the steps employed to derive such a vector can include the construction of a vector comprising a recombinase recognition site flanked by two regions of homology to a border region. In this fashion, a double crossover event causes the insertion of the recombinase recognition site into the border region of the T-DNA.
- the region of homology can be anywhere within the border region, but it must be chosen to leave the T- DNA border repeat intact and lie within the region of DNA typically transferred to plant cells.
- an antibiotic selectable marker is included to facilitate the recovery of cells containing the modified Ti-plasmid.
- the first antibiotic marker is flanked by identical recombinase recognition sites, such that the removal of the antibiotic marker by expression of the recombinase in the bacteria allows the recovery of a first modified Ti-plasmid free of antibiotic marker genes.
- a plant marker gene in the DNA constructs used for modification of the Ti-plasmid. The plant marker gene is arranged to be activated upon successful excision of the T-DNA in the plant cell by induced or introduced recombinase activity. In this fashion, loss of oncogenic function can be conveniently scored.
- FLP has been shown to be functional in tobacco and Arabidopsis (Kilby et al . , The Plant Jour. 8 (5) : 637-652, 1995, incorporated herein by reference)
- Cre-lox has been shown to be functional in tobacco (Russell et al . , Mol . Gen . Genet . 234:49-59, 1992, Odell et al . , Mol . Gen . Genet . 223:369-378, 1990, Dale and Ow, Gene 91:79-85, 1990, Dale and Ow, Proc . Na tl Acad. Sci .
- the recombinase gene is typically modified to prevent expression in bacterial cells, and restrict expression to plant cells. This is particularly important for those embodiments where the recombinase gene is contained within the border regions of the modified Ti-plasmid, since expression of the recombinase gene in bacterial strains, such as the Agrobacteri um strain comprising the modified Ti-plasmid would lead to the loss of the T-DNA.
- the recombinase gene is modified to contain an intron to restrict the expression in bacteria.
- the intron typically is modified to contain at least one stop signal to ensure no expression of the recombinase activity in bacteria.
- the modification of the Ti-plasmid can take place with any Agrobacterium species capable of conferring a phenotype that includes hormone independent growth and inability to regenerate to morphologically normal plants.
- Wild-type T-DNA or Ri-DNA can be used as well as modified forms thereof.
- a combination of oncogenes normally found within the T-DNA or Ri-DNA can be used. The actual combination will be dependent on the particular plant species to be transformed as well as convenience.
- the Agrobacterium species contemplated for use in the present invention includes A. rhizogenes , A tumefaciens (nopaline, octopine, agropine strains) A. vitis, or any other strain capable of transferring oncogenes to plant cells. In particular the use of the nopaline strain C58 is preferred.
- These Agrobacterium strains are commonly available from the American Type Culture Collection.
- the method to modify the Ti-plasmid can comprise many different steps, but in general the following steps are employed (these are illustrated diagrammatically in figures 4 - 7):
- Agrobacterium strain comprising a Ti-plasmid and recovery of a Ti-plasmid modified to contain a recombinase recognition site;
- recombinase gene itself in one of the vectors used to insert a second recombinase recognition site in a Ti-plasmid.
- the recombinase gene is modified to contain a plant intron to prevent expression in bacterial cells, and is under the control of an inducible plant promoter.
- inducible promoters two inducible promoters are used for demonstration, the cold- inducible • promoter from Brassica (Genbank Ace. N. U14665) and the tet operator / repressor system (Gatz and Quail, Proc. Natl. Acad. Sci. USA 85: 1394-1397, 1988, incorporated herein by reference) .
- glucocorticoid transcriptional factor and DNA encoding the glucocorticoid responsive elements are utilized with plant genes to provide a gene expression system that inhibits gene expression but can be de-repressed in the presence of steroid hormones.
- the recombinant DNA molecule comprising the modified T-DNA of the Ti-plasmid can additionally comprise a gene encoding a "novel trait" which could be any recombinant protein or peptide of interest, typically this "novel trait” could be a heterologous protein of commercial interest or a protein that confers an agronomically useful trait such as herbicide tolerance.
- a "novel trait” could be any recombinant protein or peptide of interest, typically this "novel trait” could be a heterologous protein of commercial interest or a protein that confers an agronomically useful trait such as herbicide tolerance.
- These traits could include insect resistance, such as the Bt insecticidal protein gene, examples of the methods for modification and use of Bt coding sequences in plants can be found in U.S. Pat. No. 5,380,381, incorporated herein by reference.
- Other strategies for control of insect predation in plants include genes encoding protease inhibitors (U.S.
- genes encoding an enzyme capable of making a nutraceutical product such as increased vitamins (e.g., International Publication No. WO 99/53041 incorporated herein by reference) or increased production of secondary metabolites of commercial value, or genes controlling the biochemistry or physiology of plant development, such as delayed or induced flowering, (e.g., homeotic genes such as LEAFY (Weigel and Nilsson, Nature 277:495-500, 1995, Session et al . , Science 289: 779-781, 2000, incorporated herein by reference) reduction of seed in fruit (e.g., International Publication No.
- a gene encoding a novel trait can be added to the plasmids used to modify the wild-type C-58. Said novel trait may be added upon modification of the right border sequences or the left border sequences.
- it is possible to add any number of genes encoding novel traits by orienting the gene encoding the novel trait relative to the recombinase recognition site such that upon activation of the recombinase enzyme the wild-type T-DNA and recombinase enzyme coding region is excised, leaving the novel trait or traits flanked by the T- DNA borders .
- Agrobacterium strains are prepared, plant cells are transformed, cells containing the oncogenic region are cultured under hormone free conditions, or minimal media conditions, and transformed cells are allowed to proliferate, while non- transformed cells are eliminated. After this period of time, the recombinase activity is induced. The recombinase activity eliminates the oncogenic region, leaving behind the novel trait and a small region of the T-DNA bounded by the Left and Right T-DNA borders. This is shown in figure 8.
- the recombinase site may be scattered throughout the genome of the plant by selecting various transformed cell lines, each with a different site of insertion. These individual plant lines may be regenerated and crossed, leading to the formation of plant lines that comprise many recombinase recognition sites, scattered throughout the genome of the plant.
- the invention has a further utility in plant genetics, namely the use of the method to introduce a multiplicity of recombinase recognition sites in a plant genome, the sites able to be utilized by a recombinase to alter the arrangement of the plant genome by the use of a recombinase activity.
- the recombinase can be introduced in a second transformation event, or can be present in the plant genome under the control of an inducible promoter such as those described herein.
- the recombinase can also be introduced by crossing with a plant engineered to express the recombinase, either constitutively or more preferably under the control of an inducible promoter or a tissue specific promoter.
- a pollen- specific or microspore specific promoter controlling the expression of the recombinase is preferred as this will lead to the production of a population of pollen cells comprising novel genetic arrangements following the introduction of the pollen-
- any number of recombinase sites can be introduced, and the method can be adapted to produce plant cells with genomes altered to only contain recombinase sites without addition coding sequences.
- Another use for an introduced recombinase site is to provide a region for site-specific integration of a novel trait by the use of recombinase mediated gene insertion.
- the region that remains in the plant chromosome can also comprise a gene from the oncogenic region, or a portion of a gene from the oncogenic region.
- the oncogenic region There are many different oncogenes within the oncogenic region, including genes encoding the formation of opines, thus it may be preferred in some cases to insert the recombinase recognition site to allow for removal of a portion of the oncogenic region while leaving one or a portion thereof of an oncogene normally found in the oncogenic region.
- transposases In addition to recombinases, the use of transposases to eliminate or excise oncogenic regions of a modified Ti- plasmid is contemplated.
- Known transposons and associated transposase activities include Ac/Ds and En/Spm elements from maize (e.g. see Federoff, N. Maize Transposable Elements . In Berg, D.E. and Howe, M.M. (eds) Mobile DNA, pp.375-411, American Society for Microbiology, Washington, D.C, 1989), Tam-1 and Tam-3 from snapdragon (e.g. see Sommer et al , Transposable Elements of Antirrhinum maj us . In Plant Transposable Elements, 0.
- Tnt-1 from tobacco (Pouteau, S. et al, Mol Gen Genet . 228:233-239, 1991), T ⁇ h-1 from petunia (Gerats A.G.M. et al , The Plant Cell 2:1121-1128, 1991) and the Tst-1 element from potato (Koster-Topfer, et al, Plant Mol . Biol 14:239-247,1990), all of which are incorporated herein by reference .
- a transposon has certain features such as a central coding region encoding a transposase enzyme and DNA sequences at the 5' and 3' end of the transposon that are recognized by the specific transposase. When the transposase acts upon these sequences, transpostion of the transposable element occurs. Artificial transposons have been constructed which comprise heterologous genes. These recombinant transposons can transpose in the presence of the transposase enzyme. In accordance with the invention, the sequences recognized by the transposase can be linked to the oncogenes in a fashion that permits the excision of the oncogenes by the transposase enzyme. The transposition or excision event irreversibly inactivates the oncogene function.
- Transposons can be modified to reduce or eliminate re-insertion into other chromosomal locations, or the arrangement of the transposase recognition sites is structured to irreversibly delete a portion of the oncogene.
- oncogenes can be modified to have transposase recognition sites between the promoter of a modified oncogene and the coding region, such that transposition effectively eliminates a portion of the coding region or promoter, thus inactivating the oncogene. Any number of strategies may be employed to utilize transposition as a means to inactivate oncogene function.
- transposase activities to specifically cause transposition (or loss) of oncogenes is contemplated within the scope of the invention. Indeed, it is possible, to modify the T-DNA to contain DNA sequences capable of being recognized by a transposase enzyme such that upon exposure to the transposase enzyme, the oncogenes are excised from the original location of insertion, with subsequent loss or low frequency of transposition to another site.
- transposons when properly modified, can be used as a means to favor the excision of the oncogenes within the scope of the invention.
- the oncogenic T-DNA region of the nopaline strain of Agrobacterium provides preferred DNA components.
- the art has described the structure and function of these genes, allowing the skilled artisan to devise specific DNA constructs and culture conditions that provide optimum transformation efficiency.
- the nopaline strain is preferred as the T-DNA of the nopaline strain C-58 is a single T-DNA region as contrasted to octopine strains that comprise a pseudo duplicated T-DNA such that there is a "right" and "left" T-DNA region.
- nopaline T-DNA there are oncogenes of known function such as oncogenes 1,2 & 4, as well as oncogenes of undefined function such as 5, 6b, as well as genes encoding nopaline synthase (nos) , agrocinopin ' e synthase (acs) , octopine, nopaline secretion (ons) and others. Combined, the activities of these oncogenes lead to the formation of crown galls.
- Oncogene 1 encodes the enzyme Indole Acetamide Synthase (IAMS) that converts tryptophan, an amino acid normally found in plant cells to indole acetamide.
- IAMS Indole Acetamide Synthase
- the function of oncogene 1, that is the conversion of tryptophan (a endogenous amino acid contained within all plant cells) to indole acetamide is described by Van Onckelen et al . , FEBS lett . 198, 357-360, 1986, incorporated herein by reference.
- Oncogene 2 encodes the enzyme Indole Acetamide
- IAMH Iron Hydrolase
- Oncogene 4 encodes isopentyl transferase and can synthesize a cytokinin activity.
- the T-DNA region contains a number of other genes that are involved in the oncogenic process and hence are considered oncogenes. Many of the genes are involved in the regulation of the formation of tumors and hence play an important role in the efficiency of tumor formation.
- the cells transformed with .the entire complement of oncogenes can grow very rapidly and under conditions that favor their growth at the expense of non-transformed cells, but only in a disorganized fashion leading to the formation of crown galls.
- the complement of oncogenes found in the C58 strain provide the genetic function required for the formation of crown gall tissue.
- oncogenes For some plant species, a portion of the normal complement of oncogenes may be employed. Indeed, one or more oncogenes may be used, and the culture media adjusted to provide a level of phytohormone that would normally be found by expression of all of the normally expressed oncogenes.
- plant cells resultant from the first transformation event may not exhibit complete hormone independent growth, but under certain conditions of culture exhibit a phenotype which is similar to hormone independent growth and incapable of regeneration to morphologically normal cells. These cells would then be subject to expression of the recombinase and culture conditions would then be selected to provide said transformed cells with the ability to regenerate to morphologically normal cells. In this fashion, the use of a limited number of oncogenes is contemplated, thus simplifying the method.
- the oncogene activities of choice are the oncogenes 1 and 2 from the Ti or Ri plasmid of Agrobacterium and the oncogene 4, which causes the production of cytokinins as well as the other oncogenes commonly found associated with these other oncogens within the Ti - or Ri-DNA region.
- the activity of these genes combined leads to hormone independent growth.
- the phytohormone activity of these genes can be substituted in many cases by endogenously applied phytohormones .
- oncogenes genes When these oncogenes genes are turned off, or silenced in one manner or another, the cells can grow normally and in many cases cells can regenerate to morphologically normal plants. Thus the silencing of the oncogenes can be used as a selectable event in tissue culture.
- the various means that can be used to silence such as repression are illustrated in figure 3.
- a second DNA is expressed that encodes a repressor capable of eliminating oncogene expression or function.
- any mechanism that effectively blocks the accumulation of the product of said oncogenes in a cell comprises repression.
- Said methods may include: the binding of a specific "repressor protein or factor” to a DNA region or "operator” within the promoter of said oncogene.
- the promoter region of the oncogene can be conveniently modified to be responsive to this repressor element.
- these DNA binding proteins described in the art include bacterial repressor elements and associated DNA binding regions (operator DNA) such as the Lac Z repressor or the tet repressor or other bacterial repressors.
- bacterial repressors may be found in the class of repressor proteins includes: LacR, GutR, DeoR, FucR and GlpR that regulate sugar catabolism in bacterial systems (van Rooijen, R.J. and de Vos, W.M., J. Biol . Chem . 265:18499-18503, 1990, incorporated herein by reference) or the Agrobacterium repressor known as accR that regulates the biosynthesis of agrocinopines and conjugal transfer (Bodman et al . , Proc . Natl Acad Sci USA 89:643-647, 1992 incorporated herein by reference) . Any other repressor may be employed within the scope of this invention.
- repressors can be employed including those found in fungi such as yeast or any other organism.
- a gene expression mechanism capable of inhibiting the expression of a gene is the LexA system (U.S. Pat. No. 4,833,080, incorporated herein by reference) .
- the LexA mechanism utilizes a repressor and a specific DNA operator sequence to control gene expression.
- Other sources may include a yeast derived system (Mett et al . , Proc. Natl. Acad. Sci. USA 90 4567 - 4571, 1993, incorporated herein by reference) a mammalian derived gene expression system (Schena et al . , Proc. Natl. Acad. Sci.
- the repressor may be capable of binding a specific DNA sequence that can be inserted into the transcriptional control sequences of the oncogene (s), the binding capable of substantially inhibiting the expression of the oncogene.
- DNA binding proteins that have been modified to bind strongly to specific DNA sequences, so called "transdominators" may also be employed.
- Other repressors may include antisense RNA directed to the oncogene.
- Other repression strategies aimed at elimination of gene expression can be employed within the scope of the method.
- Three predominant methods have been described for achieving inhibition of gene expression, anti-sense RNA, co-suppression and ribozyme technologies. Methods for utilizing antisense RNA have been described for example U.S. Pat. No. 4,801,540 and U.S. Pat. No. 5,107,065, incorporated herein by reference.
- the process of co-suppression of gene activity is described by Jorgensen et al, 1991, U.S. Pat Nos. 5,034,323 and 5,283,184, incorporated herein by reference.
- the use of ribozymes is described by Cech et al U.S. Pat Nos. 4,987,071 and 5,116,742,
- method of the present invention can be used to derive transgenic plants.
- the invention finds utility across a range of species and plant varieties. Any plant species capable of being transformed by wild type Agrobacterium can be subjected to the present method and morphologically normal transformed plants can be easily obtained. Any number of repression schemes can be used including repression of gene activity, elimination of oncogenes through recombination or transposition, or addition of a protein that negates the activity of the oncogenes. This could include a protein capable of binding the products of the oncogene, such as auxin or cytokinin binding proteins, or an enzymatic activity capable of conjugating or metabolizing products of the oncogenes, such as an auxin conjugating enzyme or a cytokinin conjugating enzyme.
- the method finds utility across a range of crops and for various purposes, including the introduction of novel genetic traits .
- the oncogene activity is repressed in a reversible fashion.
- a combination of an oncogene and a repressor gene allows normal plant cell growth and development leading to plants with normal morphological characteristics. Separation of the two genetic constructs, for example via segregation or crossing over leads to de-repression and morphologically abnormal plants which can be easily discriminated.
- the method may provide utility for maintaining certain genetic combinations.
- a gene encoding a novel trait is linked to the oncogene introduced in the first transformation step or linked to a repressor gene introduced in a second transformation event.
- more than one gene encoding a novel trait is linked to the repressor and the oncogene, thus permitting a convenient means to assemble and maintain a variety of genetic traits in a single plant line. For example, a number of novel traits could be added individually to a cell line that is derived from a crown gall. Each of the novel traits would be linked to a repressor molecule that inhibits only one or two of the oncogenes present in the cell line.
- Morphologically normal plant cells could only form upon introduction of all of the required oncogene repressors linked to the various novel traits. In this fashion, simultaneous introduction of a predetermined number of novel traits would be a prerequisite for regeneration of morphologically normal plant cells. In this fashion multiple genes could be introduced into plant cells and recovery of plants containing all of the novel traits would be inherent in the system.
- the method allows for the selection of plant cells first obtained by the natural ability of Agrobacterium to efficiently transfer DNA containing one or more active oncogenes or the entire oncogenic region, thus producing a population of plant cells containing oncogenes, which can grow under conditions insufficient for the growth of untransformed cells. Once these cells are established, a cell line useful for the introduction or any number of transgenes is derived.
- the method then employs a second DNA transfer event which utilizes a genetic construct capable of inhibiting the activity of one or more of the oncogenes such that plant cells containing the second DNA are capable of regenerating into morphologically normal plants.
- a second DNA transfer event which utilizes a genetic construct capable of inhibiting the activity of one or more of the oncogenes such that plant cells containing the second DNA are capable of regenerating into morphologically normal plants.
- selection via the use of traditional toxic selection may or may not be used.
- Certain aspects of the method also rely on the controlled expression of a DNA construct that permanently deletes the oncogenes to allow regeneration to take place.
- the DNA construct encoding the deletion function may be included in the initial construct or may be added to the plant cell via subsequent transformation event.
- the oncogenes of the tumorigenic Agrobacterium strains have been extensively studied. Generally, there are two types of oncogenes on the Agrobacterium plasmid: the tmr oncogene and the tms oncogenes.
- the tmr oncogene also known as the ipt gene
- tms (comprising tms oncogene 1 and tms oncogene 2) encode enzymes responsible for auxin overproduction in suitable hosts, leading to the production of roots.
- tms and tmr genes usually lead to the production of a form of crown galls on suitable hosts.
- oncogenes found within the T-DNA act as modifiers or potentiators of crown gall formation.
- plant cells containing oncogenes contained within an intact T-DNA exhibit hormone-independent growth in culture.
- Said plant cells in culture usually are in a de-differentiated state, e.g. callus, said callus capable of growth without exogenous phytohormones.
- oncogene function is inhibited, the plant cells are then able to differentiate into cells capable of regeneration under appropriate conditions, in some cases regeneration occurs upon the crown gall callus itself.
- the entire complement of genes encoded within the T- DNA region is used rather than modification of the region to delete one or more oncogenes. This will include the other oncogenes within the T-DNA region including those specifying the synthesis of opines and other T-DNA encoded products.
- the present method takes advantage of the fact that regeneration of normal plants from oncogene containing callus may require no additional manipulation other than elimination of oncogene activity.
- revertants e.g. shoots or roots have been have been have been observed to form from oncogene containing callus in culture. Usually these structures are formed from cells that have lost the oncogene function through mutation.
- U.S. Pat. No. 4,658,082 describes a method for the selection of such shooty revertants as a means of using in vivo infection of plant tissue to derive plants containing heterologous DNA.
- 4,658,082 does not contemplate the repression of oncogene function with a second transformation event nor does it allow one to conveniently recover plants that are morphologically normal.
- the methods described in U.S. Pat. No. 4,658,082 anticipate recovery of plants where the tumorogenic DNA (e.g. oncogenes) are randomly lost during culture or by mutation or other random events. This method does not allow efficient transformation nor does it allow for selection of plant cells that contain one or more oncogenes, the cells capable of regeneration to morphologically normal plants.
- the recovery of plants as described in U.S. Pat. No. 4,658,082 relies on the use of T-DNA conferring a shooty phenotype. The shoots are not morphologically normal.
- plants which contain one or more of the
- Ti oncogenes are phenotypically abnormal having crown gall tumors or curled and twisted leafs due to growth hormone imbalance. These abnormal plants are unsuitable for commercial applications. Accordingly, the art specifies modification of the Agrobacterium Ti plasmid in a variety of ways, typically by removal of the oncogenes, to become a tool for the introduction of DNA into plant cells. Generally, Agrobacterial transformation methods that have been used to date have used Ti plasmids in which the genes that result in the formation of cytokinins and auxins, other open reading frames within the T- DNA of unknown function and the genes for opine synthesis have been removed. Such plasmids are generally referred to as being "dis-armed”. Accordingly, an "armed" Ti plasmid is generally considered to contain a gene normally found within the T-DNA or a gene called an oncogene. The present invention contemplates the use of "armed" plant vectors.
- the present method also finds utility for the transformation of other crop species that are difficult to transform by conventional means using dis-armed plasmids and selection.
- crops include sunflower, cotton, soybean, safflower, for example.
- the method allows for the formation of a population of cells essentially completely transformed and these cells can then be induced to delete or inactivate the oncogenic activity of the T-DNA region, making regeneration possible without application of selection.
- This method provides a convenient means to recover a morphologically normal transgenic plant from any crop species capable of being transformed by wild-type Agrobacterium . The ability to generate plant cells free of selectable markers is of great value to the industry.
- the invention finds utility for transformation of a wide range of species including: Brassica oleracea species such as broccoli, cabbage, cauliflower, kale, Chinese kale, collard, and kohlrabi; Brassica rapa species including Chinese cabbage, pak choi, and turnip.
- Brassica rapa species including Chinese cabbage, pak choi, and turnip.
- the invention also finds utility for transformation of other vegetable crops such as: Tomato ⁇ Lycopersicon esculentum) , Cucumis spp. , including C. melo (melon) and C. sa tivus (cucumber) , Cucurbita spp. (squash) , including C. maxima and C.
- pepo Spinach ( Spinacia oleracea ) , Carrot (Daucus carota ) Peppers including Capsicum spp. (pepper) , including C. annuum, C. frutescens, and C. chinense, onions such as Allium spp. (onion) , including A. cepa (bulb onion) and A. fistulosum (bunching onion) , Radish (Raphanus sa tivus) , Watermelon ( Ci trullus lanatus) , and Lettuce ⁇ Lactuca sa tiva ) .
- the invention also finds utility for transformation of onamental species such as Impatiens, Pansy [ Viola x wi ttrockiana ) and Lisianthus ⁇ Eustoma grandiflorum) .
- the present method differs from the art in many ways .
- the present invention does not require the construction of a vector where the oncogenic region of the T-DNA is deleted from within the Ti-plasmid.
- the present method also allows for the use of the natural form of the Ti-plasmid rather than the use of a binary or co-integrate type plasmids.
- the present invention may utilize the entire natural DNA transfer process and in the most elemental embodiment comprises the entire oncogenic region of the Ti-plasmid and the wild-type T-DNA that carries all of the genes required for tumor formation, not just one (e.g. the cytokinin biosynthesis gene as described by Ebinuma et al , ibid) .
- Whole plants, plant explants or plant cells can be conveniently transformed with armed Agrobacterium strains.
- cells may be selected on the basis of tumor formation and growth on hormone free media, plants can not regenerate from transformed cells until the tumor forming genes are eliminated or their activity inhibited.
- the use of certain oncogenes, e.g., the oncogene 4 is used as a visual marker for transformed cells, as transformed cells form morphologically abnormal shoots.
- transformed cells are unable to undergo any differentiation and are first identified by the formation of undifferentiated tissue (e.g. crown gall).
- undifferentiated tissue e.g. crown gall
- normal plants are regenerated in a single step from this tissue by elimination of the activity of the tumor forming genes, without the need for a selectable marker.
- the present invention combines the natural T-DNA transfer process, the known activity of the oncogenic T-DNA region and the ability to conveniently identify crown gall tissue with new techniques in molecular biology to derive a novel transformation process that incorporates the efficiencies of the natural process with techniques that allow for the convenient transformation and recovery of morphologically normal plants from many different plant species.
- the transformed plants produced in this fashion will have stably inserted into their genome a DNA region composed of a right and left T-DNA border flanking a novel DNA sequence heterologous to plant cells and at least a portion of an oncogene from the oncogenic region of a Ti-plasmid. In some instances the entire complement of oncogenes normally found in the T-DNA will be present. In other embodiments only a portion of an oncogene will remain. Thus plants produced may at least contain oncogenic regions from the T-DNA but will be capable of normal growth and exhibit normal morphology. These plants are capable of transferring the inserted DNA through normal reproductive methods to progeny and can produce seed containing the inserted DNA. The sexual transfer of the inserted DNA allows for introduction of the inserted DNA into other sexually compatible plant species or varieties.
- the modification of the Ti-plasmid is carried out by the construction of a vector comprising a recombinase site and regions of homology that permit the introduction of the site by homologous recombination within the Agrobacterium strain.
- the vector used in the construction of a wild- type C-58 Ti-plasmid modified to contain, at the right border region, DNA sequences recognized by a site-specific recombinase.
- pRBC-1 Light Border Construct
- the common plasmid vector pGEM-7Zf (+) (Promega,
- pNBEAmp The ampicillin resistance gene was isolated from pBR322 using PCR primers (Seq ID Nos 25 and 26) The ampicillin gene (Seq ID No 11) was added to pNBE as shown in figure 28. The resultant plasmid was called pNBEAmp.
- the FRT recombinase recognition sites were added to pNBEAmp by annealing two single stranded DNAs (Seg. ID Nos 29 and 30), and the resultant annealed double-stranded DNA was inserted into pNBEAmp to derive pAmpFRT(R) as shown in figure 28.
- a 35S polyadenylation signal (Seq ID No 10) was constructed from the plasmid pFF19 (Timmermans et . Al., J. Biotech 14:333-344, 1990) as outlined in figure 29 by the modification of pFFl9 to form pFFl9M by the elimination of the Sma I - Sph I region of the polylinker as shown.
- a visible marker gene was included for convenience of monitoring the transformation process.
- the visible marker gene which is optional for this construct, comprised the GUS- NPTII fusion from the plasmid pGKK14, (Seq ID No.
- the GUS-NPTII fusion gene was cloned into pGEM4Z as described in figure 30, using the Bam HI and Eco RI sites to form the plasmid called pGus :NPTIIter .
- the plasmid was digested with Sac I and Eco RI and the polyadenylation signal from pFFl9-M was added as a Sac I - Eco RI fragment. This produced the plasmid pGUS-pA, which comprises the GUS gene and the 35S polyadenylation signal. This sequence of manipulations is described in figure 30.
- the next series of manipulations involved the recombination of the GUS and NPTII gene in pGUS-pA by digesting pGUS-pA with Sac I and adding the Sac I fragment from pGUS:NPTIIter to derive the plasmid pGusNPTIIpA.
- To the plasmid pGusNPTIIpA was added the ampicillin gene linked to the FRT sites from the plasmid pAmpFRT (R) as shown in figure 32. This produced the vector pAmpFRT (R) Gus, shown in figure 32.
- the first is the vector pUM21, derived from pUK21 (GenBank ace. AF223640) by addition of the basis of mobilization site from a derivative of pBR322 (pTeasy, Promega, Madison, WI . , USA) using PCR primers (Seq ID Nos. 13 & 14) to derive a Sac II - Pci I fragment (Seq ID No 12) that was cloned as shown in figure 22.
- the resulting plasmid pUM21 has the pBR322 mobilization sequence.
- ROH right region of the right border
- the plasmid pROH(R R ) was digested with Eco RI and the Eco RI fragment of pAmpFRT (R) Gus was added to derive pAmpFRT(R)GusROH(R R ) .
- This plasmid was digested with Not I and Sal I and the Not I - Xho I fragment from pTeasy-RL was added. This fragment is derived from PCR amplification of the left region of the region of homology from the right border to the T-DNA from the Ti-plasmid in the Agrobacterium strain C58 (Seq ID No 4) and was isolated by PCR amplification with two primers (Seq ID Nos.
- the vector pRBC-1 comprises, in the 5' - 3' direction, the following components: a DNA sequence representing the left portion of the region of homology for the right border of the T-DNA region of the Ti-plasmid from the Agrobacterium strain C58, a FRT recombinase recognition site, an ampicillin marker gene, a second FRT site, a promoter-less GUS-NPTII gene, a 35S polyadenylation signal, a region of DNA representing the right region of the region of homology from the right border of the T-DNA region of the Ti-plasmid from the Agrobacterium strain C58.
- the detailed restriction map is shown in figure 34.
- This example describes the introduction of the plasmid pRBC-1 into C58 Agrobacterium .
- the pRBC-1 vector as used to insert a sequence into the Ti-plasmid of the strain C58 by a combination of triparental mating (Rogers et al . , Methods
- pRBC-1 in the E . coli strain DH5FT was combined with Agrobacterium C58 and a E. coli helper strain HB101 carrying the wide host range plasmid pRK 2013. Cells are plated on minimal media and the Agrobacterium is selected for resistance to the antibiotic carbinicillin, a structurally related analog of ampicillin and rifampicin as well as sensitivity to kanamycin.
- the resultant vector comprises a wild-type C- 58 Ti-plasmid modified to contain a DNA sequence recognized by a site-specific recombinase at the right border of the T-DNA, (and optionally a marker coding region) .
- the inserted DNA also contains an ampicillin resistance gene flanked by two FRT sites .
- the FLP recombinase gene is expressed in trans to eliminate the ampicillin gene.
- the FLP recombinase gene is modified to remove the intron found in the plasmid vector pOG44, and the gene is placed under the control of a bacterial promoter such as that found in the vector pLEX (Invitrogen, www.invitrogen.com) as a Bgl II - Xho I fragment og POG44 into the Bam HI - Xho I region of the pLEX plasmid.
- the plasmid is then introduced into Agrobacterium by triparental mating as described above, or alternatively by direct DNA transfection, and the cells are plated on minimal media without antibiotics.
- the pOG44 vector can be used directly since the typical "leakage" of eukaryotic coding sequences in bacterial cells may be sufficient to provide a level of recombinase protein capable of causing excision of the amp marker.
- the preferred method is to have the recombinase coding sequence under the control of an inducible bacterial promoter.
- the cells are then replica plated on ampicillin containing media and cells are chosen that are ampicillin sensitive.
- the arrangement of DNA as a result of the activity of the FLP recombinase is verified by a combination of restriction mapping and PCR analysis.
- the plasmid containing the recombinase gene is lost since it is unable to be maintained in Agrobacterium .
- the modified Ti-plasmid is referred to as pTI-C58 RBC-1.
- the resultant Agrobacterium strain is called C58 RBC-1 and carries a nopaline Ti-plasmid modified to contain a FRT recombinase recognition site at the right border region of the T-DNA.
- This example demonstrates use of the strain C58 RBC-1 to produce transformed plant cells.
- the C58 RBC-1 strain of Agrobacterium is used to form tumors on inoculated plants to demonstrate that the modification does not inhibit the oncogenic activity of the Ti- plasmid.
- sterile seedlings of various Brassica (napus , rapa , oleracea and carinata ) species and tobacco are inoculated with an overnight cultures of the Agrobacterium strains C58 and C58 RBC-1 by wounding the stem with a sterile needle containing the Agrobacterium .
- Tumor formation is scored and no difference is observed between the rate of tumor formation with the wild-type and the C58 strain with a modified right border.
- This example illustrates construction of a vector for the introduction of a recombinase site in the left border region of the Ti-plasmid from the wild-type C-58 nopaline Agrobacterium .
- the construction of a vector to introduce a second recombinase recognition site into a C-58 Ti- plasmid (pTi-C58 RBC-1) modified to contain, at the right border regions, DNA sequences recognized by a site-specific recombinase.
- addition DNA sequences are introduced that include a recombinase encoding gene, modified for expression in plant cells, under the control of an inducible promoter.
- the vector used to introduce the sequences into the left border region of the Ti-plasmid also comprises a series of unique restriction sites convenient for the introduction of a gene encoding a novel trait into the Ti-plasmid along with the recombinase site.
- the vector is called pLBC-1 (Left Border Construct), is capable of acting as a "shuttle vector" for the introduction of genes encoding novel traits and its assembly is detailed below.
- the pLBC-1 vector was constructed for introduction of the recombinase region via homologous recombination with the pTi-C58 RBC-1 Ti-plasmid, with the region of homology being targeted to the left border region.
- a number of cloning steps were employed. The steps are outlined in figures 23 - 26.
- the "shuttle vector" pLBC-1 is used to introduce the specific DNA sequence (FRT) recognized by the recombinase into the modified Ti-plasmid pTiC58 RBC-1.
- pLBC-1 comprises: a region of homology with the left border region of the Ti- plasmid (ROH(L L ) (Seq ID No. 1), a plant expressible promoter (NosPr) (Seq ID No. 7), a recombinase recognition sequence (FRT) , a multiple cloning site, an inducible promoter (CiPr) (Seq ID No.
- the plasmid pNBE was used to clone the Amp resistance gene to derive the plasmid pAmp.
- the plasmid pAmp was digested with Eco RI and Kpn I and the NosTer fragment was added to derive pAmpTer (figure 23) .
- This plasmid was then digested with Kpn I and the modified pFLop recombinase gene was added as a Kpn I fragment.
- the pFLoP gene was constructed as shown in figure 10.
- the FLP recombinase was modified to contain a Arabidopsis eEF-lB intron (Gidekel et al., Gene 170:201-206, 1996), referred to as intron (e) , by the insertion of a synthetic intron sequence (Seq ID No 6.) into the Pst I site of the FLP recombinase (Seq ID No 5.) to derive a plant expressible FLP recombinase with a plant intron, said intron containing multiple stop signals to eliminate expression in bacterial hosts through read-trough expression.
- the plasmid that incorporates the Amp, NosTer and pFLoP gene is called pAmpTerFLoP and is shown in figure 23.
- pLBC-1 Another intermediate vector for the construction of pLBC-1 was constructed as outlined in figure 24.
- the vector pNBE is digested with Sph I and Xba I and the Sph I - Xba I fragment of pTeasy-LL is added.
- PTeasy-LL is a vector that was derived by cloning the PCR product representing the left hand-side of the left border region of homology using the PCR primers described in Seq ID Nos. 21 & 22.
- the plasmid that incorporates the pTeasy-LL fragment and the pNBE vector is called pNBE-ROH (L L ) .
- pAmpFRT(R) To construct pAmpFRT(R), pNBE and the Amp resistance gene were used to construct pNBEAmp. This plasmid was digested with Bgl II and Bam HI and a synthetic double stranded DNA comprising the FRT sites (Seq ID Nos 29 & 30) was added to derive pAmpFRT (R) .
- FIG. 25 Another series of intermediate plasmids were constructed as outlined in figure 25.
- pUM21 was used to clone the right-hand side of the region of homology from the Left Border region (ROH(L R )) following PCR amplification using primers Seq ID Nos. 23 & 24.
- the resultant plasmid was called pROH(L R ) .
- To this plasmid was added the cold-inducible promoter from Brassica (Seq ID No 9) following PCR amplification using primers Seq ID Nos. 27 & 28.
- the resultant plasmid was called pCiPrROH (L R ) .
- To this plasmid was added a Bgl II fragment from pAmpTerFLoP ( Figure 23) to derive pCiPrFLoPTerAmpROH(L R ) .
- the vector pLBC-1 contains: a region of homology with the left border region of the Ti-plasmid, a plant expressible promoter, a recombinase recognition sequence, a multiple cloning site, an inducible promoter, a modified FLP recombinase gene containing a plant intron, a polyadenylation signal, an ampicillin resistance gene and a region of homology with the left border region of the C-58 Ti-plasmid.
- the restriction map of pLBC-1 is shown in figure 27.
- This example illustrates the introduction of the plasmid pLBC-1 into C58 RBC-1 Agrobacterium .
- the pLBC-1 vector as used to insert the second recombinase site and associated DNA components into the pTi-C58 RBC-1 Ti-plasmid of the strain C58 RBC-lby a combination of triparental mating and homologous recombination as described in example 2.
- pLBC-1 in the E. coli strain DH5FT was combined with Agrobacterium C58 RBC-1 and a E. coli helper strain HB101 carrying the wide host range plasmid pRK 2013. Cells are plated on minimal media and the Agrobacterium is selected for resistance to the antibiotics carbenicillin and rifampicin and sensitivity to kanamycin.
- the resultant vector comprises a wild-type C-58 Ti- plasmid modified to contain a DNA sequence recognized by a site-specific recombinase at both the right border and left border of the T-DNA, (and optionally a marker coding region) .
- the plasmid further comprises a recombinase gene, under the control of a plant promoter inducible by cold treatment.
- the vector further contains a promoterless GUS-NPTII gene which contains at the 5' end a FRT site, and a nos promoter at the left border region, bounded at the 3' end by a FRT site, such that successful excision of the oncogenic region will eliminate the oncogenic effect and lead to the joining of the nos promoter and the GUS-NPTII gene, thus conferring a visible phenotype on the transformed cell.
- the modified Ti-plasmid is referred to as pTi-C58 CIMB (Cold Inducible Modified Borders) .
- This example illustrates the construction of a second vector for the introduction of a recombinase site in the Left border region of the Ti-plasmid from the wild-type C-58 nopaline Agrobacterium .
- the control of the expression of the recombinase enzyme contained within the modified Ti-plasmid can be by many different means.
- a cold inducible promoter was employed.
- an inducible promoter system based on the tet repressor is employed within the pLBC-1 vector.
- the vector containing this alternative gene control system is called pLBC-2.
- Said second vector pLBC-2 differs from the first vector described (pLBC-1) in the type of inducible promoter used to control the expression of the recombinase gene.
- the pLBC-2 "shuttle vector" as is used to introduce the specific DNA sequence (FRT) recognized by the recombinase into the modified Ti-plasmid pTiC58 RBC-1 as described previously.
- the shuttle vector is called pLBC-2 and comprises: a region of homology with the left border region of the Ti-plasmid (ROH(L L ) (Seq ID No. 1), a plant expressible promoter (Nos) (Seq ID No.
- a recombinase recognition sequence FRT
- a multiple cloning site an inducible promoter system
- the tet promoter operator system as shown in figure 16 and modified to contain the tCUP promoter (Foster et al, Plant Mol Biol 41: 45-55, 1999)
- FLP modified FLP recombinase gene containing a plant intron
- NosTer polyadenylation signal
- Amp ampicillin resistance gene
- ROH(L R ) Seq ID No. 2
- the arrangement of these components in pLBC-2 are shown in figure 35.
- pLBC-2 follows a parallel path to the construction outlined for pLBC-1, the primary difference between pLBC-1 and pLBC-2 is the inducible promoter system utilized to control the expression of the recombinase gene.
- tet repressor/operator system In pLBC-1 a cold-inducible promoter is used to control the expression of the recombinase gene, in pLBC-2 the tet repressor/operator system is used.
- the tet repressor/operator system has been used in plant cells and typically comprises the modification of a plant promoter to contain a DNA sequence capable of being recognized by the tet repressor protein, in this case 3 copies of the operator sequence are inserted into the 35S promoter as described by Gatz and Quail (Proc. Natl. Acad. Sci. USA 85: 1394-1397, 1988) to result in a 35S promoter capable of being repressed by the presence of a tet repressor.
- tet repressor is controlled by another plant promoter, in the present case the constitutive "tCUP" promoter is used.
- the constitutive "tCUP" promoter is used.
- 35S promoter is used, however since both the repressor and operator sequences would have been in the same vector and both comprise the 35S, another constitutive promoter was employed to eliminate the possibility for re-arrangements and duplications due to the presence of two identical sequences on the same vector.
- the tet repressor system employed in the present invention relies on the constitutive expression of the tet repressor from the "tCUP" promoter to produce repressor molecules capable of repressing gene expression from the 35S promoter modified to contain the tet operator and controlling the expression of the FLP recombinase.
- the tet repressor has been modified to more closely conform to plant codon usage and contains a nuclear localization signal.
- This modified tet repressor, referred to as Tet Rsyn is designed for expression in plant cells.
- the DNA sequence is shown in Seq ID No. 31. Accordingly the expression of the FLP recombinase is permanently shut off under thi.s arrangement of DNA components.
- the tet repression can be relieved by the presence of tetracycline, which binds to the tet repressor and causes it to dissociate from the tet operator.
- expression of the FLP recombinase can be induced by culturing in the presence of tetracycline.
- the construction of pLBC-2 comprised the following steps:
- the vector pNBE was used to clone a Sph I - Xba I fragment of the tCUP promoter to produce the vector pNBE-tCUP.
- To this vector was added the pTET2 construct which comprises the modified coding sequence for the tet repressor and the octopine synthase terminator (OCS ter) .
- OCS ter octopine synthase terminator
- the next steps in the assembly of pLBC-2 utilized a series of components used to derive pLBC-1 as described in example 3.
- the assembly of these components is shown in figure 37.
- the plasmid pROH(L R ) was combined with pAmpTerFLoP to derive pAmpTerFLoPROH (L R ) which was then combined with pROH(L L )NosFRT to derive pROH (L L ) NosPFRTFLoPTerAmpROH (L R ) .
- This plasmid was then combined with the tet repressor system contained in the plasmid pTetRepSys as shown in figure 37 to derive pLBC-2.
- the final restriction map of pLBC-2 is as shown in figure 38.
- This example illustrates the introduction of the plasmid pLBC-2 into C58 RBC-1 Agrobacterium .
- the pLBC-2 vector was used to insert the second recombinase site and associated DNA components into the pTi-C58 RBC-1 Ti-plasmid of the strain C58 RBC-1 by a combination of triparental mating and homologous recombination as described in example 2.
- the resultant Agrobacterium strain is selected for resistance to carbenicillin and rifampicin, as well as sensitivity to kanamycin and the structure of the Ti-plasmid verified.
- the resultant plasmid is called pTi-C58 TIMB (Tet Inducible Modified Borders) .
- This example illustrates transformation of plant cells with pTi-C58 CIMB.
- the modified C-58 CIMB Ti-plasmid is verified for function by inoculation on plant explants and observation of crown gall formation.
- the cold-inducible promoter is inactive at normal temperatures (e.g., 10-20°C) , but highly active at 4°C.
- Upon induction of the recombinase activity by culturing tumor tissue at 4°C overnight, excision of the region contained between the two flanking recombinase sequences occurs, leading to transgenic plant cells comprising the T-DNA border flanking the recombinase recognition DNA sequence.
- These plant cells now have an active GUS-NPTII fusion protein and the number of transformed cells are scored by staining for GUS activity or selection on kanamycin.
- This example illustrates transformation of plant cells with pTi-C58 TIMB.
- the modified C-58 TIMB Ti-plasmid is verified for function by inoculation on plant explants and observation of crown gall formation.
- Upon induction of the recombinase activity by culturing crown gall tissue in the presence of lOuM tetracycline, excision of the region contained between the two flanking recombinase sequences occurs, leading to transgenic plant cells comprising the T-DNA border flanking the recombinase recognition DNA sequence.
- These plant cells now have an active GUS-NPTII fusion protein and the number of transformed cells are scored by staining for GUS activity or selection on kanamycin.
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Priority Applications (5)
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AU31462/01A AU784571B2 (en) | 2000-02-08 | 2001-02-07 | Novel methods and constructs for plant transformation |
US10/182,616 US7112721B2 (en) | 2000-02-08 | 2001-02-07 | Methods and constructs for plant transformation |
JP2001558226A JP2003521940A (en) | 2000-02-08 | 2001-02-07 | Novel methods and constructs for plant transformation |
CA2399469A CA2399469C (en) | 2000-02-08 | 2001-02-07 | Novel methods and constructs for plant transformation |
EP01903550A EP1261725A2 (en) | 2000-02-08 | 2001-02-07 | Methods and constructs for agrobacterium-mediated plant transformation |
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US18106300P | 2000-02-08 | 2000-02-08 | |
US60/181,063 | 2000-02-08 |
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WO2001059086A2 true WO2001059086A2 (en) | 2001-08-16 |
WO2001059086A3 WO2001059086A3 (en) | 2001-12-13 |
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PCT/CA2001/000136 WO2001059086A2 (en) | 2000-02-08 | 2001-02-07 | Methods and constructs for agrobacterium-mediated plant transformation |
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EP (1) | EP1261725A2 (en) |
JP (1) | JP2003521940A (en) |
AU (1) | AU784571B2 (en) |
CA (1) | CA2399469C (en) |
WO (1) | WO2001059086A2 (en) |
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
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EP1264891A1 (en) * | 2001-05-31 | 2002-12-11 | Plant Research International B.V. | Modification of plant genomes by inducible site-specific recombination of transgenes |
WO2004081211A1 (en) * | 2003-03-12 | 2004-09-23 | National Institute Of Agrobiological Sciences | Technique of removing marker gene by transient expression of site-specific recombinase gene |
WO2005121346A1 (en) * | 2004-06-08 | 2005-12-22 | New Zealand Institute For Crop & Food Research | Transformation vectors |
WO2008074116A1 (en) | 2006-12-20 | 2008-06-26 | Alellyx S.A. | Nucleic acid molecules encoding plant proteins in the c3hc4 family and methods for the alteration of plant cellulose and lignin content |
WO2008074115A1 (en) | 2006-12-20 | 2008-06-26 | Alellyx S.A. | Nucleic acid constructs and methods for altering plant fiber length and/or plant height |
EP2450446A2 (en) | 2006-09-13 | 2012-05-09 | 22nd Century Limited, LLC | Increasing levels of nicotinic alkaloids |
US8624083B2 (en) | 2007-05-25 | 2014-01-07 | National Research Council Of Canada | Nucleic acid sequences encoding transcription factors regulating alkaloid biosynthesis and their use in modifying plant metabolism |
EP2682403A2 (en) | 2008-05-08 | 2014-01-08 | Alellyx Applied Genomics | Genes and methods for increasing disease resistance in plants |
EP2698432A1 (en) | 2012-08-17 | 2014-02-19 | University of Copenhagen | Agrobacterium rhizogenes transformation and expression of rol genes in Kalanchoë |
US8791329B2 (en) | 2005-02-28 | 2014-07-29 | 22Nd Century Limited Llc | Reducing levels of nicotinic alkaloids in plants |
CN102741416B (en) * | 2009-12-04 | 2015-05-06 | 淡马锡生命科学研究院有限公司 | Improved medium compositions, selection methods and agrobacterium strains for transformation of plants |
US9422532B2 (en) | 2006-06-19 | 2016-08-23 | 22Nd Century Limited, Llc | Nucleic acid encoding N-methylputrescine oxidase and uses thereof |
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JP7288915B2 (en) * | 2018-10-04 | 2023-06-08 | 株式会社カネカ | DNA constructs used for plant genome editing |
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CA1340766C (en) * | 1984-12-24 | 1999-09-28 | Clive Waldron | Selectable marker for development of vectors and transformation systems in plants |
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- 2001-02-07 CA CA2399469A patent/CA2399469C/en not_active Expired - Lifetime
- 2001-02-07 WO PCT/CA2001/000136 patent/WO2001059086A2/en active Application Filing
- 2001-02-07 EP EP01903550A patent/EP1261725A2/en not_active Withdrawn
- 2001-02-07 JP JP2001558226A patent/JP2003521940A/en active Pending
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Also Published As
Publication number | Publication date |
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AU3146201A (en) | 2001-08-20 |
CA2399469C (en) | 2010-12-14 |
WO2001059086A3 (en) | 2001-12-13 |
EP1261725A2 (en) | 2002-12-04 |
JP2003521940A (en) | 2003-07-22 |
AU784571B2 (en) | 2006-05-04 |
CA2399469A1 (en) | 2001-08-16 |
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