JP2003521940A - Novel methods and constructs for plant transformation - Google Patents

Abstract

(57) Summary The present invention provides a novel method for transforming crop species, a novel method for selecting and identifying transformed plant cells, and a novel method for recovering regenerated whole plants. About the method. The method also relates to the development of plants having novel properties and plants containing the novel recombinant DNA construct.

Description

Detailed Description of the Invention

[0001] Cross-Reference to Related Applications This application claims priority of US Provisional Patent Application No. 60 / 181,063 filed on Feb. 8, 2000, the disclosure herein by reference in its entirety Partial.

TECHNICAL FIELD The present invention provides novel methods and selectable markers for transforming plant species.
A method for producing a transgenic plant species that does not contain a gene.

[0003] development of background transgenic crop invention, a method for stably insert the recombinant DNA, it is necessary generally referred to as transformation process. Transformed plant cells are made by utilizing a variety of methods for inserting foreign DNA into plant cells. Further in these methods, genetic constructs containing genes encoding resistance to chemical selective agents such as antibiotics (eg kanamycin or hygromycin) or herbicides are used. By culturing a group of transformed plant cells in the presence of a medium containing a specific combination of plant hormones and nutrients capable of growing cells and regenerating into whole plants, and a selection agent. Plant cells expressing a selectable marker are selectively obtained. As described above, the method for producing transformed cells requires means for physically inserting foreign DNA and means for selecting cells that have incorporated the DNA in a stable form that can be expressed.

[0004] After the foreign DNA is stably inserted and expressed, cells containing the foreign DNA are regenerated, so that a complete plant is usually recovered after the generation of buds, embryos, and organs. The regenerated plant tissue is induced by several different means, generally by altering the culture conditions of the tissue, more commonly by the manipulation of plant hormones, and passing on foreign DNA to subsequent generations. Form a complete plant capable of forming seeds and / or pollen. In some plant species, there is a major limitation in the efficient selection and regeneration of plant cells after transfer of DNA, as opposed to the actual transfer of DNA into cells. Thus, a practical limitation in using standard transformation methods generally relates to the efficiency of selection and identification of transformed plant cells, not necessarily the transfer of DNA to plant cells. Another step in the often problematic process is the regeneration of transformed cells into whole plants in the presence of selection agents.

One of the potential problems with the selection of plant cells includes the effect of the selection agent on non-transformed cells, ie plant cells that do not contain a selectable marker. Often, the selection process causes the majority of non-transformed cells to die, reducing the regeneration efficiency or frequency of transformed cells for a variety of physiological reasons. Therefore, the selection pressure required to identify cells containing foreign DNA has often been found to be an inhibitor of efficient regeneration of plant cells.

[0006] Thus, methods that allow for selection of plant cells without direct lethal effect on non-transformed cells may provide a means for reliable recovery of transformed plant cells. . Furthermore, it may prove to be more efficient in regeneration, especially in plant species, varieties or cells which are difficult to transform by conventional means. This problem is a major limitation for routine and economical production of transformed plants.

It is believed that the transformation of plant tissue occurs only in a small percentage of the total number of cells exposed to the transformation process that actually incorporates DNA into the nuclear DNA. Upon application of the selection agent, many plant cells die and substances that interfere with the efficient regeneration of these transformed cells by affecting other nearby cells, including cells containing the selectable marker gene (eg, Ethylene or compounds related to aging)
May be released. Death of non-transformed cells may release substances that inhibit the survival of transformed cells. Therefore, a major limitation in efficient production of transformed plants lies in the recovery of transformed plant cells and subsequent regeneration of whole plants. Methods that allow the rapid and efficient recovery of plant cells containing foreign DNA under conditions where the selection process does not kill non-transformed cells (and does not subsequently inhibit regeneration) are particularly costly and time-consuming for transformation. In the known plant species, it seems to provide a simple and efficient means for recovering plants. Many plant species can be transformed with the introduced DNA, but the efficiency of the regeneration process is very low, hindering the development of transgenic crops. In addition, significant mutations in tissue culture potential tissue culture response or regeneration found within species, subspecies or varieties within a particular crop genus severely limit the effective range of current transformation processes. There is.

[0008] The Brassica species are of particular interest. Transformation of plants of the Cruciferae family by Agrobacterium and other methods has been reported. A significant portion of the scientific work describing various methodologies, techniques and studies on transgenic Brassica plants containing useful foreign DNA of commercial interest has been accumulated. A significant effort to achieve this was 1980
It has been done since the early 1980s and is still being done today. Transformation has now evolved in technology to routine genotyped species of Brassicaceae, but remains difficult in other species of Brassicaceae. In addition, there is a strong genotype impact on the successful transformation of Brassica that can successfully transform,
A technology that can be widely applied to "difficult" genotypes will have great industrial value. Although many transgenic Brassica are subjected to field evaluation, analysis of field trial data shows that the majority of transgenic Brassica (> 95%) are of the B. napus type, and most of the materials are B It has been clearly shown to be derived from a limited range of germplasm of .napus. One of the reasons for this is the difficulty of routinely obtaining transformed B.rapa and B.oleracea types and the fact that the development of commercial transgenic plants is relatively recent.

[0009] However, many reports, especially on the transformation of Brassica, detail the difficulty of routinely obtaining transformed Brassica species by Agrobacterium-mediated transformation. Many of these reports have shown success with one or two specific varieties, but do not teach detailed methods generally applicable to all Brassica species. Despite the ability to use multiple manipulations of culture conditions, some varieties have proved to be very difficult to transform by previously reported methods.

Much of the early work on Brassica transformation was performed using B. napus cv Westar (Radke et al, Theor. Appl. Genet. 75: 685-694, 1988; Moloney et a.
l., Plant Cell Reports 8: 238-242, 1989; Moloney et al., 1989, US 5,188,
958; Moloney et al., 1989, US 5,463,174). Westar was a convenient choice because it responds to the tissue culture and transformation protocols described in the above references and allows the recovery of transgenic plants. However, many of the Brassica transformation studies, or variations thereof, performed using the above methods have yielded variable results depending on the innate response of the particular plant material to the transformation protocol. As an example, transformation frequencies achieved in Brassica napus are sometimes variable and very low (Fry et al., Plant Cell Reports 6: 321-325, 1987; Mehra-Pal).
ta et al., In Proc Sth Int. Rapeseed Congress, Saskatoon, Saskatchewan,
1991; Swanson and Erickson, Theor. Appl. Genet. 78: 831-835, 1989). Variable and very low transformation frequencies were also observed in the following other Brassica species: eg B. oleracea (Christie and Earle, In Proc 5th Crucifer Genetics Works.
hop, Davis, pp 46-47, 1989; Metz et al., Plant Cell Reports 15: 287-292,
1995; Eimert and Siegemund, Plant Molec. Biol. 19: 485-490, 1992; DeBl
ock et al., Plant Physiol. 91: 694-701, 1989; Berthomieu and Jouanin, Pl.
ant Cell Reports 11: 334-338; Toriyama et al, Theor. Appl. Genet. 81: 76
9-776, 1991); B. rapa (Radke et al., Plant Cell Reports 11: 499-505, 199.
2, Mukhopadhyay et al, Plant Cell Reports 11: 506-513, 1992); B. juncea.
(Barfield and Pua, Plant Cell Reports 10: 308-314, 1991; Deepak et al.,
Plant Cell Reports 12: 462-467, 1993; Pua and Lee, Planta 196: 69-76, 19
95); B. nigra (Gupta et al, Plant Cell Reports 12: 418-421, 1993); and B.
.carinata (Narasimhulu et al., Plant Cell Reports 11: 359-362, 1992; Ba
bic, M. Sc. Thesis, Univ of Saskatchewan, 1994).

Many Brassica species, varieties and cultivated species represent a very diverse group with completely different morphologies and physiological characteristics. In addition to the oilseed Brassica,
The vegetable Brassica is a crop with significant economic value. This value can 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. Thus, an efficient transformation system for broccoli, cabbage, cauliflower, kale, and other cruciferous vegetables would be of value. Many of the Brassica vegetable species of commercial interest do not respond well to the previously described methods, or
Or it does not react at all. There is a need for transformation methods that are effective for virtually all Brassica species, especially those previously rendered cumbersome for transformation.

In addition to the obstacles faced in Brassica, other plant genera have shown similar phenomena. Many commercially important plant species or genera are difficult to transform and typically only a limited range of specific genotypes can be transformed. This includes crops such as cotton, soybean, sunflower, cereal crops such as wheat, barley, rice, corn, and many ornamental and vegetable crops. Often, a wide range of genotypes are culturable and regenerable, but the combination of selection and transformation processes precludes efficient regeneration, leading to very inefficient transformation of elite lines. Thus many transformation processes are performed with limited germplasm, followed by lineage characterization and extensive backcrossing. Many of the transgenic crop varieties produced to date are produced by the transformation of specific varieties that have been demonstrated to be capable of transformation and subsequent large-scale crossing with the optimal varieties for widespread cultivation. It was This characteristic gene transfer is
It takes time and money.

Accordingly, methods capable of efficiently transforming a wide range of crop species and individual varieties are essentially genotype independent and of a variety that would be of significant industrial benefit. Can be used to impart the novel properties of In addition, methods that avoid the use of virulence-selective agents that kill non-transformed cells as a major step in the process are species that are known to be difficult to regenerate efficiently or that are currently not possible. In a variety of plants can provide an opportunity to achieve efficient regeneration of plants.

It is recognized that one of the most efficient transformation processes is the natural infection process of Agrobacterium species. Agrobacterium is free-living
It is a Gram-negative soil bacterium. Toxic strains of this bacterium can infect plant tissues and induce the production of neoplastic tumors, commonly called crown gall. A virulent strain of Agrobacterium is a large plasmid DNA known as Ti-plasmid.
The Ti-plasmid contains the genes necessary for plasmid transfer and replication and a DNA region called T-DNA. The T-DNA region is DN
It is bounded by a T-DNA border sequence critical for the A transfer process. These T-DNA border sequences are recognized by the vir gene encoded on the Ti-plasmid, which is involved in the DNA transfer process. Thus, the native Ti-plasmid contains the vir gene and the T-DNA region and the T-DNA borders required for efficient DNA transfer to recipient plant cells. The entire complement of these genetic components, together with the gene encoded on the bacterial chromosome, allows the T-DNA region to be efficiently transmitted to plant cells, which are then encoded by the various T-DNA encoded sequences. Stably integrates and expresses the gene.

The T-DNA transferred and integrated into the nucleus of plant cells encodes several genes encoding enzymes for producing unusual amino acids (opines), and enzymes capable of producing plant hormones. And genes involved in the regulation of plant development. As such, TD transmitted to plant cells
There are a relatively large number of genes in NA, and all the functions of the genes are not completely elucidated in the genus Agrobacterium. However, the main effect of genes in T-DNA is on the level of plant cell proliferation and development. After infection with Agrobacterium, plant cells infected with T-DNA grow uncontrolled due to the activity of the T-DNA gene. These genes, referred to as “oncogenes” because of the phenotype they confer, cause transformed cells to grow in culture, independent of hormones. Expression of these various oncogenes results in the formation of goal callus, ie callus that is unable to regenerate the structure of differentiated and complex plants such as fertile shoots or whole plants.

The process of goal or tumorigenesis is very efficient, with essentially 100% of the inoculated Agrobacterium-sensitive plant tissue forming tumors. Thus, the natural Agrobacterium DNA transfer process is extremely efficient. Moreover, the physiological process of crown gall formation is also very efficient in conferring a transformed phenotype. Furthermore, many wild type Agrobacterium have a broad host range and are capable of transferring large DNA moieties, typically T-DNA transferred to plant cells is up to 25 kilobases of DNA. (For example, Nopaline strain)
Alternatively, it contains more DNA (eg octopine strain). Thus, Agrobacterium's naturally-occurring DNA transduction system allows plant cells to DN
An efficient means of transmitting A is provided, after which transformed tissue is formed and identified.

Therefore, many methods have been developed for transferring DNA to plant cells using Agrobacterium. Typically, these methods engineer Ti-plasmids that no longer contain genes involved in altered morphology (“oncogenes”), replacing these genes with recombinant genes encoding beneficial properties. It is based on doing. To this beneficial property is linked a gene encoding a selectable marker so that cells which have received the DNA can be selected. Usually, the vir gene is
Although left intact, there are also ways to alter the vir gene in various forms.

There are two main types of plant transformation systems based on Agrobacterium, the binary method and the co-integrate method.
The use of a binary transformation system is described by Schilperoort in US Pat. No. 4,940,838.
as described by et al. An example of a cointegrate system is Fraley et al.
, Biotechnology, 3: 629-635, 1985. Both cointegrate and binary systems, engineered non-tumorigenic DNA, typically DNA containing the oncogene complement of the Agrobacterium gene, typically a selectable marker and a gene encoding a novel property. Is based on replacing. Repetitive sequences at the T-DNA border are maintained in both systems and are
The transfer process is used to transfer the portion of DNA located between T-DNA boundaries into plant cells. As described above, according to these transformation methods, it is possible to avoid the problem regarding the recovery of morphologically normal plant cells, which occurs when the method of inserting the oncogene region of Ti-plasmid into plant cells is used. . Therefore, these methods rely on the presence of a selectable marker to recover transformed cells, as selection based on the activity of oncogenes is no longer available.

It is recognized that the presence of oncogenes in the DNA delivered to plant cells interferes with the regeneration of morphologically normal plants. It is also recognized that various oncogenes encoded by T-DNA play different roles, and the total activity of various oncogenes forms a crown gall.
Each of the major oncogenes independently performs a predetermined function, and the presence of only one or two functional oncogenes typically results in the formation of morphologically abnormal plants. This is in contrast to the crown gall, which is produced by the activity of all oncogenes, and crown gall (or callus derived from crown gall tissue) is unable to regenerate or form morphologically normal plants. . In fact
Crown gall tissue cannot be regenerated into differentiated plant tissue. Therefore, crown gall tissue is considered to represent the final tissue in the Agrobacterium infection process. Thus, it is recognized in the art that the full complement of oncogenes contained within T-DNA eliminates the possibility of recovering morphologically normal plants. There is.

However, it has been shown in the art that one or two oncogenes can be included in a plant transformation vector and the activity of these oncogenes can be used as a method for screening transformed cells. There is. For example, Ebunuma et al.,
(Proc. Natl. Acad. Sci. USA 94: 2117-2121, 1997, US 5,965, 791), as a means for identifying transformed plant cells (typically oncogene 4
A plant transformation vector containing the isopentyltransferase gene from Agrobacterium). In this way, the activity of the oncogene causes the production of cytokinin, a plant hormone that causes the production of shoots. In this case, a "shooty" mutant is produced (
Morphologically abnormal shoots), plant cells are selected based on abnormal shoot formation and kanamycin or herbicide resistance. As an additional feature of this method, the oncogene is bounded by a transposable element that ultimately deletes the oncogene from the transformed plant cell. The placement of the DNA in this vector is such that the beneficial gene remains in the transformed plant cell.

This method discriminates between transformed cells and non-transformed cells depending on the activity of oncogene 4, but this method requires visual discrimination of plant shoot regeneration. , This can lead to the formation of chimeric plants, so various tissue culture procedures are required to recover transgenic plants derived from a single cell.

Indeed, the method described in US Pat. No. 5,965,791 relies on the regeneration of morphologically abnormal plants as a transformation marker. Morphological abnormal induction genes cause abnormal plant formation, and in many cases somatic mutations and culture mutations,
Further analysis is required in order to obtain transgenic plants in full because they lead to effects on the same phenotype. As such, this method is limited and cannot provide a convenient means for transforming cumbersome plant species or varieties. Furthermore, this method relies on the use of a dis-armed Agrobacterium strain, the efficiency of its transformation of which is correspondingly reduced to the formation of native crown gall.

Similarly, oncogene 2 is used to identify transformed plant cells and to recover the transgenic plants after regeneration and selection of the transgenic cells under appropriate medium or appropriate selection conditions. It has been shown to be usable. According to Keller et al., (International Publication No. WO 00/37060), the isolated oncogene 2 can be used to identify transgenic plant cells after transformation and culturing in selective medium. Proven. Aberrant plantlets are produced in the presence of the oncogene 2 enzyme and a suitable substrate for the enzyme. As mentioned above, the selection of transformed plants depends on the formation of morphologically abnormal structures.

In both of these embodiments, the normal transfer process of DNA from Agrobacterium to plant cells is a binary vector system and dis-armed T.
-Results of modifying a plant transformation vector to include a DNA region. These alterations, though not fully understood in their function if not all of the native sequences found in T-DNA, are thought to play an important role in the efficiency of transduction and integration processes. Removal of most, including many open reading frames and genes is involved. These utilized oncogenes have been modified to characterize their natural role in the formation of crown gall tissue outside of the normal environment.

However, the natural T-DNA transduction process with wild-type T-DNA and subsequent formation of crown gall or plant tumors is a highly efficient process. The process is usually performed for plant species or varieties (eg, various Brassica species or cotton) that are cumbersome to conventional tissue culture protocols for introducing DNA with Agrobacterium and recovering transgenic plants. Very efficient. In many cases, crown gall is easily formed in decapitated plants or injured stems, even in plants with genotypes that are awkward to transform with typical dis-armed vectors. To be done. Crown goal formation is also easily scored, and true goals (ie those with a complete T-DNA region) are unable to form differentiated plant tissue. Spontaneous shoots or roots can be formed using several Agrobacterium strains, but usually crown gall differentiates unless one or more coding oncogenes undergoes mutation or loss of function. It does not form cells, even differentiated morphologically abnormal cells. Crown gall can also be easily cultured by simply growing it in a hormone-free minimal medium. Crown gall can be made bacteria-free by culturing it in an antibiotic solution for a certain period of time. Thus, obtaining bacterial-free transformed plant cells from many different plant species containing complete tumorigenic T-DNA is a simple process.

Thus, in addition to a simple means of identifying transformed cells (by hormone independent culture growth), plant transformation methods that can take advantage of this efficient process are Many advantages can be provided, including the efficiency of selection of transformed plant cells. However, the art teaches that it is not possible to recover morphologically normal plant cells using naturally occurring oncogene regions. Thus, a method of recovering morphologically normal plant cells after the formation of crown gall is not useful, and as a means of producing morphologically normal transformed plants from a wide range of plant species, natural The efficiency and wide host range of the T-DNA transfer process cannot be used.

SUMMARY OF THE INVENTION The present invention provides a novel method for producing and recovering morphologically normal, selectable marker-free transformed plants. The present invention utilizes the efficient and convenient T-DNA transduction process found in wild-type Agrobacterium to produce plant cells that can grow under hormone-free conditions, thereby allowing for simple culture conditions. Form cells that can be easily identified by their visual characteristics. Then, plant cells are selected from the non-transformed cells to induce regeneration into a complete plant.

The method of the present invention can avoid the selection of plant cells with antibiotics or herbicides, especially for plant species that are difficult to transform with standard Agrobacterium and selection techniques. Provide an efficient means of recovery.

A wide range of methods of the present invention utilize the transformation of plant cells with Agrobacterium oncogenes that contain oncogenes and are deficient in the growth of non-transformed cells (eg, plant growth). Produce a cell population capable of growing under conditions lacking hormones). Thereby, transformed cells are selected. Preferably, a full complement of oncogenes found in wild-type Agrobacterium T-DNA.
transform the cells.

Transformed cells can grow under conditions insufficient to grow non-transformed cells, but the presence of the oncogene causes the transformed cells to regenerate and form morphologically normal plants. Doing is usually hindered. Therefore, the transformed cells may then be further modified to remove the function of the oncogene, thereby restoring their ability to regenerate and proliferate normally, resulting in the recovery of morphologically normal plants. it can. Accordingly, in a broad aspect, the present invention provides a method for producing a transformed plant, which comprises (a) introducing a construct containing at least one Agrobacterium oncogene into a plant cell to obtain a transformed cell containing the construct. The expression of the Agrobacterium oncogene in the transformed cells allows the cells to grow under conditions insufficient to grow non-transformed cells, thereby selecting transformed cells. And (b) counteracting the effect of said at least one Agrobacterium oncogene in said transformed cell and regenerating a morphologically normal transformed plant from said transformed cell. .

In one aspect, the construct comprises, in the 5 ′ to 3 ′ direction, a first recombinase recognition site, at least one Agrobacterium oncogene, and a second recombinase recognition site. At this time, the step of canceling includes excising the Agrobacterium oncogene or a part thereof from the construct using a recombinase that recognizes the first and second recombinase recognition sites. The recombinase may be encoded by the recombinase coding sequence contained in the original construct containing the oncogene, or may be introduced into the transformed plant cell in the state contained in the second construct. The recombinase coding sequence may be under the control of an inducible promoter or may include a plant intron.

Preferably, either the oncogene-containing construct or the second construct comprises a novel property coding sequence which confers on the plant a new property when expressed in the plant. When the novel characteristic coding sequence is included in a construct containing one or more Agrobacterium oncogenes, transformed cells under conditions in which non-transformed cells cannot grow (eg, in the absence of plant growth hormone). Due to their ability to grow, cells transformed with the novel characteristic coding sequence are selected, either in the absence of plant growth hormone or under conditions of hormone levels insufficient to grow non-transformed cells. The transformed cells grow at. Therefore, the use of additional selectable markers is not necessary.

In a particularly preferred method, the oncogene-bearing construct comprises, in the 5 ′ to 3 ′ direction, a T-DNA right border sequence, a first recombinase recognition site, at least one oncogene, a second recombinase recognition site. , And a modified Agrobacterium T-DNA region containing the left border sequence of T-DNA. Preferably, the construct is introduced into plant cells by Agrobacterium-mediated transformation.

In another aspect, the recombinase recognition site is replaced with a transposase recognition site and the transposase enzyme that recognizes the transposase recognition site is
It is used to excise one or more oncogenes or parts thereof.

[0035] In yet another aspect, the counteracting step comprises contacting a construct containing an Agrobacterium oncogene with a DNA binding protein and suppressing the expression of the product encoded by the oncogene. The construct containing the Agrobacterium oncogene may comprise a DNA binding protein coding sequence encoding a DNA binding protein, or the DNA binding protein coding sequence is transformed into a plant cell in a second transformation. It may be included in the second construct introduced.

In yet another aspect, the counteracting step comprises contacting the Agrobacterium oncogene or transcript thereof with an antisense polynucleotide that suppresses transcription of the oncogene or translation of the transcript. .

The use of recombinases, transposases, DNA binding proteins, and antisense polynucleotides is just one part of a suitable method for counteracting the effects of Agrobacterium oncogenes in transformed cells according to the present invention.
Other methods for counteracting the effects of the Agrobacterium oncogene will be apparent to those of skill in the art, including the use of co-suppression or ribozyme technology.

The method of the invention is suitable for use in monocots or dicots, preferably dicots. Preferred plants include members of the mallow family, flax family, Asteraceae family, Solanaceae family, legume family, Euphorbiaceae family, Astragalus family, or Brassicaceae family. Preferably, the plant is a member of the genus Brassica, for example broccoli, cabbage, cauliflower, kale, Chinese kale, collard, kohlrabi, Chinese cabbage, thai rhinoceros (pak choi).
Alternatively, it is a turnip, but is not limited thereto.

The present invention also provides plants, plant seeds, plant embryos, plant cells, or plant tissues produced according to the methods of the present invention.

The invention also provides various vectors, constructs, and modified Ti-plasmids useful for practicing the methods of the invention.

In one aspect, the invention provides for the TD of Ti-plasmid in the 5'-3 'direction.
A DNA sequence homologous to the left part of the right border region of the NA region, a recombinase recognition site,
And DNA homologous to the right part of the right border region of the T-DNA region of Ti-plasmid
A DNA vector containing the region of

In another aspect, the present invention relates to the Ti-plasmid TD in the 5′-3 ′ direction.
A DNA sequence homologous to the left part of the left border region of the NA region, a recombinase recognition site,
And DNA homologous to the right part of the left border region of the T-DNA region of Ti-plasmid
A DNA vector containing the region of

The DNA vector may further contain an antibiotic resistance marker useful for selection in Agrobacterium.

In another aspect, the present invention provides a modified Ti-plasmid containing a recombinase recognition site 3 ′ to the right border of T-DNA.

In yet another aspect, the present invention provides a modified Ti-plasmid containing a recombinase recognition site 5 ′ to the left border of T-DNA.

In yet another aspect, the present invention provides a modified Ti-plasmid containing a recombinase recognition site 3 ′ to the right border of T-DNA and a recombinase recognition site 5 ′ to the left border of T-DNA. provide.

The modified Ti-plasmid may further comprise a recombinase coding sequence expressible in plant cells and / or may further comprise a scorable marker coding sequence expressible in plant cells. You can leave.

Particularly preferred vectors and plasmids include, as described herein, the following: modified Ti-plasmid pTi-C58 CIMB; modified T.
i-plasmid pTi-C58 TIMB; modified Ti-plasmid pTi-
C58 RBC-1; DNA vector pRBC-1; DNA vector pLB
C-1; and DNA vector pLBC-2.

The invention further provides a construct for transforming a plant cell, which comprises in the 5'to 3'direction a first recombinase recognition site, at least one oncogene, and a second recombinase recognition site. To do. The construct may further include a recombinase coding sequence that encodes a recombinase. Preferably, the construct is
A novel characteristic coding sequence located either on the 5 ′ side of the first recombinase recognition site or on the 3 ′ side of the second recombinase recognition site, the expression of said sequence in a plant revealing a novel characteristic to the plant. The new property code sequence to be provided is further included. The recombinase coding sequence may be under the control of an inducible promoter or may include a plant intron.

A particularly preferred construct has, in the 5 ′ to 3 ′ direction, a T-DNA right border sequence, a first recombinase recognition site, at least one oncogene, a second recombinase recognition site, and a T-DNA left border sequence. Containing modified Agrobacterium T-DN
Area A.

In another aspect, the transposase recognition site may be replaced with a recombinase recognition site and the transposase coding sequence may be replaced with a recombinase coding sequence.

The invention also provides a construct comprising a DNA binding protein coding sequence encoding at least one oncogene and a DNA binding protein. Preferably,
The construct further comprises a novel property coding sequence, wherein expression of the sequence in the plant confers on the plant a new property.

The invention also provides a plant, plant seed, plant embryo, plant cell or plant tissue comprising a construct or vector according to the invention.

The invention further provides Agrobacterium cells comprising a construct, vector or Ti-plasmid according to the invention.

The present invention also relates to a method for modifying a Ti-plasmid so that it contains a recombinase or transposase recognition site, wherein (a) a first DNA vector is added to an Agrobacterium cell containing the Ti-plasmid. A DNA sequence homologous to the left part of the right border region of the T-DNA region of the Ti-plasmid, a recombinase or transposase recognition site, and the T
The first DNA vector containing a region of DNA homologous to the right part of the right border region of the T-DNA region of the i-plasmid in the 5'to 3'direction was used as the DNA vector and the Ti-
Agrobacterium containing a modified Ti-plasmid having a recombinase or transposase recognition site near the T-DNA right border sequence in the T-DNA region, which is introduced under sufficient conditions for homologous recombination with the plasmid. And (b) in the Agrobacterium cell from step (a) a second DNA vector, the left part of the left border region of the T-DNA region of the modified Ti-plasmid. A second DNA vector containing a DNA sequence homologous to, a recombinase or transposase recognition site, and a region of DNA homologous to the right part of the left border region of the T-DNA region of the modified Ti-plasmid in the 5'to 3'direction. Is introduced under sufficient conditions that homologous recombination occurs between the second DNA vector and the modified Ti-plasmid,
A further modified Ti-plasmid, the TD of the further modified Ti-plasmid
A recombinase or transposase recognition site is provided near the left border sequence of the T-DNA in the NA region, and the T in the T-DNA region of the further modified Ti-plasmid is provided.
-Providing a method comprising obtaining Agrobacterium cells containing a further modified Ti-plasmid with a recombinase or transposase recognition site in the vicinity of the DNA right border sequence.

Preferably, the Ti-plasmid further comprises within the T-DNA region a recombinase or transposase enzyme coding sequence under the control of an inducible promoter. The Ti-plasmid is scoreable and expressible in plant cells
A marker gene may be further included in the T-DNA region.

DETAILED DESCRIPTION OF THE INVENTION The definitions are set forth below for a clear and consistent understanding of the specification and claims, including the scope given to the terms used herein.

The singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

A “coding sequence” or “coding region” is the amino acid sequence of a protein or part of a gene encoding a functional RNA such as tRNA or rRNA.

A “complementary” or “complementary sequence” is a nucleotide sequence that forms a double strand by hydrogen bonding with another nucleotide sequence according to the Watson-Crick base pairing rules. For example, the complementary nucleotide sequence for 5'-AAGGCT-3 'is 3
It is'-TTCCGA-5 '.

“Expression” refers to the transcription of a gene into structural RNA (rRNA, tRNA), or transcription of the gene into messenger RNA (mRNA) and subsequent translation into protein.

Polynucleotides are said to be ", if they perform substantially the same biological function.
Functionally equivalent ".

Polynucleotides are “heterologous” to each other 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 that organism in the specified form and configuration.

A polynucleotide or polypeptide is "homologous" if they are identical when the sequences of nucleotides or amino acid residues in the two sequences are placed in maximal correspondence, respectively, as described herein. Or “identical” sequences. Sequence comparisons between two or more polynucleotides or polypeptides are generally performed by comparing two sequence portions over all comparison windows and identifying and comparing similar local regions of sequence. The comparison window is generally about 20 to about 200 contiguous nucleotides or contiguous amino acid residues. By comparing the two optimally arranged sequences in all of the comparison windows,
"Percentage sequence identity" in polynucleotides and polypeptides
Or “sequence homology percentage”, eg 50, 60, 70, 80,
Sequence identity of 90, 95, 98, 99, or 100 percent can be determined, where the polynucleotide or polypeptide sequence portion of the comparison window is the (additional sequence for optimal placement of the two sequences). Alternatively, it may contain additions or deletions (ie gaps) as compared to a reference sequence that does not contain deletions.

Percentages are calculated as follows: (a) Determine the number of positions where the same nucleobase or amino acid residue is present in both sequences and give the number of matched positions; (b) Match Divide the number of positions made by the total number of positions in the comparison window; (c) Multiply the answer by 100 to give the percent sequence identity.

Optimal placement of sequences for comparison can be accomplished by computer implementation or inspection of known algorithms. The readily available sequence comparison and multiple sequence placement algorithms are
Local Alignment Search Tool (BLAST) (Altschul, SF et al. 1990. J. Mol.
Biol. 215: 403; Altschul, SF et al. 1997. Nucleic Acids Res. 25: 3
389-3402) and ClustalW programs. BLAST on the internet ht
It is available at tp: //www.ncbi.nlm.nih.gov, and the ClustalW version is
It is available at http: // www2. ebi. ac. uk. Other suitable programs include Wis
consin Genetics Software Package (Genetics Computer Group (GCG), 575 Sci
ence Dr., Madison, WI) GAP, BESTFIT, FASTA, and TFASTA. As used herein and in the claims, the "percentage sequence identity" or "percentage sequence homology" of an amino acid sequence ensures that the default value of the BLASTX program available as described above values)) and the optimal arrangement of sequences.

The term “stringent conditions” or “stringent hybridization conditions” refers to a probe having a higher detectable frequency than other sequences (
Conditions that hybridize to the target sequence at a frequency of at least twice background or more). Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 ° C. below the thermal melting point (Tm) of that particular sequence at a given ionic strength and pH. T
m is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions are those in which the salt concentration is less than about 1.0 M Na ion at pH 7.0-8.3, typically about 0.01-1.0 M Na ion concentration (or other Salt) of at least about 30 ° C. for short temperature probes (eg 10-50 nucleotides) and at least about 60 ° C. for long temperature probes (eg longer than 50 nucleotides). In addition, stringent conditions can also be obtained by adding a destabilizing agent such as formamide. Typical examples of low stringency conditions include hybridization at 37 ° C., 30% formamide, 1M NaCl, 1% SDS in buffer, and washing at 50 ° C., 2 × SSC. Typical high stringency conditions include 37 ° C, 50% formamide,
Hybridization with 1 M NaCl, 1% SDS, and 60 ° C. 0
． Washing with 1 × SSC is included. Hybridization techniques 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 (1) a component that is substantially or essentially a component that normally accompanies or interacts with the material when found in its naturally occurring environment. Or (2) cells that are naturally free of materials that have been artificially transformed into a composition, and / or materials found in that environment, if the material is in its naturally occurring environment. Of material placed in a certain position. Isolated material optionally includes material that is not found with the material in which it naturally occurs. Changes to synthetic materials may be made to or taken from the material in its natural state. For example, a naturally occurring nucleic acid becomes an isolated nucleic acid when modified by an artificial synthetic method performed in the cell from which the nucleic acid is derived or transcribed from the modified DNA. Similarly, a naturally occurring nucleic acid becomes an isolated nucleic acid when introduced by artificial means at a location in the genome where the nucleic acid is not naturally present.

Two DNA sequences are "operably linked" if the nature of the two DNA sequences to join carries out their normal function without interfering with the ability of the sequences. For example, a promoter region is operably linked to a coding sequence if the promoter is capable of effecting transcription of the coding sequence.

A “polynucleotide” is two or more deoxyribonucleotides (DNA) or ribonucleotides (RNA).

A “construct” is a nucleic acid molecule isolated from a naturally-occurring gene, or a nucleic acid molecule that has been modified to contain otherwise linked and juxtaposed nucleic acid molecules that are not naturally occurring. .

[0072]   A "polypeptide" is a sequence of two or more amino acids.

A “promoter” is a cis-acting DNA sequence generally located upstream of the start site of a gene to which RNA polymerase binds and initiates correct transcription.

A “recombinant” polynucleotide, eg, a recombinant DNA molecule, is a novel nucleic acid sequence formed in vitro by ligation of two or more heterologous DNA molecules (eg, cloned into its cloning site or polylinker). Recombinant plasmid containing one or more foreign DNA inserts).

A “recombinase recognition site” is a sequence of nucleotides that is recognized and triggered by a site-specific recombinase enzyme.

“Transformation” is the application of a recombinant DNA derived from another cell of a different genotype from the outside, and the DNA of the cell is integrated by incorporating the DNA into the genome of the applied cell. Means to instruct and modify.

A “transgenic” organism, such as a transgenic plant, is a foreign DNA
Is the introduced organism. A "transgenic plant" includes all its progeny, hybrids, and hybrids that retain exogenous DNA, whether sexually or asexually reproduced.

A “vector” can be any of a number of nucleic acid sequences into which the desired sequence can be inserted by restriction enzyme treatment and ligation. The vector is typically its own origin of replication; one or more unique restriction endonuclease recognition sites that can be used to insert foreign DNA; usually a selectable marker such as a gene encoding antibiotic resistance; Often, it contains a recognition sequence (e.g., a promoter) for expressing the inserted DNA. Ordinary vectors include plasmids,
Includes phages, fasmids, and cosmids.

A “transposase recognition site” is naturally the 5 ′ end and 3 ′ of a transposable element.
A sequence of nucleotides found at the ends and which is acted upon by the transposase enzyme during transposition of the transposable element.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Novel means for producing transgenic plants and novel DNs according to the invention
Methods and compositions are provided for transgenic plants having the A composition.

In the most preferred embodiment of the invention, the oncogene region itself is deleted,
Alternatively, the function of the oncogene is permanently inhibited by the use of suppressive strategies that disrupt the genetic interruption. In another aspect of the invention, the oncogene itself has been engineered to respond to a novel suppressive strategy. In yet another aspect of the invention, a selected oncogene is used rather than the entire complement of genes normally transmitted to plant cells via wild-type Agrobacterium.

Wild-type Agrobacterium is generally known to transform many different crop species with high efficiency. Many Agrobacterium species and strains have been described, the transmission of T-DNA and subsequent oncogenic processes are generally dependent on the function of the conserved and introduced oncogenes, which are T. -Many contained in the DNA region. Also, many Agrobacterium strains, such as nopaline or succin amopine, are capable of transforming with high efficiency any genotype of many different plant species, in effect Brassica species (ie, forming tumors). it can)
It is known. The method of the present invention takes advantage of this efficiency as a means to initially derive a population of transformed cells from any species or genotype that can be infected by Agrobacterium. These cells preferably contain the full complement of oncogenes and are incapable of regenerating morphologically normal plants from these cells. Preferably, the oncogene is introduced into plant cells by Agrobacterium-mediated transformation, and the Agrobacterium used herein is capable of being grown in a suitable medium independent of hormones. Is a stock.

In a most preferred embodiment of the method, the Agrobacterium strain is modified by the addition of a recombinase recognition site flanking the oncogene region and a recombinase gene expressible in plant cells under the control of an inducible promoter. -Modified to include a plasmid. In this aspect, the introduction of the recombinase gene causes the recombinase gene to be expressed in the transformed plant cell, which causes the deletion of the oncogene region, and the plant cell can be regenerated into a complete plant body. . A preferred method further comprises introducing a new DNA sequence outside the region excised by the recombinase in the Ti-plasmid but within the repetitive sequence of the T-DNA border of the Ti-plasmid. In this form, deletion of the oncogene region from the inserted DNA produces a plant cell in which the novel DNA sequence is stably inserted into the plant genome.

This method does not require regeneration from the first transformation and utilizes regeneration of normal plants as the selection step in the second transformation, so that a typical transformation protocol Many of the less efficient conventional processes and features can be eliminated. The outline of this method is shown in FIG.

The method also provides excellent advantages with respect to selection. First, there is no need to use a selection agent as part of the procedure. Initial transformation relies on hormone-independent growth of transformed cells by expression of oncogenes. These transformed cells can be selected by simply plating in a hormone-free medium. This process scores the infectivity of Agrobacterium (scor
e) was spent for years. Only transformed cells (oncogene-bearing cells) grow in hormone-free medium. Thus, obtaining all of the transformed cell populations is straightforward. As part of the method of the present invention, these cell populations are further engineered by transient expression (which activates a DNA construct capable of causing a deletion of the oncogene region), typically with additional Regenerable cells are usually generated directly from the cell population originally containing the oncogene region, without the need for hormones or long-term complex tissue culture.

By eliminating the selection step, it is not necessary to balance the toxic effect of the antibiotic with the death of the non-transformed cells (there is no subsequent negative effect on the transformed cells), resulting in high regeneration efficiency. Can be provided. This special process (selection vs. regeneration) has been difficult for many plant species, especially Brassica genotypes. In fact
Selectable markers based on letting non-transformed cells to death are efficient, but require considerable culture manipulation and time to use them.

The novel method of the present invention does not require tissue culture experiments for new genotypes and minimizes long-term regeneration protocols, and thus has many excellent effects in developing transgenic lines. I will provide a.

The attributes of the system include: (a) Removing selectable markers as a selection process is based on regeneration.

(B) Transformation independent of genotype. It has been shown that wild type Agrobacterium transforms almost all variants within a genus or species. Even crops such as sunflower that are difficult to transform will benefit from this approach.

(C) Minimal tissue culture procedure. Removal of the toxic selection agent allows the use of simpler tissue culture protocols.

(D) Rapid regeneration of transformed plants. Transgenic plants can be rapidly recovered by the minimal need for tissue culture operations and the need for selection.

(E) Reducing the effort of regenerating transgenic plants. By the method of the present invention,
Multiple independent transformations can be a simple culture in liquid or solid culture and a single regeneration induction.

Thus, the method of the present invention was found to be useful for producing transgenic plants in many plant species.

According to the present invention, (1) to produce plant cells initially having oncogene activity which are convenient for selecting transformed plant cells, (2) then T-DN
A variety of methods can be used to modify the oncogene region of A to regenerate and regenerate morphologically normal plants.

The initial step of forming tumor cells can be done in several different ways.
These methods include direct inoculation of whole plants, inoculation of sterile plant tissue or plant explants, or inoculation of decapitated plantlets. Tumor cells may simply be cultured in minimal medium or, in some cases,
Directly grow on inoculated plants.

Once the oncogene function has been inhibited or eliminated, the entire population of cells has been transformed, so standard regeneration protocols for that plant species need not be applied and no selection agent needs to be applied . In this form, it is a simple matter to change the conditions of tissue culture to guide cells to regenerate plants, and all regenerated plants have been transformed. Regeneration of transgenic shoots may occur directly on infected plants, i.e., inoculated with bud-cut plants, after inactivating the oncogene region, the crown gall tissue is removed. Can directly form shoots.

Such transformation and regeneration methods are in contrast to virulence selection techniques, which involve first performing a transformation process on plant cells and then placing them in the presence of a phytotoxic agent. It is a technique in which a transgene is linked to a selectable marker that detoxifies the phytotoxic agent. Thus, regeneration relies on selecting transformed cells within a population of untransformed cells. The present invention forms a population of substantially all transformed cells and allows regeneration to occur without the selective pressure of phytotoxic agents.

A first aspect of the present invention is the naturally occurring oncogene found in wild-type T-DNA of Agrobacterium, and Agrobacterium, to produce transformed plant cells in the first place. It is envisioned to use the naturally occurring high efficiency plant transformation process of the genus, which results in expression of the oncogene in transformed cells, which results in the cells proliferating independent of hormones. Therefore, transformed cells can be easily identified.

In fact, the wild-type Agrobacterium transformation process that leads to the formation of gall is often efficient, with dis-armed strains of Agrobacterium (eg T-DNA lacking oncogenes). Tumors can be formed on a wide variety of plant species and varieties, including plants that are generally awkward to transform with). Therefore, plant cells having tumorigenic T-DNA can be easily obtained and identified without the need for the use of toxicity selection agents.

In the second step, a novel strategy for removing oncogene regions or for removing oncogene function is to transform transformed cells capable of regenerating morphologically normal plants. Used to produce.

In the most preferred embodiment of the invention, the Ti-plasmid is modified to contain a recombinase recognition site and a recombinase gene that can be expressed under suitable conditions in plant cells. When the gene is expressed, the oncogene region or part thereof is excised, the tumor phenotype is removed and the whole plant cell can be regenerated from the originally transformed plant cell. By including the DNA sequence encoding the novel characteristic in the Ti-plasmid, it is possible to recover a complete plant containing the novel characteristic inserted in the plant genome.

In another aspect of the invention, cells transformed with the oncogene region of T-DNA are then subjected to a second transformation with a DNA construct that inhibits the activity of the oncogene, As a result, morphologically normal plants can be easily induced and formed from the cell population that initially expressed the oncogene.

The above two aspects of the method do not use direct selection on a population of transformed cells. By inhibiting the oncogene, the cells can be regenerated into a morphologically normal plant, and therefore, by this method, from the transformed cells containing the oncogene,
Morphologically normal transformed plants can be recovered. By avoiding the use of selective agents that cause death of non-transformed cells and relying on the conversion of morphologically abnormal to morphologically normal conditions, a simple and efficient morphology, complete plant Body recovery occurs.

In the most preferred embodiment of the invention, the use of site-specific recombinases is envisaged as a means for eliminating the activity of oncogenes. The use of recombinases can provide a direct means to eliminate oncogene function in transformed cells. In addition, the beneficial transgene is located between the Agrobacterium T-DNA border sequences, but outside the region excised by the site-specific recombinase.
, The cells obtained after exposure to the recombinase contain only the transgene. The cells can then be regenerated into morphologically normal plants.

For example, T-DNA may be modified (eg, by site-directed mutagenesis or homologous recombination as a means of introducing new DNA sequences) to create a site that can be recognized by a site-specific recombinase. Can be provided. These DNA sequences are typically short (less than 40 base pairs, and often much shorter) and flanked within the T-DNA border sequence (ie, within the portion of the DNA that is transmitted to the plant cell).
region) and the oncogene is contained between these two sequences. Induction of recombinase activity results in specific excision of the oncogene, leaving the T-DNA border sequence (and any novel transgenes associated) in place. The recombinase activity can be determined by stable transformation, transient expression of DNA, eg viral infection of said cells with recombinant viruses containing biolistics or recombinase genes, or by various means such as electroporation. It can be induced by introducing an enzyme. In the most preferred embodiment, the recombinase gene is contained within the Ti-plasmid but is under the control of a plant inducible promoter so that expression of recombinase (and deletion of the oncogene region) contains the oncogene region. The resulting cells are placed under suitable induction conditions.

The steps used to obtain such a vector may include the construction of a vector containing a recombinase recognition site flanked by two homology regions to the border region. In this form, due to the double crossover, the recombinase recognition site is T-
It is inserted in the border region of DNA. The homology region can be located anywhere within the border region, but it leaves the border repeat sequence of T-DNA intact, such that it is located within the region of DNA commonly transmitted to plant cells. Must be selected. Conveniently, an antibiotic selectable marker is included to facilitate recovery of cells containing the modified Ti-plasmid. It is convenient to carry out the modification stepwise, typically to modify the first boundary such as the right boundary region and to recover the modified Ti-plasmid containing the recombinase sequence at the right boundary. Then, a second round of modification was performed on this modified right border Ti-plasmid, and a recombinase site was introduced into the left border region in the same manner as above using the homology region for the left border region, and Both modified Ti-plasmids are recovered.

For convenience, further modifications are envisioned. In one variation, the first antibiotic marker is flanked by identical recombinase recognition sites, so that when the antibiotic marker is removed by expression of the recombinase in bacteria, the first antibiotic marker does not contain the antibiotic marker gene. One modified Ti-plasmid can be recovered. By using such a strategy, after removing the first antibiotic marker introduced by the first homologous recombination, two recombinase sites can be introduced using the same marker. Also, the plant construct gene can be included in the DNA construct used for modification of the Ti-plasmid. The plant marker gene is arranged to be activated upon successful excision of T-DNA in plant cells by the activity of the induced or introduced recombinase.
In this form, the loss of oncogene function is conveniently scored.
be able to.

Many site-specific recombinases have been described in the literature (Kilby et al.,
Trends in Genetics, 9 (12): 413-418, 1993). Three widely used recombinase systems include: Zygosaccharomyes rouxii p
The activity identified as R encoded by the SR1 plasmid, Saccharomyces cere
FLP encoded by 2m circular plasmid derived from visiae, and phage P
Cre-lox derived from 1. All of these recombinase systems have been shown to function in heterologous hosts. For example, R has been shown to function in tobacco cells (Onouchi et al., Nucl. Acids. Res. 19 (23): 6373-6378, 1991.
, Which is incorporated herein by reference. FLP has been shown to function in tobacco and Arabidopsis (Arabidopsis) (Kilby et al.,
The Plant Jour. 8 (5): 637-652, 1995, incorporated by reference herein, Cre-lox has been shown to function in tobacco (Russell et al. ,
Mol. Gen. Genet. 234: 49-59, 1992, Odell et al., Mol. Gen. Genet. 223: 3
69-378, 1990, Dale and Ow, Gene 91: 79-85, 1990, Dale and Ow, Proc. Natl
Acad. Sci. USA 88: 10558-10562, 1991, Haaren and Ow, Plant Molecular Bi
ology 23: 525-533, 1993, incorporated herein by reference). Therefore, the use of site-specific recombinases to direct homologous recombination in higher cells is well documented. Within the scope of the present invention, the excision function of the site-specific recombinase is envisioned as a means to help eliminate the activity of oncogenes.

The recombinase gene is generally modified so that it is not expressed in bacterial cells, and its expression is restricted to plant cells. This means that the recombinase gene has a modified T
This is of particular importance in the embodiment where it is contained within the border region of the i-plasmid. This is because expression of the recombinase gene in a bacterial strain such as an Agrobacterium strain containing the modified Ti-plasmid leads to deletion of T-DNA. in this way,
In these cases, the recombinase gene was modified to contain introns and restricted bacterial expression. Introns are typically modified to contain at least one termination signal to ensure that recombinase activity is not expressed in bacteria.

The modification of the Ti-plasmid can be performed in any Agrobacterium species that is capable of growing independently of hormones and conferring a phenotype that is unable to regenerate morphologically normal plants. . Wild-type T-DNA or Ri-RNA, as well as modified forms thereof, can be used. Combinations of oncogenes normally found in T-DNA or Ri-DNA can be used. The actual combination will depend on the particular plant species being transformed, as well as its convenience. Agrobacterium species envisioned for use in the present invention include A. rhizogenes, A tumef
aciens (noparin, octopine, agropine strains), A. vitis, or any other strain capable of transmitting oncogenes to plant cells. Above all, the use of Nopaline strain C58 is preferred. These Agrobacterium strains are commonly referred to as the American Type Culture Collection.
Available from.

The method of modifying the Ti-plasmid can include many different steps, but generally the following steps are used (these are illustrated in Figures 4-7):-Ti- Construct a vector containing a homology region for the T-DNA region of the plasmid, which is adjacent to the recombinase recognition site; -introducing the vector containing the recombinase recognition site into a Ti-plasmid containing Agrobacterium strain In order to do so, mating by three parents (triparental mati
ng) to recover the Ti-plasmid modified to contain the recombinase recognition site; -This procedure is repeated again with different homology regions, and the Ti-plasmid with the two recombinase recognition sites in the desired positions. To get

It is common practice in the art to utilize homologous recombination in bacteria to obtain an extrachromosomal element with a new DNA sequence inserted as a result of the recombination. In this case, two homology regions are used flanking the recombinase recognition site, ensuring that only the region between the two homologous sequences is inserted into the Ti-plasmid. It has been found convenient to include an antibiotic resistance marker for the selection of stable recombinants. As mentioned above, antibiotic markers can be flanked by recombinase recognition sites to delete the marker once inserted into the Ti-plasmid. In most recombinase enzymes, the deletion of a sequence bounded by two recombinase sites results in the formation of a single recombinase site.

It has also been found convenient to include the recombinase gene itself in one of the vectors used to insert a second recombinase recognition site into the Ti-plasmid. The recombinase gene has been modified to contain a plant intron, is not expressed in bacterial cells, and is under the control of a plant inducible promoter.
In the case illustrated, two types of inducible promoters are used for demonstration: a low temperature inducible promoter from Brassica (Genbank Acc. N. U14665), and tet.
Operator / Repressor System (Gatz and Quail, Proc. Natl. Acad. Sci
USA 85: 1394-1397, 1988, incorporated herein by reference). Other inducible or regulated promoters known in the art can be used within the scope of the invention. For example, Mett et al., (Proc. Natl. Acad.
Sci. USA 90 4567-4571, 1993, incorporated herein by reference), describes a gene expression system derived from the yeast metallothionin gene. They demonstrated that a chimeric plant promoter containing a DNA sequence designated ACE1 was repressed by a metallo-active transcription factor and could be derepressed in the presence of copper ions. Furthermore, a mammalian-derived gene expression system has been shown to function in plant cells (Proc. Natl.
Acad. Sci. USA 88: 10421 10425, 1991, incorporated herein by reference). In this gene expression system, DNA encoding a glucocorticoid transcription factor and a glucocorticoid response factor is used together with a plant gene,
This provides a gene expression system that suppresses gene expression but can be derepressed in the presence of steroid hormones.

Recombinant DNA molecules containing modified T-DNA of the Ti-plasmid have "new properties".
The gene encoding the "novel characteristic" can be any useful recombinant protein or peptide, and typically this "novel characteristic" is Can be a heterologously beneficial protein or a protein that imparts agronomically useful properties such as herbicide tolerance. These properties include insect resistance such as the Bt insecticidal protein gene and specific examples of modified methods and Bt coding sequences used in plants are found in US Pat. No. 5,380,381, which is incorporated by reference. It is made a part of the disclosure content of this specification. Other strategies for controlling insect predation in plants include genes encoding protease inhibitors (U.S. Patent No. 5,436,392, incorporated herein by reference),
Insect venom proteins (US Pat. Nos. 5,441,934 and 5,177,308, incorporated herein by reference), and various other proteins are included. Still other properties include: genes encoding fungal or disease resistance (eg, Defense-related proteins in higher plants. Bowles, DJ,
Annual Review of Biochemistry 1990 59: 873-907; or US Pat. No. 5,703,04
No. 4 and No. 5,607,919, all incorporated herein by reference), genes that encode quality-related characteristics such as reducing ethylene production to delay fruit ripening (e.g., US Pat. No. 5,998,702; 5,859,330;
And No. 5,916,250, incorporated herein by reference) and increased vitamins (eg, International Publication No. WO 99/53041, incorporated herein by reference) ), Or a gene encoding an enzyme capable of producing a nutraceutical product, such as increasing the production of commercially valuable secondary metabolites, or delaying or inducing flowering (For example, LEA
Homeotic genes such as FY (Weigel and Nilsson, Nature 277: 495-500,
1995, Session et al., Science 289: 779-781, 2000, incorporated herein by reference)) and reduce seeds in fruits (e.g. International Publication No. WO
97/40179, incorporated herein by reference), and the like, which control the development of plants biochemically or physiologically. The scope of the invention is not limited to the novel properties. In fact, if it is possible to transmit a large DNA region to Agrobacterium, it is considered that even a large genomic DNA fragment containing many genes can be transmitted by this system.

It is clear that the gene encoding the novel property can be added to the plasmid used to modify wild type C-58. The novel property can be added when modifying the right border sequence or the left border sequence. Thus, the activation of the recombinase enzyme cuts out the wild-type T-DNA and the recombinase enzyme coding region, leaving a novel property adjacent to the T-DNA border, leaving the novel property-encoding gene at the recombinase recognition site. Any number of novel property-encoding genes can be added by orienting to one another.

After recovering the modified Ti-plasmid, an Agrobacterium strain is prepared, a plant cell is transformed, and a cell containing an oncogene region is cultured under a hormone-free condition or a minimal medium condition, Transformed cells are grown and non-transformed cells are removed. After this period, recombinase activity is induced. Recombinase activity removes the oncogene region, leaving a novel feature and a small region of T-DNA flanked by T-DNA left and right borders. This is shown in FIG.

It should be noted that it may be desirable to leave only the recombinase site in the plant DNA after excising the oncogene region. In this form, the recombinase site can be utilized to introduce additional genes using recombinase-mediated integration of DNA. Furthermore, the recombinase sites may be scattered throughout the plant genome by selecting various transformed cell lines, each with a different insertion site. These individual plant lines may be regenerated and crossed to form plant lines containing multiple recombinase recognition sites scattered throughout the plant genome. The recombinase enzyme can then be utilized to rearrange, overlap, or delete regions of the plant genome to create new genotypes and genetic combinations. Thus, the present invention includes an additional utility in plant genetics: multiple recombinase recognition sites in the plant genome (which can be utilized by recombinases to relocate the plant genome utilizing recombinase activity. Site) is used. The recombinase may be introduced in the second transformation or it may be present in the plant genome under the control of an inducible promoter as described herein. Further, the recombinase is introduced by crossing with a plant that is constitutively engineered to express recombinase, or more preferably a plant that is engineered to express recombinase under the control of an inducible promoter or a tissue-specific promoter. May be. Pollen or microspore specific promoters controlling the expression of the recombinase are preferred. This is because this promoter produces pollen cell populations containing a new genetic arrangement after inducing recombinase expression in pollen in plant cells whose genome contains many recombinase sites.

The use of the Cre-lox recombinase system to cause chromosomal recombination has been described in the art (Qin et al, Proc. Natl. Acad Sc).
i., USA 91: 5, 1706-1710, 1994), it seems that no method for simply inserting a large number of recombinase recognition sites into a plant genome without using a selection marker is supposed. The need for a selectable marker limits the number of recombination events that can be produced in possession of the recombinase site, thus limiting the usefulness of the procedure. In the present invention, any number of recombinase sites can be introduced,
This method can be used to produce plant cells with an altered genome only containing the recombinase site, without the addition of coding sequences. Another use of introduced recombinase sites is to utilize recombinase-mediated gene insertion to provide regions for site-specific integration of novel properties.

The region remaining in the plant chromosome may include a gene derived from the oncogene region or a part of the gene derived from the oncogene region. Within the oncogene region, there are many different oncogenes, including genes that code for the formation of opines, so a recombinase recognition site was inserted to remove a portion of the oncogene region, while Retaining one or a portion of the oncogenes normally found in the region is preferred in some cases.

In addition to recombinase, it is envisaged to use transposase to remove or excise the oncogene region of the modified Ti-plasmid. Known transposons and related transposase activities include: Ac / Ds and En / Spm factors from maize (eg, Federoff, N. Maize).
Transposable Elements. In Berg, DE and Howe, MM (eds) Mobile DNA,
pp. 375-411, American Society for Microbiology, Washington, DC, 1989
Tam-1 and Tam-3 from snapdragons (see, for example, Sommer et al,
Transposable Elements of Antirrhinum majus. In Plant Transposable Eleme
nts, 0. Nelson, ed, Plenum Press, New York, pp. 227-235, 1988), tobacco-derived Tnt-1 (Pouteau, S. et al, Mol Gen Genet. 228: 233-239, 199
1), Tph-1 derived from petunia (Gerats AGM et al, The Plant Cell 2:
1121-1128, 1991), and a potato-derived Tst-1 factor (Koster-Topfer, et al,
Plant Mol. Biol 14: 239-247, 1990), all incorporated by reference herein.

A transposon has a central coding region that encodes a transposase enzyme, and exhibits certain features having a DNA sequence recognized by a specific transposase at the 5 ′ and 3 ′ ends of the transposon. Transposase is the DNA of these
Acting on the sequence causes transposition of the transposable element. An artificial transposon containing a heterologous gene has been constructed. These recombinant transposons can transpose in the presence of the transposase enzyme. According to the present invention, a sequence recognized by transposase can be linked to an oncogene in a form such that the oncogene can be excised by the transposase enzyme. Transposition or excision irreversibly inactivates the function of oncogenes. The transposon may be modified so that reinsertion into other chromosomal sites does not occur easily or does not occur, or a part of the oncogene is irreversibly deleted so that the transposase recognition site The arrangement may be constructed. For example, the oncogene can be modified to have a transposase recognition site between the promoter of the modified oncogene and its coding region, such that transposition effectively causes a portion of the coding region or promoter. Remove and inactivate the oncogene. Any of several strategies can be used to exploit transposition as a means of inactivating the function of oncogenes.

The use of transposase activity to specifically cause transposition (or deletion) of oncogenes is contemplated within the scope of the present invention. In fact, T-DNA can be modified to contain a DNA sequence that can be recognized by the transposase enzyme,
As a result, when exposed to the transposase enzyme, the oncogene is excised from its original insertion site, followed by deletion or infrequent translocation to another site. Thus, when properly modified, the transposon can be used as a means of favoring excision of an oncogene within the scope of the present invention.

The oncogenic T-DNA region of the Agrobacterium nopaline strain has the preferred DN
Provide the A component. The structure and function of these genes are described in the art and one of ordinary skill in the art can devise specific DNA constructs and culture conditions that provide optimal transformation efficiency. Nopaline strain is Nopaline strain C-5
Since the T-DNA of 8 is a single-stranded T-DNA region, it is preferable as compared with the octopine strain in which the "right" and "left" T-DNA regions are present because it contains pseudo double-stranded T-DNA . In the T-DNA of C-58 nopaline, an oncogene having a known function such as oncogenes 1, 2, and 4 and an oncogene whose function is not specified such as 5 and 6b, and nopaline synthase are contained. There are coding genes (nos), genes coding for agrocinopine synthase (acs), octopine, genes coding for nopaline secretion (ons) and other genes. The combined activity of these oncogenes forms the crown gall.

Oncogene 1 is indoleacetamide synthase (I), an enzyme that converts tryptophan, an amino acid normally found in plant cells, into indoleacetamide.
AMS) code. The function of oncogene 1, ie conversion of tryptophan (an endogenous amino acid contained in all plant cells) to indoleacetamide, is described in Van Onckelen et al., FEBS lett. 198, 357-360, 1986. Which is incorporated herein by reference.

Oncogene 2 is an enzyme that converts indoleacetamide to indoleacetic acid,
It encodes indoleacetamide hydrolase (IAMH). Oncogene 2
For the function of A., ie, the ability to convert indole acetamide to indole acetic acid, Tomashow et al., Proc. Natl. Acad Sci. USA 81, 5071-5075, 1984.
, And Schroder et al., Eur. J. Biochem. 138, 387-391, 1984, which are hereby incorporated by reference. Specifically, oncogene 2 cooperates with oncogene 1 to synthesize indoleacetic acid, a plant growth regulator, from tryptophan by a pathway found in bacterial cells but not in plant cells. The activity of related oncogenes is shown in A. rhizogenes, A. vitis (Canad
ay, J. et al., Mol. Gen. Genet. 235: 292-303, 1992), and Pseudomonas s.
avastanoi (Yamada et al., Proc. Natl. Acad. Sci. USA, 82: 6522-6526, 198
5), which are incorporated herein by reference.

Oncogene 4 encodes isopentyltransferase and is capable of synthesizing cytokinin activity.

In addition to these well-defined individual oncogenes, the T-DNA region is involved in the oncogenic process and contains several other genes that are considered oncogenes. Many of these genes are involved in the regulation of tumorigenesis and play important roles in the efficiency of tumorigenesis. Thus, cells transformed with the entire complement of the oncogene will be sacrificed in the untransformed cells, but in a tissue-disturbed form, under conditions favorable to the growth of the transformed cells. Proliferate very quickly, forming crown gall. The oncogene complement found in strain C-58 collectively provides the genetic functions required for the formation of crown gall tissue.

In some plant species, some of the usual complements of oncogenes can be used. In fact, more than one oncogene can be used, the medium of which is adjusted to provide the levels of phytohormones normally found by the expression of all of the normally expressed oncogenes. Thus, the plant cells obtained by the first transformation do not completely show hormone-independent growth, but under certain culture conditions have a phenotype similar to hormone-independent growth. , Shows a phenotype in which morphologically normal cells cannot be regenerated. These cells are then exposed to recombinase expression and, after selection of culture conditions, provide the transformed cells with the ability to regenerate morphologically normal cells. This form simplifies the method as it envisages the use of a limited number of oncogenes.

In these aspects of the invention, the oncogene activity selected is oncogenes 1 and 2 from the Agrobacterium Ti- or Ri-plasmid, and oncogene 4 that causes the production of cytokinins, and Ti-. Or Ri
-Other oncogenes commonly found in the DNA region in association with these oncogenes. The combined activity of these genes results in hormone-independent growth. The plant hormone activity of these genes can often be replaced by endogenously applied plant hormones.

These oncogenes were somehow erased or silenced
At times, cells proliferate normally and are often capable of regenerating morphologically normal plants. Thus, oncogene silencing can be used as a selectable event in tissue culture. Various means that can be used for silence, such as suppression, are illustrated in FIG. In this aspect of the invention,
A second DNA is expressed that encodes a repressor capable of removing oncogene expression or function.

There are many ways in which a gene can be silenced or its activity suppressed. In the context of the present invention, any mechanism that effectively inhibits accumulation of the product of said oncogene in a cell includes suppression. The method involves binding a particular "repressor protein or factor" to a DNA region or "operator" within the oncogene promoter. The promoter region of the oncogene can be conveniently modified to respond to this repressor element. Examples of these DNA binding proteins described in the art include bacterial repressor factors and associated DNA binding regions (operator DNA) such as the Lac Z repressor or tet repressor or other bacterial repressors. Other examples of bacterial repressors can be found in the class of repressor proteins and include: LacR, Gu that regulates sugar catabolism in bacterial systems.
tR, DeoR, FucR, and GlpR (van Rooijen, RJ and de Vos, WM, J. Bio
l. Chem. 265: 18499-18503, 1990, incorporated herein by reference), or the Agrobacterium repressor known as accR (Bodman), which regulates biosynthesis and conjugative transduction of agrocinopine. et al., Proc. Natl Acad S
ci USA 89: 643-647, 1992, incorporated herein by reference). Any other repressor can be used within the scope of the invention. Other sources of repressors can be used including those found in fungi such as yeast or any other organism. The LexA system is an example of a gene expression mechanism capable of suppressing gene expression (US Pat. No. 4,833,080).
No., incorporated herein by reference). The LexA mechanism utilizes repressors and specific DNA operator sequences to control gene expression.
Other sources include: Yeast-derived systems (Mett et al., P
Roc. Natl. Acad. Sci. USA 90 4567-4571, 1993, incorporated herein by reference), mammalian gene expression systems (Schena et al., Proc. Na
tl. Acad. Sci. USA 88: 10421 10425, 1991, incorporated herein by reference), and artificial systems. For example, US Pat. No. 5,198,346 (incorporated by reference herein) produces novel DNA binding proteins, especially repressors of gene expression that can be used within the scope of the present invention. It describes how to do it. In the present invention, the repressor can bind to a specific DNA sequence that can be inserted into a transcriptional regulatory sequence of an oncogene, and this binding can substantially suppress the expression of the oncogene.

It is also possible to use other DNA binding proteins which have been modified to bind tightly to specific DNA sequences, so-called "transdominators". Other repressors may include antisense RNA for oncogenes. Other suppression strategies aimed at eliminating gene expression can be used within the scope of the invention. Three good ways to achieve repression of gene expression are described, antisense RNA, co-repression, and ribozyme technology. Methods utilizing antisense RNA are described, for example, in US Pat. No. 4,801,540.
And US Pat. No. 5,107,065, which are incorporated herein by reference. The process of co-suppression of gene activity is described by Jorgensen et al, 199.
1, U.S. Pat. Nos. 5,034,323 and 5,283,184, which are incorporated herein by reference. The use of ribozymes is described in Cech et al, US Pat. Nos. 4,987,071 and 5,116,742, which are incorporated by reference herein.

Thus, there are many ways in which transgenic plants can be obtained using the methods of the invention. Therefore, the present invention was found to be useful beyond the range of plant species and varieties. Any plant species that can be transformed with wild-type Agrobacterium can be subjected to the method of the present invention, and a morphologically normal transformed plant can be easily obtained. Any of several suppression schemes can be used, such as suppression of gene activity, removal of oncogenes by recombination or transposition, or addition of proteins that counteract oncogene activity. This includes a protein capable of binding to the product of an oncogene, such as an auxin or cytokinin binding protein, or an enzymatic activity capable of conjugating or metabolizing the product of an oncogene, For example, an auxin-conjugating enzyme or a cytokinin-conjugating enzyme is included. Thus, the method was found to be useful for various purposes beyond the scope of crops, such as the introduction of new genetic traits.

In some embodiments of the invention, oncogene activity is repressed in a reversible form. Thus, the combination of an oncogene and a repressor gene allows the growth of normal plant cells and the development of plants with normal morphological characteristics. For example, the segregation of two genetic constructs by segregation or crossing-over leads to derepression and easily distinguishable morphologically abnormal plants. Thus, the method can provide utility for maintaining certain genetic combinations.

In another aspect of the invention, the gene encoding the novel property is linked to an oncogene introduced in the first transformation step or is a repressor introduced in the second transformation step. It is linked to the gene. In another aspect of the invention, the one or more genes encoding the novel trait are linked to the repressor and the oncogene so that convenient means bring together various genetic traits in a single plant line, Can be maintained. For example, some novel properties can be added individually to cell lines derived from crown gall. Each of the novel properties is linked to a repressor molecule that suppresses only one or two oncogenes present in the cell line. Morphologically normal plant cells can only form when they have introduced all of the necessary oncogene repressors linked to various novel properties. In this form, the simultaneous introduction of a certain number of new properties is a prerequisite for the regeneration of morphologically normal plant cells. In this form, multiple genes can be introduced into plant cells and the recovery of plants containing all of the novel properties is essential in the system.

Thus, in the production of transgenic plants, oncogenes and T-DN
New utility of the A oncogene region is obtained. In particular, the method allows for the selection of plant cells initially obtained by the natural ability of Agrobacterium to efficiently transmit DNA containing one or more active oncogenes or entire oncogene regions, Produces a population of oncogene-containing plant cells capable of growing under conditions insufficient to grow non-transformed cells. Once these cells are established, one obtains a cell line or any number of transgenes that are effective for transfection.

The method then performs a second DNA transfer utilizing a genetic construct capable of suppressing the activity of one or more oncogenes, such that the plant cells containing the second DNA are , It is possible to regenerate morphologically normal plants. Depending on the particular plant and tissue culture method used, selection may or may not be made utilizing conventional virulence selection.

[0139] Certain aspects of the method also rely on the regulation of expression of DNA constructs that permanently delete oncogenes and cause regeneration. The DNA construct encoding the function of the deletion may be included in the initial construct or added to the plant cell via subsequent transformation.

The oncogenes of oncogenic Agrobacterium strains have been extensively studied. In general, there are two oncogenes on Agrobacterium plasmids: tm
r oncogene and tms oncogene. The tmr oncogene (also known as the ipt gene) encodes an enzyme that synthesizes isopentyladenosine 5'-monophosphate, a cytokinin plant hormone that induces shoot formation in appropriate hosts. The oncogene, termed tms (including tms oncogene 1 and tms oncogene 2), encodes an enzyme involved in auxin overproduction leading to root formation in the appropriate host. The combination of the tms and tmr genes usually forms some form of crown gall on the appropriate host.

Some of the other oncogenes found in T-DNA function as modifiers or potentiators of crown gall formation. Normal,
Plant cells containing the oncogene contained within the complete T-DNA show hormone-independent growth in culture. The plant cells in culture are usually callus that is in a dedifferentiated state and can grow, for example, without exogenous plant hormones.
However, if the function of the oncogene is suppressed, the plant cell can then differentiate into regenerable cells under appropriate conditions, and in some cases regenerability on the crown gallcallus itself. Occur.

The combination of the activities of the tms and tmr genes may be sufficient for hormone-independent growth, but apparently the entire complement of oncogenes.
) Was effective in the formation of crown goals. Thus, in the most preferred and broadly applicable aspect of the present invention, the entire complement of the gene encoded within the T-DNA region is modified such that one or more oncogenes are deleted. It is used rather than modified. This includes T-DNA
Other oncogenes within the region are included, such as genes that direct the synthesis of opine and other products encoded by T-DNA.

The method of the present invention takes advantage of the fact that regeneration of normal plants from callus containing oncogenes does not require any additional manipulation other than removal of oncogene activity. . A precedent for this exists in the art as a "revertant" and it has been observed, for example, that shoots or roots form from callus in culture containing oncogenes. Usually, these structures are formed from cells lacking oncogene function due to mutations. In fact, U.S. Pat.No. 4,658,082
The publication describes a method for selecting such shooty revertants as a means of utilizing in vivo infection of plant tissues to obtain plants containing heterologous DNA. However, U.S. Pat.No. 4,658,082 does not assume that the function of the oncogene is suppressed by the second transformation, nor does it contemplate the convenient recovery of morphologically normal plants. . U.S. Pat.No. 4,65
The method described in 8,082 has been previously described for the recovery of plants in which oncogenic DNA (eg, oncogenes) is lost randomly during culture or due to mutations or other random events. (Anticipate). This method does not allow efficient transformation or selection of plant cells containing one or more oncogenes that are capable of regenerating into morphologically normal plants. The recovery of plants described in US Pat. No. 4,658,082 relies on the use of T-DNA which confers a shooty phenotype. This shoot is not morphologically normal.

In general, plants containing one or more Ti-oncogenes are abnormal in their phenotype and have crown gall tumors or curled and twisted leaves due to growth hormone imbalance. These abnormal plants are unsuitable for commercial application. Therefore, the art directs the modification of the Agrobacterium Ti-plasmid in various ways, typically by removing the oncogene, which
It will be a tool for introducing DNA into plant cells. In general, the Agrobacterium-based transformation methods used today have eliminated genes that form cytokinins and auxins, other open reading frames in T-DNA with unknown function, and genes that synthesize opine. Ti-plasmid is used. Such plasmids are commonly referred to as "dis-armed".
) ”. Thus, "armed" Ti-plasmids are generally considered to contain genes commonly found in T-DNA, or genes called oncogenes. The present invention contemplates the use of "armed" plant vectors.

Since the first step of the process is the transformation of plants by an efficient process of natural crown gall formation, the method of the present invention is performed by conventional means and selection with dis-armed plasmids. It has been found to be useful for the transformation of other crop species that are difficult to transform. These crops include, for example, sunflower, cotton, soybean, and safflower. As a result, this method allows the formation of essentially completely transformed cell populations, which are then induced to delete or inactivate the oncogene activity of the T-DNA region. , Can be played without applying selection. This method provides a convenient means of recovering morphologically normal transgenic plants from any crop species that can be transformed with wild type Agrobacterium. The ability to produce plant cells that do not contain selectable markers is of great industrial value.

In particular, the present invention has been found to be useful for transforming a wide variety of species including the following species: broccoli, cabbage, cauliflower, kale,
Chinese kale, collard, and kohlrabi
Brassica oleracea species such as i); Brassica rapa species such as Chinese cabbage, pak choi, and turnip. The present invention has also been found to be useful for transforming other vegetable crops exemplified below: Tomato (Lycopersicon esculentum), C. m
Cucumis species such as elo (melon) and C. sativus (cucumber), Cucurbita species (pumpkin) such as C. maxima and C. pepo, spinach (Spinacia oleracea),
Pepper containing Capsicum species (capsicum) such as carrot (Daucus carota), C. annuum, C. frutescens, and C. chinese, A. cepa (valve onion (bulb
onion)) and A. fistulosum (bunching onion)
Onions, including llium species (onions), radishes (Raphanus sativus), watermelons (Citrullus lanatus), and lettuce (Lactuca sativa). The present invention has also been found to be useful for transforming ornamental species such as the genus Impatiens, Pansy (Pansy) (Viola x wittrockiana), and Lisianthus (Lisianthus) (Eustoma grandiflorum). It was

The method of the present invention differs from the art in many ways. The present invention is T-
It does not require the construction of a vector in which the oncogene region of DNA has been deleted from within the Ti-plasmid. Also, the method of the present invention allows the use of the native form of Ti-plasmid, rather than the use of binary or co-integrate type plasmids. The present invention can take advantage of the entire natural DNA transfer process, and in the most basic embodiment, the complete oncogene region of the Ti-plasmid and all but not one of the genes required for tumorigenesis (eg, Ebinuma et al, ibid., Cytokinin biosynthesis genes). Complete plants, plant explants, or plant cells,
It can be easily transformed with an armed Agrobacterium strain. In the present invention, cells can be selected on the basis of tumorigenesis and growth on hormone-free media, until the tumorigenic gene is removed or the oncogene activity is suppressed, Plants cannot be regenerated. Certain oncogenes, such as oncogene 4, are used in the art as a visible marker for transformed cells because the transformed cells form morphologically abnormal shoots. In the methods of the invention, transformed cells are unable to undergo differentiation and are initially identified by the formation of undifferentiated tissue (eg crown gall). In the method of the present invention, normal plants are regenerated from this undifferentiated tissue in a single step by eliminating the activity of oncogenic genes without the need to use selectable markers.

Thus, in one aspect, the invention features a natural T-DNA transduction process,
The known activity of the oncogene T-DNA region and the ability to easily identify the crown gall tissue are combined with the new technology of molecular biology, which enables simple transformation and many To obtain a novel transformation process that incorporates the efficiency of natural processes into the technology capable of recovering morphologically normal plants from plant species.

Transformed plants produced in this form have in their genome tumors derived from the oncogene region of the Ti-plasmid and the T-DNA right and left borders flanking a novel DNA sequence homologous to the plant cell. A DNA region containing at least a part of the gene is stably inserted. In some cases, there is an entire complement of the oncogene normally found in T-DNA. In other embodiments, only a portion of the oncogene remains. The plant thus produced may contain the oncogene region derived from T-DNA to a minimum, but can grow normally and exhibit a normal morphology. These plants can transmit the inserted DNA to their progeny by a usual breeding method, and can produce seeds containing the inserted DNA. Insert DNA
Sexual transmission of the insert DNA can be introduced into other plant species or varieties that are sexually compatible.

The following examples serve to illustrate the method and do not limit the scope of the invention.

Example 1 The construction of a modified wild type Agrobacterium Ti-plasmid is described in the following example. This example is a T derived from wild type C-58 nopaline Agrobacterium.
The construction of a vector for introducing a recombinase site into the right border region of the i-plasmid is described.

The modification of the Ti-plasmid is carried out by constructing a vector containing a recombinase site and a homology region capable of introducing the site into the Agrobacterium strain by homologous recombination. In this example, the vector is used in constructing a wild-type C-58 Ti-plasmid modified to contain a DNA sequence recognized by a site-specific recombinase in the right border region.

[0153] To alter the Ti- plasmid, pRBC-1 (R ight B order C onstruct)
The vector referred to was constructed for homologous recombination with the wild-type C-58 Ti-plasmid. Several cloning steps were used. The steps used are outlined in Figures 28-33. The assembly of the various DNA components involved in these steps is as follows:

As shown in FIG. 21, the general plasmid vector pGEM-7Zf (+) (Promega, Madison, WI, USA) contains Nco I, Bgl II,
And modified by adding an Eco RV site to create pNBE. The ampicillin resistance gene was cloned into pBR322 using PCR primers (SEQ ID NOs: 25 and 26).
Isolated from. The ampicillin gene (SEQ ID NO: 11) is shown in FIG.
Added to pNBE. The resulting plasmid is called pNBEAmp.

The FRT recombinase recognition site was added to pNBEAmp by annealing two single stranded DNAs (SEQ ID NOs: 29 and 30) and the resulting annealed double stranded DNA was inserted into pNBEAmp. As shown in 28, pAmpFRT (R) was obtained.

In addition to pAmpFRT (R), several other DNA components were isolated. As shown in FIG. 29, pFF19 was modified by removing the Sma I -Sph I region of the polylinker to create pFF19M, which gave the 35S polyadenylation signal (SEQ ID NO: 10) to the plasmid pFFl9 (Timmermans et . al., J. Biotech 14: 333-344,
1990). In addition, a visible marker gene was included for convenience in monitoring the transformation process. An optional visible marker gene for this construct is the GUS-NPTII fusion gene (SEQ ID NO: 8; Datla, RSS, Hammerlindl, JK, Pelcher, LE, Crosby, WL) derived from plasmid pGKK14.
, and G. Selvaraj, 1991, Gene 101: 239-246), which is Dr. Gopalan.
Selvaraj, Plant Biotechnology Institute, National Research Council of Ca
Courtesy of nada, Saskatoon, Saskatchewan. A map of the gene is provided in Figure 12.

The GUS-NPTII fusion gene was cloned into pGEM4Z using the Bam HI and Eco RI sites to form a plasmid designated pGus: NPTIIter as described in FIG. This plasmid was restricted with Sac I and Eco RI and the polyadenylation signal from pFF19-M was added as a Sac I-Eco RI fragment. This created the plasmid pGUS-pA containing the GUS gene and the 35S polyadenylation signal. This operation procedure is described in FIG.

The next series of procedures was derived from pGUS: NPTIIter by restriction of pGUS-pA with Sac I.
By adding the Sac I fragment, the GUS and NPTII genes were recombined in pGUS-pA, resulting in the plasmid pGusNPTIIpA. As shown in FIG. 32,
The ampicillin gene linked to the FRT site derived from the plasmid pAmpFRT (R) was added to the plasmid pGusNPTIIpA. This created the vector pAmpFRT (R) Gus, as shown in FIG.

Assembly of final vector used for homologous recombination with Ti-plasmid
) Used several other components. First, the vector pUM
21, which was derived from a derivative of pBR322 (pTeasy, Promega, Madison, WI., USA) using PCR primers (SEQ ID NOS: 13 and 14), as shown in FIG. ) Is added to obtain a cloned Sac II-Pci I fragment (SEQ ID NO: 12), pUK21 (GenBank acc
AF223640). The resulting plasmid pUM21 has the mobilization sequence of pBR322.

A homology region (ROH) (SEQ ID NO: 3) derived from the right region of the right border (ROH R _R ) was isolated by PCR using the primers specified in SEQ ID NO: 17 and 18, and this fragment was As shown in FIG. 33, pROH (R _R ) was obtained by cloning as an Eco RI-Kpn I fragment in pUM21.

The plasmid pROH (R _R ) was restricted with Eco RI and the Eco RI fragment of pAmpFRT (R) Gus was added to obtain pAmpFRT (R) GusROH (R _R ). This plasmid was used as Not I and Sal I
Restriction treatment was carried out with and the Not I-Xho I fragment derived from pTeasy-RL was added. This fragment was obtained by PCR-amplifying the left region of the homology region derived from the right border of the T-DNA derived from the Ti-plasmid of Agrobacterium strain C58 (SEQ ID NO: 4) and the two primers (SEQ ID NO: 19). And 20) and isolated by PCR amplification followed by Not I and Xho I restriction. The fragment in pTeasy-RL has an FRT recombinase recognition site in addition to the homology region. Figure 3
The pTeasy-RL fragment was cloned into pAmpFRT (R) GusROH (as detailed in the steps outlined in Section 3).
PRBC-1 is formed by inserting it into R _R and incorporating it.

The vector pRBC-1 contains the following components in the 5'-3 'direction: the left part of the homology region to the right border of the T-DNA region of the Ti-plasmid derived from Agrobacterium strain C58. DNA sequence represented, FRT recombinase recognition site, ampicillin marker gene, second FRT site, promoterless GUS-NPTII
Gene, 35S polyadenylation signal, DNA region representing the right part of the homology region derived from the right border of the T-DNA region of the Ti-plasmid derived from Agrobacterium strain C58. A detailed restriction map is shown in FIG.

Example 2 This example describes the introduction of plasmid pRBC-1 into C58 Agrobacterium.

In this example, the pRBC-1 vector was bred by three parents (Rogers et al., M.
ethods Enzymology 118: 627-636, 1986) and homologous recombination
Used to insert sequences in the Ti-plasmid of strain 58. In this example, pRBC-1 in the E. coli DH5FT strain is combined with the E. coli helper HB101 strain having Agrobacterium C58 and the broad host range plasmid pRK2013. Cells are plated in minimal medium and Agrobacterium selected for resistance to the antibiotics carbinicillin and rifampicin, which are structurally related analogs of ampicillin, and for sensitivity to kanamycin.

Agrobacterium cells that are resistant to the appropriate antibiotic are selected and the integrity of the DNA inserted in the right border region is confirmed by PCR and restriction analysis. The resulting vector contains the wild-type C58 Ti-plasmid modified at the right border of T-DNA to contain the DNA sequence (and any marker coding region) recognized by the site-specific recombinase.
The inserted DNA further contains an ampicillin resistance gene flanked by two FRT sites.

To remove the resistance gene, the FLP recombinase gene was trans (in
trans) and remove the ampicillin gene. To achieve this, F
The LP recombinase gene is modified to remove the intron found in plasmid vector pOG44 and the gene is placed under the control of a bacterial promoter. For example, the vector pLEX (Invitrogen, www. Invitrogen.com) contains POG44 Bgl II-
What appears as the Xho I fragment is changed to the Bam HI-Xho I region of the pLEX plasmid.

The plasmid is then introduced into Agrobacterium by mating with the three parents mentioned above or by direct DNA infection and the cells are seeded in minimal medium without antibiotics. Alternatively, the typical “leakage (le) of eukaryotic coding sequences in bacterial cells
The pOG44 vector can be used directly, as "akage)" can be sufficient to provide a level of recombinase protein capable of causing excision of the amp marker. A preferred method is to place the recombinase coding sequence under the control of a bacterial inducible promoter.

After that, the cells were transferred to a medium containing ampicillin by the replica plate method (replica
plate), select cells that are sensitive to ampicillin. The placement of the DNA resulting from FLP recombinase activity is confirmed by a combination of restriction mapping and PCR analysis. The plasmid containing the recombinase gene is lost because it cannot be maintained in Agrobacterium. The modified Ti-plasmid is
It is called pTI-C58 RBC-1.

The Agrobacterium strain obtained was called C58 RBC-1 and was designated T-DNA.
Contains the nopaline Ti-plasmid modified to contain the FRT recombinase recognition site in the right border region of E. coli.

Example 3 This example demonstrates that the C58 RBC-1 strain is used to produce transformed plant cells.

The C58 RBC-1 strain of Agrobacterium was used to form tumors on inoculated plants, demonstrating that the modification does not inhibit the tumorigenic activity of the Ti-plasmid.

To form tumors, various Brassica species (napus, rapa, oleracea, and
carinata) and tobacco seedlings of Agrobacterium C58 and C58 RB by injuring the stem with a sterile needle containing Agrobacterium.
Inoculate a culture of strain C-1 overnight. Tumor formation is scored and no difference in tumorigenicity between the wild type and the C58 strain with modified right border is observed.

Example 4 This example illustrates the construction of a vector for introducing a recombinase site into the left border region of a Ti-plasmid derived from wild type C-58 nopaline Agrobacterium.

In this example, the construction of the vector was carried out on a C-58 Ti-plasmid (pTi-C58 RBC-1) modified to contain the DNA sequence recognized by the site-specific recombinase in the right border region, Introducing a second recombinase recognition site. In addition to the recombinase recognition sequence, an additional DNA sequence containing the recombinase-encoding gene modified for expression in plant cells is introduced under the control of an inducible promoter. The vector used to introduce the sequence of interest into the left border region of the Ti-plasmid further contains a series of unique restriction sites that are convenient for introducing the novel property encoding gene into the Ti-plasmid along with a recombinase site. The vector is referred pLBC-1 and (L eft B order C onstruct) , can function as a "shuttle vector (shuttle vector)" to introduce new characteristics encoding gene, for the assembling (assembly) is , Detailed below.

The pLBC-1 vector was constructed to introduce the recombinase region by homologous recombination with the Ti-plasmid pTi-C58 RBC-1, the homology region of which targets the left border region. Several cloning steps were used. The steps are outlined in Figures 23-26. The various Ds involved in these steps
The assembly of NA components is as follows:

In the assembly of the DNA components detailed here, “shuttle vector” pLBC
-1 is a modified DNA sequence (FRT) which is recognized by recombinase
Used to introduce i-plasmid pTiC58 RBC-1. pLBC-1 includes the following: homology region with the left border region of Ti-plasmid (ROH ( _LL )) (SEQ ID NO: 1), plant-expressible promoter (NosPr) (SEQ ID NO: 7), Recombinase recognition sequence (FRT), multiple cloning site, inducible promoter (CiPr)
(SEQ ID NO: 9), a modified FLP recombinase gene containing a plant intron (
FLoP), polyadenylation signal (NosTer), ampicillin resistance gene (Amp) (SEQ ID NO: 11), and homology region (ROH ( _LR )) with the right side of the left border region of the C-58 plasmid (SEQ ID NO: 2). The arrangement of these components is shown in FIG.

The following steps were used to assemble these components: plasmid pN
BE was used to clone the Amp resistance gene, resulting in plasmid pAmp. The plasmid pAmp was restricted with Eco RI and Kpn I and the NosTer fragment was added to obtain pAmpTer (FIG. 23). Then, this plasmid was subjected to restriction treatment with KpnI, and the modified pFLop recombinase gene was added as a KpnI fragment. The pFLoP gene was constructed as shown in Figure 10. In this figure, FLP recombinase is FLP
By inserting a synthetic intron sequence (SEQ ID NO: 6) into the Pst I site of recombinase (SEQ ID NO: 5), an Arabidopsis eEF-referred to as intron (e)
The FLP recombinase, which has been modified to contain the IB intron (Gidekel et al., Gene 170: 201-206, 1996) and has a plant intron and can be expressed in plants, is obtained. The intron contains multiple termination signals and is read-through.
gh) expression eliminates expression in a bacterial host. The plasmid incorporating the Amp, NosTer, and pFLoP genes, called pAmpTerFLoP, is shown in FIG.

Another intermediate vector for constructing pLBC-1 is constructed as outlined in FIG. In this series of steps, the vector pNBE is restricted with Sph I and Xba I and the Sph I -Xba I fragment of pTeasy-LL is added. PTeasy-LL is a vector obtained by cloning a PCR product representing the left side of the left border homology region using the PCR primers shown in SEQ ID NOs: 21 and 22.
The plasmid incorporating the pTeasy-LL fragment and the pNBE vector is called pNBE-ROH ( _LL ).

Nos promoter was added to pNBE-ROH (L _L ) as an Xba I -Xho I fragment, and pROH (
To obtain a _L L) NosPro. The Nos promoter was isolated by PCR amplification as shown in Figure 11, using the primers of SEQ ID NOs: 15 and 16.

PROH (L _L ) NosPro was restricted with Sph I and Xho I and cloned into pAmpFRT (R) to obtain pROH (L _L ) NosPrFRT as shown in FIG. Plasmid pAmpFRT (R)
Was obtained as shown in FIG. To construct pAmpFRT (R), pNBE and Amp resistance gene were used to construct pNBEAmp. This plasmid was designated Bgl II and B
Restriction treatment with am HI and addition of synthetic double-stranded DNA containing FRT site (SEQ ID NOs: 29 and 30) gave pAmpFRT (R).

Another series of intermediate plasmids was constructed as outlined in FIG. In this series of steps, pUM21 was used to perform PCR amplification with the primers of SEQ ID NOs: 23 and 24, followed by the right side of the homology region (R
OH (L _R )) was cloned. The resulting plasmid is called pROH ( _LR ). After PCR amplification using the primers of SEQ ID NOS: 27 and 28, a cold-inducible promoter (SEQ ID NO: 9) derived from Brassica was added to this plasmid. The resulting plasmid is called pCiPrROH ( _LR ). PAmpTerFLo was added to this plasmid.
A BglII fragment derived from P (FIG. 23) was added to obtain pCiPrFLoPTerAmpROH ( _LR ).

This plasmid, pCiPrFLoPTerAmpROH (L _R ), was restricted with Not I and Bam HI, and the Not I -Bam HI fragment derived from pROH (L _L ) NosPrFRT was added to obtain pLBC-1. This is shown in FIG. The vector pLBC-1 contains the following: a modified FLP containing a homology region to the left border region of the Ti-plasmid, a promoter expressible in plants, a recombinase recognition sequence, multiple cloning sites, an inducible promoter, a plant intron. Recombinase gene, polyadenylation signal, ampicillin resistance gene, and homology region to the left border of the C-58 Ti-plasmid. A restriction map of pLBC-1 is shown in FIG.

Example 5 This example illustrates the introduction of plasmid pLBC-1 into C-58 RBC-1 Agrobacterium.

In this example, the pLBC-1 vector was cloned from the C58 RBC-1 strain pTi-C58 RBC-1 Ti by the combination of three parental crosses described in Example 2 and homologous recombination.
Used to insert a second recombinase site and related DNA components into the plasmid. In this example, pLBC-1 in the E. coli DH5FT strain was cloned into Agrobacterium C58 RBC-1 and the broad host range plasmid pRK20.
E. coli helper HB101 strain having 13 is combined. Cells were plated in minimal medium for resistance to the antibiotics carbinicillin and rifampicin,
And Agrobacterium for sensitivity to kanamycin.

Agrobacterium cells resistant to the antibiotic are selected and the integrity of the DNA inserted in the left border region is confirmed by PCR and restriction analysis. The resulting vector was a wild-type C58 Ti- modified to contain a DNA sequence (and any marker coding region) recognized by a site-specific recombinase at both the right and left borders of T-DNA. Contains plasmid. The plasmid further comprises a recombinase gene under the control of a plant promoter which is induced by cold treatment. The vector contains a promoterless GUS-NPTII gene having an FRT site at the 5'end, and 3
'It further contains a nos promoter bounded at the FRT site at the end, so that
Successful excision of the oncogene region, removal of the tumorigenic effect, linking the nos promoter to the GUS-NPTII gene, thereby conferring a visible phenotype on the transformed cells. The modified Ti-plasmid is pTi-C58 CIMB (Cold Inducible Modified Bo
rders).

Example 6 This example illustrates the construction of a second vector for introducing a recombinase site into the left border region of a Ti-plasmid derived from wild type C-58 nopaline Agrobacterium.

It will be appreciated that regulation of the expression of the recombinase enzyme contained within the modified Ti-plasmid can be done by many different means. In the previous example, a cold-inducible promoter was used. In this example, the tet repressor-based inducible promoter system is used in the pLBC-1 vector. The vector containing such an alternative gene regulation system is called pLBC-2. In this example, the construction of this second vector was carried out by modifying the C-58 Ti-plasmid (pTi-C58) to contain the DNA sequence recognized by the site-specific recombinase in the right border region.
RBC-1) is to introduce a second recombinase recognition site. The second vector, pLBC-2, differs from the first vector (pLBC-1) in the type of inducible promoter used to control the expression of the recombinase gene.

To modify the Ti-plasmid, a vector was constructed for homologous recombination with the pTi-C58 RBC-1 Ti-plasmid. Several cloning steps were used. The steps are outlined in Figures 36-37. These steps involved in the assembly of the various DNA components are as follows: In the assembly of the DNA components detailed here, pLBC-2 "shuttle vector" is a modified Ti-plasmid pTiC58 RBC described above. -1 is used to introduce a specific DNA sequence (FRT) recognized by the recombinase. This shuttle vector is called pLBC-2 and contains the following: homology region (ROH ( _LL )) (SEQ ID NO: 1) to the left border region of the Ti-plasmid, promoter expressible in plants (Nos) (SEQ ID NO: 7), recombinase recognition sequence (FRT), multiple cloning sites, inducible promoter system, tet promoter operator system modified to contain the tCUP promoter shown in FIG. 16 (Foster et.
al, Plant Mol Biol 41: 45-55, 1999), modified FL containing a plant intron.
P recombinase gene (FLoP), polyadenylation signal (NosTer), ampicillin resistance gene (Amp) (SEQ ID NO: 11), and homology region (ROH ( _LR )) to the right side of the left border region of C-58 plasmid (SEQ ID NO: 2). The arrangement of these components in pLBC-2 is shown in Figure 35.

Construction of pLBC-2 followed a procedure similar to the construction outlined for pLBC-1 and described in pLBC-1.
The main difference between pLBC-2 and pLBC-2 is the inducible promoter system used to regulate the expression of the recombinase gene.

In pLBC-1, a cold-inducible promoter is used to regulate the expression of the recombinase gene, and in pLBC-2, the tet repressor / operator system is used. te
The t repressor / operator system has been utilized in plant cells and typically involves modifying the plant promoter to contain a DNA sequence recognizable by the tet repressor protein, in this case Gatz. and Quail (Proc
Natl. Acad. Sci. USA 85: 1394-1397, 1988), three copies of the operator sequence were inserted into the 35S promoter, resulting in a 35S promoter that can be repressed by the presence of the tet repressor. To be

Expression of the tet repressor is regulated by another plant promoter, which in this case uses the constitutive "tCUP" promoter. Typically, the 35S promoter is used, but both repressor and operator sequences are present in the same vector, and since both sequences contain 35S, another constitutive promoter is used to identify the same. The possibility of rearrangement and duplication due to the presence of two identical sequences on the vector is eliminated.

Thus, the tet repressor system used in the present invention relies on constitutive expression of the tet repressor from the “tCUP” promoter, which was modified to contain the tet operator, 35S. A repressor molecule capable of suppressing the expression of a gene derived from a promoter and capable of regulating the expression of FLP recombinase is produced. The tet repressor has been modified to closely match plant codon usage and to contain a nuclear localization signal. Tet modified in this way
The repressor is called Tet Rsyn and is designed to be expressed in plant cells. The DNA sequence is shown in SEQ ID NO: 31. Therefore, FLP recombinase expression is permanently abrogated under such an arrangement of DNA components. But this te
tet repression binds the tet repressor and tet the tet repressor.
It can be removed by the presence of tetracycline which dissociates from the operator. In this form, FLP recombinase expression can be induced by culturing in the presence of tetracycline.

Construction of pLBC-2 involved the following steps: The vector pNBE was used to clone the Sph I -Xba I fragment of the tCUP promoter to produce the vector pNBE-tCUP. To this vector was added the pTET2 construct containing the modified coding sequence of the tet repressor and the octopine synthase terminator (OCS ter). The resulting plasmid is called ptCUPTetRsynOCSTer. To this construct was added the modified 35S promoter containing the tet operator sequence found in plasmid pUCA7-TX. The sequence of the modified 35S promoter is Gatz et al., Mol Gen Genet 227: 229-237, 199.
Can be found in 1. The resulting plasmid is called pTetRepSys. These steps are shown in FIG.

The next step in the assembly of pLBC-2 utilized the set of components used to obtain pLBC-1 described in Example 3. The assembly of these components is shown in FIG. Plasmid pROH (L _R ) was ligated with pAmpTerFLoP to give pAmpTerFLoPROH (L _R ), which was then ligated with pROH (L _L ) NosFRT to give pROH (L _L ) NosPFRTFLoPTerAmpROH (L _R ). Then, this plasmid was transformed into plasmid pTetRepSys as shown in FIG.
Ligation with the tet repressor system contained in pLBC-2. The final restriction map of pLBC-2 is shown in FIG.

Example 7 This example illustrates the introduction of plasmid pLBC-2 into C58 RBC-1 Agrobacterium.

The pLBC-2 vector was transformed into the pTi-C58 RBC-1 Ti-plasmid of the C58 RBC-1 strain by the combination of three parental crosses described in Example 2 and homologous recombination.
Used to insert a second recombinase site and related DNA component. The resulting Agrobacterium strains were selected for resistance to carbinicillin and rifampicin and for sensitivity to kanamycin,
Confirm the structure of the Ti-plasmid. The resulting plasmid is pTi-C58 TIMB (Te
t Inducible Modified Borders).

Example 8 This example illustrates the transformation of plant cells with pTi-C58 CIMB.

The modified C-58 CIMB Ti-plasmid confirms function by inoculating plant explants and observing crown gall formation. Cold-inducible promoters are inactive at normal temperatures (eg 10-20 ° C) but have high activity at 4 ° C. Induction of recombinase activity by culturing tumor tissue overnight at 4 ° C. results in excision of the region contained between two adjacent recombinase sequences, including the T-DNA border adjacent to the recombinase recognition DNA sequence. Transgenic plant cells are formed. These plant cells have an active GUS-NPT-II fusion protein and the number of transformed cells is scored by staining for GUS activity or selection on kanamycin.

Example 9 This example illustrates the transformation of plant cells with pTi-C58 TIMB.

The modified C-58 TIMB Ti-plasmid confirms function by inoculating plant explants and observing the formation of crown gall. Induction of recombinase activity by culturing crown gall tissue in the presence of 10 μM tetracycline results in excision of a region contained between two adjacent recombinase sequences, resulting in a T-DNA border adjacent to the recombinase recognition DNA sequence. Transgenic plant cells are formed which contain These plant cells contain active GUS-NP
Having the T-II fusion protein, the number of transformed cells is scored by staining for GUS activity or selection on kanamycin.

All references mentioned herein are representative of the level of art to which this invention pertains. To the extent that all publications mentioned herein are inconsistent with this specification, to the extent that each individual publication is specifically and individually incorporated by reference herein, Are made a part of the disclosure content of this specification by reference.

While the present invention has been described in detail by way of explanations and examples for the purpose of clarifying and understanding, changes and modifications can be made without departing from the scope or spirit of the invention defined by the claims. It will be appreciated that can be added.

[0203]

[Sequence list]

[Brief description of drawings]

FIG. 1 illustrates a general approach for recovering transformed plant cells and whole plants using the method of the invention.

FIG. 2 illustrates the induction of a modified Ti-plasmid containing a recombinase recognition site.

FIG. 3 illustrates the use of suppression strategies that can be used to eliminate oncogene function in plant cells transformed with the oncogene.

FIG. 4 illustrates induction of a modified Ti-plasmid containing a recombinase recognition site at the right border of T-DNA. Figure 7 shows the use of the ampicillin marker gene to first select for which homologous recombination has occurred.

FIG. 5 illustrates the use of a recombinase enzyme that cuts out the ampicillin marker gene to obtain a modified Ti-plasmid containing a recombinase recognition site at the right border of T-DNA.

FIG. 6 illustrates induction of a modified Ti-plasmid containing a recombinase recognition site, a recombinase gene, and an ampicillin marker at the T-DNA left border.

FIG. 7: Use of the ampicillin marker gene to select for modified Ti-plasmids containing recombinase sites at the T-DNA left and right borders and further containing bacterial marker and plant expressible recombinase genes. Is illustrated.

FIG. 8 illustrates the expression results of recombinase gene in plant cells transformed with modified Ti-plasmid. The oncogene region is deleted and plant DNA contains only genes that encode novel properties.

9A and 9B illustrate regions of homology used for construction of modified Ti-plasmids. The region of the nopaline Ti-plasmid used is shown. ROH refers to regions of homology, _{"- R L, -R R, -L} L and -
" _LR " refers to the subregions (or portions) of homology (right region (9B) and left region (9A)) used in constructing the vector for homologous recombination in Agrobacterium.

10A-10C provide restriction enzyme maps and notations of modified FLP recombinase DNA sequences used to construct modified Ti-plasmids.

11A-11B provide a restriction map and notation of the source (11A, pBin19) of the modified NOS promoter DNA sequence (PCR product, 11B) used to construct the modified Ti-plasmid. .

FIG. 12 provides a restriction map and notation of the GUS: NPTII fusion DNA sequence used to construct the modified Ti-plasmid.

FIG. 13 provides a restriction enzyme map and notation of the cold-inducible promoter DNA sequences used to construct the modified Ti-plasmids.

FIG. 14 provides a restriction map and notation of the CaMV 35S DNA sequence used to construct the modified Ti-plasmid.

FIG. 15 provides a restriction map and notation of the constitutive “EntCUP2” promoter DNA sequence used to construct the modified Ti-plasmid.

16A-16B provide a restriction map and notation of the tet repressor / operator DNA sequences used to construct the modified Ti-plasmids.

FIG. 17 provides a restriction map and notation of ampicillin bacterial marker DNA sequences used to construct modified Ti-plasmids.

18A-18B provide a restriction map and notation of the FRT recombinase recognizing DNA sequence used to construct the modified Ti-plasmid.

FIG. 19: Homologous recombination plasmid pRBC for inserting a recombinase sequence into the right border region of the T-DNA of the C58 nopaline Ti-plasmid.
1 provides a simplified restriction map and notation of the components used to obtain -1.

FIG. 20: Homologous recombination plasmid pLBC for inserting a recombinase sequence into the left border region of the T-DNA of the C58 nopaline Ti-plasmid.
1 provides a simplified restriction map and notation of the components used to obtain -1.

FIG. 21 provides a restriction map and representation of the modified pGEM7Zf (+) used to construct the modified Ti-plasmid.

FIG. 22 provides a restriction map of the pUM21 vector used to construct the modified Ti-plasmid and describes the construction of the pUM21 vector.

FIG. 23 illustrates the steps used to construct one of the intermediate vectors used to modify the left border region of the T-DNA of the C58 Ti-plasmid, the intermediate vector pAmpTerFloP. The structure is illustrated.

FIG. 24 shows one of the intermediate vectors (pROH ( _LL ) NosPr used to modify the left border region of the T-DNA of the C58 Ti-plasmid.
Figure 3 illustrates the steps used to construct a FRT).

FIG. 25 illustrates the steps used to construct one of the intermediate vectors used to modify the left border region of the T-DNA of the C58 Ti-plasmid.

FIG. 26 depicts the steps used to construct one of the intermediate vectors used to modify the left border region of the T-DNA of the C58 Ti-plasmid (of pLBC-1). Final derivative).

FIG. 27 provides a detailed restriction map of the homologous recombination plasmid pLBC-1 designed to insert a recombinase sequence into the left border region of the T-DNA of the C58 nopaline Ti-plasmid. The plasmid contains flanking regions of two homologies, and a recombinase recognition site (FRT) and a cold-inducible promoter (CiPr) that controls the expression of the FLP recombinase gene are located between these regions, Cold-inducible promoter
Plant intron (FLoP) with nos terminator (NosTer) linked to ampicillin resistance gene for selecting that homologous recombination has occurred
) Has been modified to contain.

FIG. 28 illustrates the steps used to construct one of the intermediate vectors (pAmpFRT (R)) used to modify the right border region of the T-DNA of the C58 Ti-plasmid. To do.

FIG. 29 illustrates the steps used to construct one of the intermediate vectors used to modify the right border region of the T-DNA of the C58 Ti-plasmid (35S polyadenylation signal). Construction).

FIG. 30 illustrates the steps used to construct one of the intermediate vectors (pGUS-pA) used to modify the right border region of the T-DNA of the C58 Ti-plasmid. .

FIG. 31. One of the intermediate vectors (pGUSNPTIIpA) used to modify the right border region of the T-DNA of the C58 Ti-plasmid.
3 illustrates the steps used to construct the.

FIG. 32 shows one of the intermediate vectors (pAMPFRT (R) Gus) used to modify the right border region of the T-DNA of the C58 Ti-plasmid.
) Illustrates the steps used to construct

FIG. 33 illustrates the steps used to construct one of the intermediate vectors used to modify the right border region of the T-DNA of the C58 Ti-plasmid (of pRBC-1). Final derivative).

FIG. 34 provides a detailed restriction map of the homologous recombination plasmid pRBC-1 designed to insert a recombinase sequence into the right border region of the T-DNA of the C58 nopaline Ti-plasmid. The plasmid contains two homologous flanking regions between which two recombinase recognition sites (FRT) flank the ampicillin resistance gene to select for homologous recombination. As well as the "promoterless" GUS-NPTII fusion gene, which can be activated upon recombination in the modified Ti-plasmid.

FIG. 35. Homologous recombination plasmid pLBC for inserting a recombinase sequence into the left border region of T-DNA of C58 nopaline Ti-plasmid.
A simplified restriction map and notation of the components used to obtain -2 is given. This plasmid contains the tet repressor / operator system for controlling recombinase expression.

FIG. 36 illustrates the steps used to construct pTetRepSys, a plasmid encoding the tet repressor system.

FIG. 37 illustrates the construction of vector pLBC-2. C58
Details of the steps used to create the homologous recombination vector for inserting DNA into the left border region of the T-DNA of Ti-plasmid are shown.

FIG. 38 depicts a detailed restriction map of the homologous recombination plasmid pLBC-2 designed to insert a recombinase sequence into the left border region of the T-DNA of the C58 nopaline Ti-plasmid. The plasmid contains two homologous flanking regions, between which is located a recombinase recognition site (FRT) as well as a tet repressor / operator system that controls expression of the FLP recombinase gene. The tet repressor / operator system has been modified to contain a plant intron (FloP) with a nos terminator (NosTer) linked to an ampicillin resistance gene to select for homologous recombination to occur. .

FIG. 39 shows the vector pRBC-1 in C58 Agrobacterium.
, The steps used to introduce pLBC-1 and pLBC-2 and the recovery of the modified Ti-plasmid are illustrated.

[Procedure amendment]

[Submission date] November 7, 2002 (2002.1.7)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12R 1:01) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI) , FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE) , SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, E, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC , LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Erund Hanner, Johann Canada, Case 1 2 Tea 8, Ontario State, Orleans, Abignon Coat 1118 (72) Inventor Wu, Ton Canada, Jay 8T7R8, Quebec, Gatineau, Du Sanary 15 F Term (reference) 2B030 CA15 CA17 CA19 4B024 AA08 CA04 CA09 DA01 DA05 EA04 FA02 GA11 4B065 AA11X AA11Y AA88X AA99Y AB01 AC14 BA02 CA53

Claims (64)

[Claims] 1. A method for producing a transformed plant, comprising: (a) introducing a construct containing at least one Agrobacterium oncogene into a plant cell to obtain a transformed cell containing the construct, Expression of the at least one Agrobacterium oncogene in the transformed cells allows the cells to grow under conditions insufficient to grow non-transformed cells, thereby selecting for transformed cells. And (b) canceling the effect of the at least one Agrobacterium oncogene in the transformed cell and regenerating a morphologically normal transformed plant from the transformed cell. 2. The construct comprises, in the 5 ′ to 3 ′ direction, a first recombinase recognition site, the at least one Agrobacterium oncogene, and a second recombinase recognition site; the canceling step comprising: 2. The method of claim 1, comprising excising the at least one Agrobacterium oncogene or a portion thereof from the construct with a recombinase that recognizes first and second recombinase recognition sites. 3. The method of claim 2, wherein the construct further comprises a recombinase coding sequence that encodes the recombinase. 4. The method of claim 2, wherein the canceling step comprises introducing into the transformed cell a second construct containing a recombinase coding sequence encoding the recombinase. 5. The novel characteristic coding sequence, wherein said construct is located on either the 5 ′ side of said first recombinase recognition site or the 3 ′ side of said second recombinase recognition site, said sequence in a plant. The method according to claim 2 or 3, wherein the expression of P. lactis further comprises a novel trait coding sequence that confers a novel trait on the plant. 6. The method of claim 4, wherein the second construct is a novel characteristic coding sequence, the expression of said sequence in a plant further comprising a novel characteristic coding sequence conferring a novel characteristic to said plant. The method described. 7. The method of claim 3 or 4, wherein the recombinase coding sequence is under the control of an inducible promoter. 8. The method of claim 3 or 4, wherein the recombinase coding sequence comprises a plant intron. 9. The method of claim 2, wherein the recombinase is selected from the group consisting of FLP recombinase, Cre recombinase, and R recombinase. 10. The construct comprises, in the 5 ′ to 3 ′ direction, a T-DNA right border sequence, the first recombinase recognition site, the at least one oncogene, the second recombinase recognition site, and T-. The method according to claim 1, which is a modified Agrobacterium T-DNA region containing a DNA left border sequence. 11. The construct comprises, in the 5 ′ to 3 ′ direction, a first transposase recognition site, the at least one oncogene, and a second transposase recognition site; the canceling step comprising: The method of claim 1, comprising excising the at least one oncogene or a portion thereof from the construct using a transposase that recognizes a second transposase recognition site. 12. The method of claim 11, wherein the construct further comprises a transposase coding sequence encoding the transposase. 13. The method of claim 11, wherein the step of canceling comprises introducing into the transformed cell a second construct comprising a transposase coding sequence encoding the transposase. 14. The construct comprises 5 of the first transposase recognition sites.
A novel characteristic coding sequence located either on the'side or on the 3'side of the second transposase recognition site, the expression of said sequence in a plant imparting a novel characteristic to said plant. 13. The method of claim 11 or 12, further comprising: 15. The method of claim 13, wherein the second construct is a novel characteristic coding sequence, the expression of said sequence in a plant further comprising a novel characteristic coding sequence conferring a novel characteristic to said plant. The method described. 16. The method according to claim 12 or 13, wherein the transposase coding sequence is under the control of an inducible promoter. 17. The method according to claim 12 or 13, wherein the transposase coding sequence comprises a plant intron. 18. The construct comprises, in the 5'to 3'direction, a T-DNA right border sequence, the first transposase recognition site, the at least one oncogene, the second transposase recognition site, and T-. The method according to claim 11, which is a modified Agrobacterium T-DNA region containing a DNA left border sequence. 19. The method of claim 1, wherein the counteracting step comprises contacting the construct with a DNA binding protein to suppress expression of a product encoded by the at least one oncogene. 20. The construct wherein D encodes the DNA binding protein
20. The method of claim 19, further comprising a NA binding protein coding sequence. 21. The method of claim 19, wherein said canceling step comprises introducing into said transformed cell a second construct comprising a DNA binding protein coding sequence encoding said DNA binding protein. 22. The construct according to claim 19 or 20, wherein the construct is a novel characteristic coding sequence, the expression of said sequence in a plant further comprising a novel characteristic coding sequence conferring a novel characteristic on said plant. the method of. 23. The method of claim 21, wherein the second construct is a novel characteristic coding sequence, the expression of said sequence in a plant further comprising a novel characteristic coding sequence conferring a novel characteristic on said plant. The method described. 24. The step of canceling comprises contacting the at least one oncogene or a transcription product thereof with an antisense polynucleotide that suppresses transcription of the at least one oncogene or translation of the transcription product. ,
The method of claim 1. 25. The method according to claim 1, wherein the plant cell is a dicotyledonous plant cell. 26. The method according to claim 25, wherein the plant cell is a member of a member of the mallow family, the flax family, the Asteraceae family, the Solanaceae family, the legume family, the Euphorbiaceae family, the Oleaceae family, or the Brassicaceae family. 27. The method of claim 25, wherein the plant cell is a member of the Brassica genus. 28. The plant cell is a broccoli, cabbage, cauliflower, kale, Chinese kale, collard, kohlrabi, Chinese cabbage, Thai rhinoceros (pak choi) or turnip cell. . The method of claim 25. 29. The method of claim 1, wherein the construct is introduced into the plant cell by Agrobacterium-mediated transformation. 30. A plant, a plant seed, a plant embryo, a plant cell, or a plant tissue, produced by the method according to any one of claims 1 to 29, or a progeny thereof. 31. A DNA sequence homologous to the left part of the right border region of the T-DNA region of the Ti-plasmid, a recombinase recognition site, and Ti in the 5′-3 ′ direction.
A DNA vector containing a region of homologous DNA in the right part of the right border region of the T-DNA region of the plasmid. 32. A DNA sequence homologous to the left part of the left border region of the T-DNA region of the Ti-plasmid in the 5'-3 'direction, a recombinase recognition site, and Ti.
A DNA vector containing a region of homologous DNA in the right part of the left border region of the T-DNA region of the plasmid. 33. The DNA vector of claim 31 or 32, further comprising an antibiotic resistance marker useful for selection on Agrobacterium. 34. A modified Ti-plasmid containing a recombinase recognition site 3 ′ to the right border of T-DNA. 35. A modified Ti-plasmid containing a recombinase recognition site 5 ′ to the left border of T-DNA. 36. A modified Ti-plasmid containing a recombinase recognition site on the 3'side of the T-DNA right border and a recombinase recognition site on the 5'side of the T-DNA left border. 37. The modified Ti-plasmid according to any one of claims 34 to 36, wherein the plasmid further comprises a recombinase coding sequence expressible in plant cells. 38. The modified Ti-plasmid according to any one of claims 34 to 37, further comprising a scorable marker coding sequence expressible in plant cells. 39. A modified Ti-plasmid pTi-C58 CIMB. 40. A modified Ti-plasmid pTi-C58 TIMB. 41. A modified Ti-plasmid pTi-C58 RBC-1. 42. A DNA vector pRBC-1. 43. A DNA vector pLBC-1. 44. A DNA vector pLBC-2. 45. A construct for transformation of plant cells, which comprises in the 5'to 3'direction a first recombinase recognition site, at least one oncogene and a second recombinase recognition site. 46. The construct of claim 45, further comprising a recombinase coding sequence that encodes a recombinase. 47. A novel characteristic coding sequence located on either the 5 ′ side of the first recombinase recognition site or the 3 ′ side of the second recombinase recognition site, the expression of said sequence in plants comprising: 47. The construct of claim 45 or 46, further comprising a novel property coding sequence that confers a new property on the plant. 48. The construct of claim 46, wherein the recombinase coding sequence is under the control of an inducible promoter. 49. The construct of claim 46, wherein the recombinase coding sequence comprises a plant intron. 50. The recombinase is selected from the group consisting of FLP recombinase, Cre recombinase, and R recombinase.
The construct described in. 51. The construct comprises, in the 5 ′ to 3 ′ direction, a T-DNA right border sequence, the first recombinase recognition site, the at least one oncogene, the second recombinase recognition site, and T-. 46. The construct of claim 45, which is a modified Agrobacterium T-DNA region containing a left DNA border sequence. 52. A first transposase recognition site, in the 5 ′ to 3 ′ direction,
A construct comprising at least one oncogene and a second transposase recognition site. 53. The construct of claim 52, wherein the construct further comprises a transposase coding sequence encoding a transposase. 54. A novel characteristic coding sequence located on either the 5'side of the first transposase recognition site or the 3'side of the second transposase recognition site, the expression of said sequence in plants comprising: 54. The construct of claim 52 or 53, further comprising a novel property coding sequence that imparts a new property to the plant. 55. The construct of claim 53, wherein the transposase coding sequence is under the control of an inducible promoter. 56. The construct of claim 53, wherein the transposase coding sequence comprises a plant intron. 57. The construct comprises, in the 5'to 3'direction, a T-DNA right border sequence, the first transposase recognition site, the at least one oncogene, the second transposase recognition site, and T-. 53. The construct of claim 52, which is a modified Agrobacterium T-DNA region containing a left DNA border sequence. 58. A construct comprising a DNA binding protein coding sequence encoding at least one oncogene and a DNA binding protein. 59. The construct of claim 58, which further comprises a novel characteristic coding sequence, the expression of said sequence in a plant conferring a novel characteristic on said plant. 60. A plant, plant seed, plant embryo comprising the construct according to any one of claims 45 to 59 or the vector according to any one of claims 31 to 33 and 42 to 44. , Plant cells, or plant tissue. 61. The construct of any one of claims 45-59, claim 3.
The vector according to any one of claims 1 to 33 and 42 to 44, or claims 34 to 41.
An Agrobacterium cell containing the Ti-plasmid according to any one of 1. 62. A method of modifying a Ti-plasmid to contain a recombinase or transposase recognition site, comprising the step of: (a) adding a first DNA vector to an Agrobacterium cell containing the Ti-plasmid, A DNA sequence homologous to the left part of the right border region of the T-DNA region of the Ti-plasmid, a recombinase or transposase recognition site, and a region of DNA homologous to the right part of the right border region of the T-DNA region of the Ti-plasmid. The first DNA vector containing 5'to 3'direction was introduced under sufficient conditions to cause homologous recombination between the DNA vector and the Ti-plasmid, and T-
Obtaining an Agrobacterium cell containing a modified Ti-plasmid having a recombinase or transposase recognition site near the T-DNA right border sequence in the DNA region; and (b) the Agrobacterium derived from step (a) In the cell, a second DNA vector, a DNA sequence homologous to the left part of the left border region of the T-DNA region of the modified Ti-plasmid, a recombinase or transposase recognition site, and T-of the modified Ti-plasmid. DNA homologous to the right part of the left border region of the DNA region
A second DNA vector containing the region of 5'to 3'direction is introduced under sufficient conditions to cause homologous recombination between the second DNA vector and the modified Ti-plasmid, A modified Ti-plasmid, comprising a recombinase or transposase recognition site near the left border sequence of T-DNA in the T-DNA region of the further modified Ti-plasmid, and the TD of the further modified Ti-plasmid.
A method comprising obtaining Agrobacterium cells containing a further modified Ti-plasmid with a recombinase or transposase recognition site near the T-DNA right border sequence in the NA region. 63. The method of claim 62, wherein the Ti-plasmid further comprises within the T-DNA region a recombinase or transposase enzyme coding sequence under the control of an inducible promoter. 64. The method according to claim 62 or 63, wherein the Ti-plasmid further comprises a scoable marker gene expressible in plant cells within the T-DNA region.