PLANT TRANSFORMATION

Abstract: A plant containing an engineered DNA construct including an oriT DNA sequence and heterologous DNA.

Description: PLANT TRANSFORMATION Background of the Invention This application is a continuation-in-part of Buchanan- ollaston et al. U.S.S.N. 926,629, filed November 3, 1986. This invention relates to the integration of. heterologous DNA into the genome of plant cells. By the term "heterologous DNA", we mean DNA (a gene or other DNA) which is non-plant derived, modified, synthetic, derived from a different plant strain or species, or derived from a different location in the plant's genome. The productivity of modern agriculture is in large measure due to the development of improved crop varieties. This has been accomplished through classical plant breeding, where compatible varieties of crops are cross-bred to yield desired improvements. This method suffers from certain limitations, such as imprecision and unpredictability of results; long time frames for breeding programs; and the limitation of the available gene pool to those plants able to cross with each other (i.e., those of the same species). Certain naturally-occurring bacterial plasmids are known to carry genetic sequences that enable transfer of plasmids from one bacterial cell to another, including the tra, mob and oriT loci. The tra gene cluster is responsible for cell-to-cell contact and the formation of a mating bridge for the transfer of DNA. The mob DNA sequence encodes proteins which nick and unwind plasmid DNA to enable its replicative transfer. These proteins specifically recognize oriT nucleotide sequences as their target, with specific protein molecules recognizing specific oriT sequences (See Guiney, D.G. et al. , (1985) The Origin of Plasmid DNA - 2 - Transfer During Bacterial Conjugation; In: Helinski, D.R., et al. (eds) "Plasmids in Bacteria", Plenum Press, New York, pp. 521-534). Summary of the Invention We have discovered that compatible oriT and mob DNA sequences which promote the transfer of DNA from one bacterial cell to another in bacterial (particularly Gram-negative) conjugation can also bring about the transfer of DNA (particularly desired heterologous genes) from bacterial cells to plant cells, in a manner analogous to known techniques involving the T-DNA (particularly the Ti border DNA) of J tumefaciens. Our discovery makes possible the production of a wide range of new and useful plants. For example, the discovery may make possible the transformation of plant cells without the need to rely on A^ tumefaciens, and could thus open up the possibility of transforming even plant cells which cannot be infected by A_ tumefaciens (e.g., certain monocotyledenous plants such as corn and other cereal grains, and certain dicotyledenous plants as well). Our discovery also makes possible the transformation of A. tumefaciens - infectible plants using vectors which contain oriT and mob functions, but not Ti .border DNA. Thus self-mobilizable broad host range plasmids such as RSFIOIO, which contain oriT and mob DNA sequences but no Ti border sequences, can be used in conjunction with A. tumefaciens to transform any plant which can be infected by A. tumefaciens. Accordingly, the invention features, in general, a plant containing an engineered DNA construct including an oriT DNA sequence and heterologous DNA. Preferably, the DNA construct is integrated into a chromosome of the plant, and preferably the construct, in the plant, contains, in sequential order, a first portion of the oriT DNA sequence, the heterologous DNA, and the balance of the oriT DNA sequence. In other preferred embodiments, the construct, in the plant, further contains a mob DNA sequence between the two portions of the oriT DNA sequence. Plants of the invention can be made by transforming a plant cell, under transforming conditions, with a first plasmid containing heterologous DNA and an oriT DNA sequence, and with a mob DNA sequence compatible with the oriT sequence, the mob DNA sequence being carried either on the first plasmid or on a second plasmid, neither the first nor the second plasmid including any DNA which is derived from or identical to Ti border DNA or A. tumefaciens. As used herein, a plant cell "transformed" with a heterologous gene means a plant cell in which the gene has gotten into the plant cell and is expressed and stably maintained in that cell (preferably, but not necessarily, by integration into a chromosome of the plant). OriT and mob sequences are "compatible", as that term is used herein, where proteins encoded by the mob sequence recognize the oriT site (generally about 100-500 base pairs in length) and are capable of nicking the plasmid DNA bearing the heterologous gene and the oriT sequence to effect linearization of the plasmid DNA for its transfer into the plant cell. As mentioned above, the compatible mob sequence can be on the same plasmid as the oriT sequence and the heterologous gene (which must be on the same plasmid), or on another plasmid. In preferred embodiments, the plant is a monocotyledenous or dicotyledenous plant; and the heterologous DNA includes a gene selected from the group consisting of non-plant genes, modified genes, synthetic genes, and genes from a different plant strain or species. In the plant cell transformation method of the invention, in some preferred embodiments all three plasmid components are carried on one plasmid. In other preferred embodiments of the method, the plasmid DNA is carried in a bacterial cell, preferably one of a species which is capable of growing in the rhizoplane or rhizointerstices of the plant species from which the plant cell is derived. Bacteria growing in the rhizoplane are those which are in direct contact with the roots and root hairs of the plant, while bacteria growing in the rhizointerstices are those growing inside the roots or root hairs, in the interstices between cells. A suitable host bacterial strain is potentially available for any plant which is associated with bacteria growing in the plant's rhizoplane or rhizointerstices, and such bacteria are known for virtually all plant species. For example, Rhizobium meliloti is a bacterial species known to associate closely with alfalfa; Rhizobium japonicum with soybeans; and Azospirillum brasilense with corn. Additionally, Agrobacterium tumefaciens is known to associate with a number of dicotyledenous plant species. Such bacteria are desired carriers of the plasmid DNA because they can contain vir gene clusters capable of causing the bacteria to recognize the presence of plants (probably by virtue of plant secretions), which recognition is a necessary first step in transformation; tra genes, which are needed for conjugation of bacterial cells, are not needed, since it - 5 - is plant transformation, not bacterial conjugation, which is occurring. Bacteria which are closely associated with a plant are also advantageously likely to contain other elements which aid in the recognition and transformation of plants. Since not all bacteria which are closely associated with plants contain the requisite vir genes, the bacterial species chosen should be not only one which is associated with the plant, but also should contain vir genes; if it does not contain vir genes, the DNA used to transform it must include, as well as oriT and mob DNA, vir genes derived, e.g., from the A. tumefaciens Ti plasmid according to conventional techniques. (See Stachel and Nester (1986) EMBO J. 5_: 1445-1454 for a restriction map of the vir gene cluster.) As an example of the necessity of vir genes, we have found that some vir~ strains of A^ tumefaciens cannot serve as a host for transformation into plants of plasmids including a heterologous gene and oriT and mob sequences, and lacking Ti border DNA. It is also known that Rhizobium meliloti is a plant-associated bacterial species, yet we have found that R. meliloti cannot serve as a host for the transformation of plants with a plasmid including a heterologous gene and oriT and mob functions, at least in part because R. meliloti lacks vir genes. Preferably, the plasmid DNA also includes a DNA sequence encoding a selectable marker protein, and the method includes the step of selecting transformants on the basis of the presence of the selectable marker protein (which can itself be the desired product of the heterologous gene) . Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. - 6 - Description of the Preferred Embodiments We now describe preferred embodiments of the invention, after briefly describing the drawings. Drawings Figs. 1, 7, and 8 are diagrammatic representations of plasmids of the invention. Fig. 2 is a diagrammatic representation of an intermediate gene fusion used in the construction of the plasmids of Figs. 1, 7, and 8. Figs. 3 and 5 are diagrammatic representations of plasmids " containing synthetic Ti border DNA. Fig. 4 is a diagrammatic representation of a plasmid containing neither Ti DNA nor mob and oriT sequences. Fig. 6 is a diagrammatic representation of the region of pSUP104 containing the oriT and mob loci. Plasmid Components As is mentioned above, the plasmids of the invention contain various DNA regions and sites, now discussed in greater detail. Selectable Marker Because transformation of plant cells with plasmids containing heterologous genes is a relatively rare event, plasmids of the invention preferably contain a DNA region which encodes a selectable marker protein for identification of transformants. This marker protein can be any protein which can be expressed in plant cells and which enables phenotypic identification of plant cells which express the protein. Preferred marker proteins are proteins which provide resistance to one or more antibiotics; currently most preferred is the protein aminoglycoside phosphotransferase, which inactivates antibiotics such as kanamycin, neo ycin, and - 7 - G418; transformants are those plant cells able to grow in the presence of antibiotic. Other examples include chloramphenicol acetyl transferase, which provides resistance to chloramphenicol; dihydrofolate reductase, which provides resistance to methotrexate; and the protein hygromycin-phosphotransferase encoded by the aph IV gene which confers resistance to the antibiotic hygro ycin B. Herbicide resistance can also be used as the selectable marker. As is mentioned above, herbicide resistance can constitute both the selectable marker and the desired function itself, in which case no additional heterologous gene need be inserted into the vector. Many of the herbicides currently commercially available worldwide have the undesired effect of being to some degree deleterious to crop plants as well as to weeds, particularly crop plants which have matured to the point of foliation. For this reason, farmers sometimes apply herbicides to farmland prior to both weed growth and crop foliation, a procedure which is potentially wasteful (weeds may not have grown to a significant degree anyway), and not amenable to selective herbicide application. Using the vectors of the invention, any protein which induces herbicide resistance can be produced, by expression of the introduced gene, in all of the plant tissues, including the herbicide-vulnerable leaves. This foliar expression permits herbicide application at any time during crop growth, including the period after the appearance of leaves on the crop plant, and thus will be particularly useful in conferring resistance to post-emergent herbicides. - 8 - Representative commercially available herbicides to which resistance can be induced include 2,2-dichloropropionic acid ("Dalapσn"), glyphosate (N-phospho-mεthyl glycine; "Roundup"), cyanazine, chlorsulfuron, L-phosphinothricin, atrazine, the imidazolines, and 6-chlorσ-N-ethyl-N-(l-methyl)- l,3,5-triazine-2,4-diamine ("Gesaprim 500 "). Mechanisms by which vector encoded protein-mediated herbicide resistance can be induced in plants according to the invention include the following: Detoxification --This involves either enzymatically altering the herbicide molecule by removing a substituent rendering the molecule non-toxic; cleaving the molecule; or adding a substituent to the molecule to render it non-toxic. The first mechanism, removal of a substituent, is the mechanism by which a- bacterial dehalogenase inactivates, by removal of the chlorine atoms, the herbicide Dalapon. The bacterial plasmid carrying the gene for dehalogenase has been isolated and is described in Beeching et al. (1983) J. Gen. Microbiol. 129, 2071. An example of the addition of a substituent to detoxify a substance toxic to plants is the enzymatic addition of a phosphate group to kanamycin by aminoglycoside phosphotransferase, described above. Modification of Herbicide Target - This involves introducing into the plant a mutant gene which codes for a form of the herbicide-targeted enzyme which is not susceptible to poisoning by the herbicide. Increase in Target - This involves introducing into the plant a gene encoding the enzyme poisoned by the herbicide, to increase the amount of the enzyme produced by the plant. - 9 - Various bacteria are able to use the detoxification mechanism to render themselves resistant to herbicides. These bacteria are a convenient source of herbicide resistance-conferring genes. Such genes are generally isolated from such bacteria (generally soil bacteria) by first selecting bacteria either able to grow on the herbicide as a nutrient source or able to detoxify the herbicide. The enzymes responsible for herbicide detoxification in these bacteria are characterized and the genes encoding the enzymes are then isolated according to- standard methods. Polyadeny1ation Site Eukaryotic (e.g., plant) messenger RNA's must be polyadeny1ated for efficient translation and processing. Polyadenyl tiσn requires a recognition site for polyadenylation enzymes near the 3' end of the DNA region encoding the selectable marker. DNA Sequences for Plasmid Transfer DNA derived from or substantially identical to (i.e., structurally similar enough to function in the same way) the mob and oriT loci of a self-mobilizable or self-transmissible bacterial plasmid (the latter contain tra genes and the former do not) is used to effect the transfer of the plasmids of the invention into plant cells. As reviewed in Guiney et al., id., the role of these loci in conjugative plasmid DNA transfer, particularly from one Gram-negative bacterium to another, is well known. As discussed above, we have discovered that the oriT-mob system is useful for the transfer of DNA from bacteria into plant cells as well. Many naturally-occurring plasmids contain mob and oriT sequences, including those of the P-type, Q-type and W-type incompatability groups. Proteins - 10 - encoded by the mob of one plasmid generally will only recognize the oriT site of that or a closely related plasmid, hence compatible mob and oriT sequences should be used together. A preferred source of mob and oriT sequences is the Q-type incompatability group of plasmids, e.g., RSFIOIO and its derivatives. These sequences may also be derived from P-type plasmids, e.g., RK2. Other plasmids from which the compatible mob and oriT sequences can be obtained are the hundreds of other known large self-transmissible plasmids. Generally, these plasmids are too large to be used in toto and to be useful in the invention, either the mob and oriT sequences are removed, by conventional methods, or extraneous DNA is removed, leaving a smaller plasmid containing oriT and mob. Nucleotide sequences of the oriT loci of several plasmids, including RSFIOIO and RK2, are given in Willetts and Wilkins (1984) Microb. Rev. 48:24-41. Regulatory Sequences Transcription of both the selectable marker gene and the desired heterologous gene (if different from the marker gene) , in order to lead to efficient expression, is preferably under the control of regulatory sequences normally expressed in plant cells, e.g., promoters such as those for the nopaline synthase (NOS) or octopine synthase (OS) genes of the T-DNA of Agrobacterium tumefaciens. Examples of other suitable promoter sequences include those of the ribulose biphosphate carboxylase small subunit gene, the nitrate reductase gene, the glutamine synthase gene, and the 35S and 19S promoters of Cauliflower Mosaic Virus ("CAMV"). Synthetic, engineered, or altered natural promoters can also be used. In some instances, it will be desirable - 11 - to use promoters which are regulated, e.g., promoters active only at certain times in the plant's development. It is desirable that the plasmids of the invention contain less than the entire structural gene normally under control of each promoter used, to ensure that metabolic energy of the transformed plant is not wasted producing protein encoded by the structural gene. Site for Desired Heterologous Gene The site for the insertion of a desired heterologous gene can be any site at which an endonuclease can act to cut the plasmid for insertion of the desired heterologous gene. Preferably the site is unique in the plasmid, so that the endonuclease cuts the plasmid only in the desired location. Desired Heterologous Gene The desired heterologous gene can be any gene which is capable of being expressed in the plant, and which encodes a protein which enhances a beneficial feature of the plant, or provides a new beneficial feature. Examples of such genes are those encoding resistance to herbicides, resistance to diseases such as tomato fusarium wilt, proteins which can be produced to improve the protein content or other nutritive value of the plant, and genes encoding biopesticides. Although the easiest modification to carry out is one involving a biochemical function which is controlled by a single protein, and is thus encoded by a single gene, more than one heterologous gene can be introduced, and the expression of such multiple genes controlled in a coordinate manner so as to introduce more complex biochemical functions into plants. Examples include multi-enzymatic pathways, e.g., energy-generating reactions and biosynthetic pathways. - 12 - As mentioned above, transcription of the desired heterologous gene, like transcription of the selectable marker gene, is preferably under the control of a regulatory sequence normally expressed in plant cells. (Also, as mentioned above, there need be no additional heterologous gene if the marker itself has utility; e.g., if it confers herbicide resistance on the plant.) The fusion of the heterologous gene to the regulatory sequence can be carried out prior to the insertion of the heterologous gene into the vector, using conventional techniques. Alternatively, the gene can be inserted into the vector by itself, and the regulatory sequence inserted upstream from the gene separately, prior to or following the insertion of the gene, using conventional methods. Plasmid Construction Referring to Fig. 1, plasmid pJP181 of the invention contains the aminoglycoside phosphotransferase gene from the transposon Tn5, encoding resistance to the antibiotics kanamycin and G418 (the kan r gene), under the transcriptional control of the NOS promoter from the Agrobacterium tumefaciens Ti plasmid; a polyadenylation site; the oriT and mob sequences from the broad host-range plasmid RSFIOIO (Scholz, P., et al., (1985) Replication Determinants of the Broad Host-Range Plasmid RSFIOIO, in Helinski, E.R. et al. (eds.). "Plasmids in Bacteria", Plenum Press, New York, pp. 243-259.); and a unique Hindlll or Sail site for insertion of a heterologous gene. Plasmid pJPlδl was constructed as follows, beginning with the construction of a fusion of the kan r gene to the NOS promoter and polyadenylation site. This fusion was derived from plasmid pVW104, the construction of which is described in - 13 - Buchanan-Wollaston et al. , "Plant Transformation Vector", U.S.S.N. 845,547, assigned to the same assignee as the present application, and hereby incorporated by reference. The fusion can also be derived from plasmid pVW125 (ATCC No. 39929, also described in Buchanan-Wollaston, id) . The fusion allows expression of the kan r gene in plant cells, for use as a selectable marker. The Bcll-Bglll fragment of pVW104 (Fig. 2), containing the kan r gene and polyadenylation site attached to the NOS promoter, was cloned into the BamHI site of pSUP104 (a plasmid derived from RSFIOIO) to yield plasmid pJP181 (Fig. 1). Plasmid pSUP104 is described in Simon et al. (1983) "Vector Plasmids for in vivo and in vitro Manipulations of Gram-Negative Bacteria" in Molecular Genetics of the Bacterial Plant Interaction (Puhler, ed., Berlin 1983), and is available from Agrigenetics Corporation. pJP181 is similar in construction to pVW144 (Fig. 3), described in Buchanan-Wollaston, id, except that pVW144, unlike pJP181, contains a single copy of a 25-bp synthetic border sequence derived from A. tumefaciens Ti DNA, which is capable of effecting transformation of plant cells. When used in the "binary" transformation method described below, we found that pJP181, like pVW144, was able to transform tobacco cells to kanamycin resistance despite the absence in pJP181 of natural or synthetic DNA derived from transforming DNA of A^. tumefaciens. To investigate the mechanism for the plant-transforming capability of pJP181, analogous vectors were constructed using, rather than pSUP104, a different plasmid, pR 290 (ATCC No. 37168; which is derived from RK2, ATCC No. 37125) as the backbone. The Bcll-Bglll fragment of - 14 - pVW104 and corresponding BamHI fragment of pVW144 (which contains the selectable marker and the 25 bp synthetic border sequence) were separately inserted into the Bglll site of pRK290, to create plasmids pVW170 (Fig. 4) and pVW171 (Fig. 5), respectively. When pVW170 and pVW171 were used in a "binary" transformation, pVW171 was, by virtue of the synthetic border sequence, able to transform tobacco cells, but pVW170 was not. We hypothesized that the ability of pJP181 to transform plant cells was due to the presence of certain RSFlOlO-derived- sequences on this vector, not present on pRK290. A likely DNA sequence present in RSFIOIO but not pR 290 is one containing both the oriT and mob loci. (The predecessor of pRK290, RK2, does contain oriT and mob sequences, but the mob sequences were removed in the construction of pRK290.) This hypothesis was tested by the construction, described below, of a vector containing only a specific DNA segment of RSFIOIO, known to contain the oriT and mob loci. The oriT and mob loci of RSFIOIO are known to be contained on a 2.9 kb Aval fragment, which also contains the oriV sequence (the vegetative origin of replication) and part of the repB gene (see Fig. 6). This 2.9 kb fragment was cloned into the EcoRI site of pVW170 to yield pVW210 (Fig. 7) and pVW214 (Fig. 8), differing only in the orientation of the insert. Both plasmids pVW210 and pVW214 were found to be capable of transforming plant cells by the "binary" method described below. This indicates that the oriT and mob loci of pSUP104 are responsible for transfor atio , Additional Vectors We have constructed several other vectors which include the RSFIOIO oriT and mob sequences, and which contain a different selectable marker (the hygromycin B resistance gene ("hyg "); Gritz et al. (1983) Gene 2_5:179-180) and/or different promoters (the 19S and 35S promoters from CAMV) . These vectors, which were constructed analogously to pJP181, described above, are also suitable for transformation of plants with heterologous DNA. For all constructs utilizing the hyg gene, this gene was modified as described in Buchanan-Wollaston et al . , id, to remove an extraneous ATG codon. One additional vector of the invention, pVW206, in which the hyg gene is under the control of the NOS promoter, was constructed by cloning the Bcll-Sall fragment of pVW189 (described in Buchanan-Wollaston et al., id) into pSUP104. Plasmid pVW206 was found to be capable of transforming plant cells by the "binary" method described below. Other vectors were constructed utilizing the CAMV 35S promoter (Hohn et al. (1982) Current Topics in Microbiol. Immunol. 9_6:193-236) , in particular the 0.42 kb EcoRI-SauIIIA fragment (nucleotides 7017-7437 - Hohn et al. ) described in Buchanan-Wollaston et al. , id. pJP194 contains a fusion of the kan gene to the 35S promoter and a polyadenylation site; pJP195 contains a fusion of the hyg gene to the 35S promoter and a polydenylation site, both vectors having a pSUP104 backbone, and lacking Ti-border sequences. Plasmids pJP194 and pJP195 were found to be capable of transforming plant cells by the "binary" method described below. Additional vectors were constructed utitilizing the CAMV 19S promoter (strain CM1841); the sequence of the CAMV 19S promoter is given in Table I, below. A 129 bp fragment containing this promoter was isolated from plasmid pBLlOl by first removing a 0.5 kb EcoRI fragment and then cutting with Mnll. Two vectors utilize this promoter fragment: pJP304, which contains a fusion of the kan gene to the 19S promoter and a polydenylation site; and pJP305, which contains a fusion of the hyg R gene to the 19S promoter and a polyadenylation site. Both these fusions have a pSUP104 backbone, and lack Ti border sequences. TABLE I: CAMV 195 Promoter E C H M 0 N B R F 0 I 1 2 AATTCAATAAGGTTAATTCCTAATTGAAATCCGAAGATAAGATTCCCACACACT TTATTCCAATTAAGGATTAACTTTAGGCTTCTATTCTAAGGGTGTGTGA E D E M C D C N R E P L V 1 1 1 TGTGGCTGATATa^AAAAGGCTACTACCTATATAAACACATCTCTGGAGACTGAGAAAATCAGACCTCCAAGCAT ACACCGACTATAGTTTTTCCGA'TGATGGATATATTTGTGTAGAGACCTCTGACTCTTTTAGTCTGGAGGTTCGTA Plasmid pJP181 has a unique HindiII site and Sail site and plasmid pVW210 has a unique Bglll site into which a desired heterologous gene can be inserted. - 17 - using conventional techniques. Similar sites exist, or can be created by standard techniques, on plasmids pVW206, pJP194, pJP195, pJP304, and pJP305. Plant Transformation The plasmids of the invention can be used to transform plant protoplasts or non-protoplast cells, using a "binary" cocultivation technique. The following is an example of this technique, using A^ tumefaciens as the bacterial delivery system. Plasmids containing oriT and mob genes (pJP181, pVW210, pVW206, pJP194, and pJP195) were transferred to an A^ tumefaciens strain, e.g., LBA4404, which carries a Ti plasmid, pLBA4404, deleted of the T-DNA region, so that cocultivation does not result in plant tumor formation. (pLBA4404, and binary cocultivation, are described in Hoekema et al. (1983) Nature 303, 5913.) pLBA4404 does, however, retain the native Ti vir (for virulence) functions, at least some of which are essential for the transfer of a hybrid plasmid of the invention from A^ tumefaciens LBA4404 to the host plant cell. The A^ tumefaciens containing the hybrid plasmid was then cocultivated with plant cells to insert the hybrid plasmid into the plant cells, where the plasmid DNA integrated into the plant cell chromosome. Plant transformation was accomplished using the "quick dip" method, which involves the dipping of plant explants in a bacterial (e.g., 1__ tumefaciens) culture in which the bacteria contain the transforming plasmid, and then incubating the infected plant parts on medium that selects for kanamycin resistance and promotes shoot regeneration; the method is described in Horsch et al. (1985) Science 227, 1229. Plant Regeneration Following selection of transformants, the plant cells (protoplasts or other cells) are cultured under conditions effecting the regeneration of mature plants. Such methods are known, e.g., for the regeneration of tobacco plants from callus culture. See, for example, "Plant Tissue Culture: Methods and Applications in Agriculture", Trevor A. Thorpe, ed. , Academic Press, New York, 1981; particularly the article by O.L. Gamborg and J.P. Shyluk at pp. 21-24, ' and the chart at page 33. The resulting mature plant, the chromosomes of which contain integrated DNA of the vectors of the invention, express the desired heterologous gene. As is mentioned above, it is believed, although not yet known with certainty, that in at least some of the transformed plant cells, transforming plasmid DNA is integrated into a plant cell chromosome such that the chromosome has integrated into it the heterologous DNA flanked by two portions of the oriT sequence. From studies of bacterial conjugation, it is well known that oriT sequences are nicked before transfer of plasmid DNA from one cell into another, and that the transfer of DNA is initiated at the oriT sequence. The nicking site or the oriT sequence of RSFIOIO has been localized to a 153bp sequence of RSFIOIO (Willetts and Wilkins, id.) and to an 80 bp sequence within that 153 bp sequence (Derbyshire et al. M.G.G., 206:154, 1987). We believe that, similarly, the engineered vectors of our invention are nicked at the oriT site prior to transfer into a plant cell, which results in a linear DNA molecule which includes a portion of the oriT DNA sequence adjacent to heterologous DNA followed by the balance of the oriT DNA sequence. It is this sequence which, we believe, is integrated into the host plant genome following transformation. Other Embodiments Other embodiments are within the following claims. For example, oriT and mob sequences may be obtained from other self- obilizable plasmids, for example, the plasmids RK2 and pPHl. The sequence of the RK2 oriT region is given in Willetts and Wilkins, id., and the RK2 mob genes are located in one of the tra clusters (Guiney, id) . Other promoter sequences can be used, e.g., those described in Buchanan-Wollaston et al. , id.

Claims: - 20 - Claims 1. A plant containing an engineered DNA construct comprising an oriT DNA sequence and heterologous DNA. 2. The plant of claim 1 wherein said engineered DNA construct comprises, in sequential order, a first portion of said oriT DNA sequence, said heterologous DNA, and the balance of said oriT DNA sequence. 3. The plant of claim 1 in which said construct further contains a mob DNA sequence between said first portion of said oriT DNA sequence and the balance of said oriT DNA sequence. 4. A plant cell transformed with a first plasmid comprising heterologous DNA and an oriT DNA sequence, and a mob DNA sequence compatible with said oriT sequence, said mob DNA sequence being carried either on said first plasmid or on a second plasmid, neither said first nor said second plasmid including any DNA which is derived from or identical to Ti border DNA of A. tumefaciens. 5. A plant made by the process of culturing the plant cell of claim 4 under conditions effecting the regeneration of said plant. 6. A plant cell produced by a method comprising bringing a plant cell, under transforming conditions, into contact with a) a first plasmid comprising 1) a heterologous gene, and 2) an oriT DNA sequence, and b) a mob DNA sequence compatible with said oriT sequence, said mob DNA sequence being carried either on said first plasmid or on a second plasmid. neither said first nor said second plasmid including any DNA which is derived from or identical to border DNA of A. tumefaciens. 7. A plant made by the process of culturing the plant cell of claim 6 under conditions effecting the regeneration of said plant. 8. A plant having a chromosome into which is integrated DNA of a plasmid which includes heterologous DNA and an oriT DNA sequence and does not contain any DNA which is derived from or is substantially identical to Ti border DNA of A. tumefaciens■ 9. The plant of any of claims 1, 2, 3, 5, 7, or 8 in which said plant is a monocotyledenous plant. 10. The plant of any of claims 1, 2, 3, 5, 7, or 8 in which said plant is a dicotyledenous plant. - 22 - 11. The plant of any of claims 1, 2, 3, 5, 7, or 8 in which said heterologous genes include a gene encoding a selectable marker. 13. The plant of claim 1 wherein said heterologous DNA comprises a gene selected from the group consisting of non—plant genes, modified genes, synthetic genes, and genes from a different plant strain or species. 14. A method of -transforming a plant cell with heterologous DNA, said method comprising bringing said plant cell, under transforming conditions, into contact with a) a first plasmid comprising 1) said heterologous DNA, and 2) an oriT DNA sequence, and b) a mob DNA sequence compatible with said oriT sequence, said mob DNA sequence being carried either on said first plasmid or on a second plasmid, neither said first nor said second plasmid including any DNA which is derived from or identical to Ti border DNA of A. tumefaciens. 15. The method of claim 14 wherein said three plasmid DNA components are all carried on said first plasmid. 16. The method of claim 14 wherein said plant cell is of a dicotyledenous plant. - 23 - 17. The method of claim 14 wherein said plasmid DNA, when brought into contact with said plant cell, is contained within a bacterial cell. 18. The method of claim 17 wherein said bacterial cell is capable of growing in the rhizoplane or rhizointerstices of the plant species from which said cell is derived. 19. The method of claim 17 wherein said bacterial cell is Gram-negative and said plant species is dicotyledenous. 20. The method of claim 14 wherein said oriT and mob DNA sequences are both derived from the same naturally- occurring Gram-negative bacterial plasmid. 21. The method of claim 20 wherein said naturally-occurring plasmid is a P-type, Q-type, or W-type plasmid. 22. The method of claim 21 where said naturally- occuring plasmid is RK2 or RSFIOIO. 23. The method of claim 14 wherein said oriT and mob DNA sequences are carried on different plasmids. 24. The method of claim 14 wherein said plasmid DNA further comprises a DNA sequence encoding a selectable marker protein, and said method further comprises selecting transformants on the basis of the presence of said selectable marker protein. - 24 - 25. A plasmid comprising 1) heterologous DNA, and 2) an oriT DNA sequence, said plasmid not containing any DNA which is derived from or substantially identical to Ti border DNA of A. tumefaciens, transcription of said heterologous DNA being under the control of a regulatory sequence normally expressed in plants. 26. The plasmid Of claim 25, said plasmid further comprising a mob DNA sequence compatible with said oriT sequence.

Inventors: BUCHANAN-WOLLASTON VICKY, CANNON FRANK S, BUCHANAN-WOLLASTON, VICKY, CANNON, FRANK, S.
Applicants: BIOTEKNIKA INTERNATIONAL, BIOTECHNICA INTERNATIONAL, INC.
Application year: 1987
Main classification: C12N15
IPC codes: A01H5, C12N5, C12N15