CA1340714C - Plant structural gene expression - Google Patents

Abstract

A DNA vector comprises T-DNA having a plant structural gene inserted therein under control of a T-DNA promoter. The DNA vector is useful in genetically modifying a plant cell to introduce the plant structural gene thereto.

Description

~'LANT STRUCTURAL GENE EXPRESSION
BACKGROUND
Shuttle Vectors Shuttle vectors, developed by Ruvkun and Ausubel (1981) Nature 289:85-88, provide a way to insert foreign genetic materials into positions of choice in a large plasmid, virus, or genome. There are two main problems encountered when dealing with large plasmids or - genomes. Firstly, the large plasmids may have many sites for each restriction enzyme. Unique, site-specific cleavage reactions are not- reproducible and multi-site cleavage reactions followed by ligation lead to great difficulties due to the scrambling of the many fragments whose order and orientation one does not want changed. Secondly, the transformation efficiency with large DNA plasmids; is very low. Shuttle vectors allow one to overcome these difficulties by facilitating the insertion, often _i.n vitro, of the foreign genetic material into a smaller plasmid, then transferring, usually by in_ vivo techniques, to the larger plasmid.
A shuttle vector consists of a DNA molecule, usually a plasmid, capable of being introduced into the ultimate recipient: bacteria. It also includes a copy of the fragment of the recipient genome into which the foreign genetic material is to be inserted and a DNA
segment coding for a selectable trait, which is also inserted into the recipient genome fragment. The selectable trait ("marker") is conveniently inserted by transposon mutagenesis or by restriction enzymes and ligases.
The shuttle vector can be introduced into the ultimate recipient: cell, typically a bacterium of the genus Acrrobacterium by a tri-parental mating (Ruvkun and Ausubel, supra), direct transfer of a self-mobilizable vector in a bi-parental mating, direct uptake of exogenous DNA by P~grobacterium cells ("transformation", la 1340'~1~
using the conditions of M. Holsters et al (1978) Molec.
Gen. Genet. 163:181--187), by spheroplast fusion of Ag~robacterium_ with another ~~ .~ 3 ~4 d '~ 1 ~
bacterial cell; by uptake of liposome-encapsulated DNA; or infection with a shuttle vector that is based on a virus that is capable of being packaged _in vitro. A tri-parental mating involves the mating of a strain containing a mobilizable plasmid, which carries genes for plasmid mobilization and conjugative transfer, with the strain containing the shuttle vector. If the shuttle vector is capable of being mobilized by the plasmid genes, the shuttle vector is transferred to the recipient cell containing the large genome, e.g.
the Ti or Ri plasmids o,F Agrobacterium strains.
After the shuttle vector 'is introduced into the recipient cell, possible events include a double cross over with one recombinational event on either side of the marker. This evens: will result in transfer of a DNA segment containing the marker to the recipient genome replacing a homologous segment lacking the insert. To select for cells that have lost the original shuttle vector, the shuttle vector must: be incapable of replicating in the ultimate host cell or be incompatible with an independently selectable plasmid pre-existing in the recipient cell. One common means of arranging this is to provide in the third parent another plasmid which is incompatible with the shuttle vector and which carries a different drug resistance marker.
Therefore, when one selects for' resistance to both drugs, the only surviving cells are those in which the marker on the shuttle vector has recombined with the recipient genome. If the shuttle vector carries an extra marker, one can then screen for and discard cells that are the result of a single cross-over between the shuttle vector and the recipient plasmid resulting in cointegrates in which the entire shuttle vector is integrated with the recipient plasmid.
If the foreign genetic material is inserted into or adjacent to the marker that is selected for, it will also be integrated into the recipient plasmid as a result of the same double recombination. It might also be carried along when inserted into the h~~mologous fragment at a spot not within or adjacent to the marker, but the greater the distance separating the foreign genetic material from the marker, the more likely will be a recombinational event occurring between the foreign genetic material and marker, preventing transfer of the foreign ~lenetic material.
Shuttle vectors have proved useful in manipulation of Agrobacterium plasmids: see D. J. GariFinkel ~e_t _al. (1981) Cell 27:143-153, A. J. M.
Matzke and M. D. Chilton (1981) J. Molec. Appl. Genet. _1:39-49, and J. Leemans _et _al.
(1981) J. Molec. Appl. GE~net. 1.;149-164, who referred to shuttle vectors by the term "intermediate vectors""

~.~~0'~14 Agrobacterium--Overview Included within the gram-negative bacterial family Rhizobiaceae in the genus Agrobacterium are the species A. turnefaciens and A. rhizogenes. These species are respectivel~~ the causal agents of crown gall disease and hairy root disease of plants. Crown gall is characterized by the growth of a gall of dedifferentiated tissue. Hairy root is a teratoma characterized by inappropriate induction of rooi:s in infected tissue. In both diseases, the inappropriately growing plant tisssue usually produces one or more amino acid derivatives, known as opines, not normally produced by the plant which are cataboTized by the infecting bacteria. Known opines have been classified into three families whose type members are octopine; nopaline, and agropine. The cells of inappropriately growing tissues can be grown in culture, and, under appropriate conditions, be regEm erated into whole plants that retain certain transformed phenotypes.
Virulent strains of Agrobacterium harbor large plasmids known as Ti (tumor-inducing) plasmids in A._ tumefaciens and Ri (root-inducing) plasmids in A. rhizogenes. Curing a strain of these plasmids results in a loss of pathogenicity. The Ti plasmid contains a region, referred to as T-DNA
(transferred-DNA), which in tumors is found to be integrated into the genome of the host plant. The T-DNA encodes several transcripts. Mutational studies have shown that some of these are involved in induction of tumorous growth.
Mutants in the genes for tml, tmr, and tms, respectively, result in large tumors (in tobacco), a propensity to generate roots, and a tendency for shoot induction. The T-DNA also encodes the gene for at least one opine synthetase, and the Ti plasmids are ~~ften classified by the opine which they caused to be synthesized. Each of the T-DNA genes is under control of a T-DP~A promoter.
The T-DNA promoters resemble eukaryotic promoters in structure, end they appear to function only in the transformed plant cell. The Ti ~lasmid also carries genes outside the T-DNA region. These genes are involved in functions which include opine catabolism, oncogenicity, agrocin sensitivity, replication, and autotransfer to bacterial cells. The Ri plasmid is organized in a fashion analogous to the Ti plasmid. The set of genes and DNA sequences responsible for transforming the plant cell are hereinafter collectively referred to as the transformation-inducing principle (TIP). The designation TIP therefore includes both Ti <ind Ri plasmids. The integrated segment of a TIP is termed herein "T-ETNA", whether derived from a Ti plasmid or an Ri plasmid. Recent general reviews of AQrobacterium-caused disease include those by D.J. Merlo (1982), Adv. Plant Pathol. _1:139-178 L.W. Ream and M.P. Gordon (1982), Science 218:854-85~9, and M.W. Bevan and M.D. Chilton (1982), Ann. Rev. Genet. 16:357-384: G. Kahl and J.
Schell (1982) Molecular Biology of Plant Tumors.
Agrobacterium--Infection of Plant Tissues Plant cells c:an be transformed by Agrobacterium in a number of methods known in the art which include but are not limited to co-cultivation of plant cells in culture with A_grobacterium, direct infection of a plant, fusion of plant protoplasts with AQrobacterium spheroplasts, direct transformation by uptake of free DNA by plant cell protoplasts, transformation of protoplasts having partly regenerated cell walls with intact bacteria, transformation of protoplasts by liposomes containing T-DNA, use of a virus to carry in the T-DNA, microinjection, and the like. Any method will suffice as long as the gene is reliably expressed, and is stably transmitted through mitosis and meiosis.
The infection of plant tissue by Aarobacterium is a simple technique well known to those skilled in the art (for an example, see D. N. Butcher et al. (1980) in Tissue Culture Methods for Plant Pathologists, eds.:
D.S. Ingrams and J'.P. Helgeson, pp. 203-208). Typi-cally a plant is wounded by any of a number of ways, which include cutting with a razor, puncturing with a needle, or rubbing with abrasive. The wound is then inoculated with a solution containing tumor-inducing bacteria. An alternative to the infection of intact plants is the inoculation of pieces of tissues such as potato tuber disks. (D. K. Anand and G.T. Heberlein (1977) Amer. J. Bot. 64:153-158) or segments of tobacco stems (K. A. Barton et al (1983) Cell 32:1033-1043).
After induction, t:he tumors can be placed in tissue culture on media lacking phytohormones. Hormone A340'~1~
independent growth is typical of transformed plant tissue and i;s in great contrast to the usual conditions of growth of such tissue in culture (A. C. Braun (1956) Cancer Res. 16:53-56).

5 Aarobacterium is also capable of infecting isolated cells and ce:Lls grown in culture, Marton et al (1979) Nature 277:1:29-131, and isolated tobacco mesophyll protoplasts. In the latter technique, after allowing time for paritial regeneration of new cell walls, Aqrobacterium cells were added to the culture for a time and then kil:Led by the addition of antibiotics. Only those cells taxpose~d to A. tumefaciens cells harboring the Ti plasm:id were capable of forming calli when plated on media laclcing hormone. Most calli were found to contain an enzymatic: activity involved in opine anabolism. Other 'workers (R. B. Horsch and R.T. Fraley (18 January :L983) 15th Miami Winter Symposium) have reported transformations by co-cultivation, leading to a high rate (goeater than 10%) of calli displaying hormone-independent growth, with 95% of those calli making opine:a. M.:R» Davey et al (1980) in Ingram and Helgeson, su,~~a_. p;p» 209-219, describe the infection of older cells i~hat h.ad been regenerated from protoplasts.
Plant protoplasts can be transformed by the direct uptake of TI1? plas:mids. M.R. Davey et al (1980) Plant Sci. Lett. lt3_:307-313, and M.R. Davey et al (1980) in Ingram and Helgeso:np supra, were able to transform Petunia protoplasts with the Ti plasmid in the presence of poly-L-~~ha-or:nithine to a phenotype of opine synthesis and hormone-independent growth in culture. It was later shown (J. Draper et al (1982) Plant and Cell Physiol. 23:451-458, M.R. Davey et al (1982) in Plant Tissue Cultu~__°e 1982, ed: A. Fujiwara, pp. 515-516) that polyethelene glycol stimulated Ti uptake and that some T-DNA sequences were integrated into the genome. F.A.
Krens et al (1982) Nature 296:72-74, reported similar 13~0~1~
5a results using polyethelene glycol following by a calcium shock, though their data suggests that the integrated T-DNA included flanking Ti plasmid sequences.
An alternative method to obtain DNA uptake involves the use of liposomes. The preparation of DNA containing liposomes is taught by Papahadjopoulos in U.S. Patents 4,078,052 and 4,235,871. Preparations for the introduc-tion of Ti-D:NA via,:liposomes have been reported (T.
Nagata et al (1982) in Fujiwara, s, u~ra, pp. 509-510, and T. Nagat~a (1981) Mol. Gen. Genet. 184:161-165). An analogous system involves the fusion of plant and bacterial cells after removal of their cell walls. An example of tihis technique is the transformation of Vinca protoplast by Aarobacterium spheroplasts reported by S.
Hasezawa et ;al (1981) Mol. Gen. Genet. 182:206-210.
Plant protoplasts can take up cell wall delimited Agrobacteriwm_ cells (S. Hasezawa et al (1982) in Fujiwara, su~~ra pp. 517-518).
T-DNA cyan be transmitted to tissue regenerated from a fusion of 'two protoplasts, only one of which had been transformed (G. J. Wullems et al (1980) Theor. Appl.
Genet. 56:20.3-208). As detailed in the section on Regeneration of Plants, T-DNA can pass through meiosis and be transmitted to progeny as a simple Mendelian trait.

~~~~'~lt~
Agrobacterium--Re4eneration of Plants Differentiated plant tissues with normal morphology have been obtained from crown gall tumors. A. C. Braun and H. N. Wood (1976) Proc. Natl. Acad.
Sci. USA 73:496-500, grafted tobacco teratomas onto normal plants and were able to obtain normally appearing shoots which could flower. The shoots retained the ability to make opines and to grow independently of phytohormones when placed in culture. In the plants screened, these tumor phenotypes were not observed to be transmitted to progeny; apparently being lost during meiosis (R. Turgeon et ~1. (1976) Proc. Natl. Acad. Sci. USA 73:3562-3564).
Plants which had spontaneouly lost tumorous properties, or which were derived from teratoma seed, were initially shown to have lost all their T-DNA (F.-M.
Yang et al. (1980) In Vitro 16:87-92, F. Yang et al. (1980) Molec. Gen. Genet.
177:707-714, M) Lemmers et al. (1980) J. Mol. Biol. 144:353-376). However, later work with plants that had become revere ants after hormone treatment (lmg/1 kinetin) showed that plants which had gone through meiosis, though losing T-DNA genes responsible for the transformed phenotype, could retain sequences homologous to both ends of T-DNA (F. Yang and R. B. Simpson (1981) Proc. Natl. Acad. Sci. UiSA 78:4151-4155). G. J. Wullems et _al. (1981) Cell 24:719-724, further demonstrated that genes involved in opine anabolism were capable of passing throuigh meiosis though the plants were male sterile and that seemingly unaltered T-DNA could be inherited in a Mendelian fashion (G.
Wullems et al. (1982) in A) Fujiwara, su ra . L. Otten et al. (1981) Molec.
Gen. Genet. 183:209-213, used Tn7 transposon-generated Ti plasmid mutants in the tms (shoot-inducing) locus to create tumors which proliferated shoots.
When these shoots were regenerated into plants, they were found to form self-fertile flowers. The resultant seeds germinated into plants which contained T-DNA and made opines. Similar experiments with a tmr (root-inducing) mutant showed that full-length 'T-DNA could be transmitted through meiosis to progeny, that in those progeny no,paline genes could be expressed, though at variable levels, and that cotrans~Formed ,yeast alcohol dehydrogenase I gene was not expressed (K. A. Barton ~e_t _al. (1983) (Cell _32:1033-1043). It now appears that regenerated tissues which lack T-DNA sequences are probably descended from untransformed cells which "contaminate" the tumor (G. Ooms _et _al.
(1982) Cell 30:589-597).
Roots resulting from transformation from _A, rhizogenes have proven relatively easy to regenerate into plantlets (M.-D. Chilton et al. (1982) Nature 295:432-434.
._ -6-1340'14 Ag~robacterimm--Genes on the TIP Plasmids:
A number of genes have been identified within the T-DNA of the: TIP plasmids. About half a dozen octopine plasmid T-DNfA transcripts have been mapped (S. B. Gelvin et al (1982) Proc., Natl. Acad. Sci. USA 79:76-80, L.
Willmitzer ea al 1;1982) EMBO J. 1:139-146) and some functions have been assigned (J. Leemans et al (1982) EMBO J. 1:147-152). The four genes of an octopine type plasmid that. have been well defined by transposon mutagenesis include tms, tmr, and tml (D. J. Garfinkel et al (1981) Cell 27:143-153). Ti-plasmids which carry mutations in theses genes respectively incite tumorous calli of Nicotiana tabacum which generate shoots, proliferate roots,. and are larger than normal. In other hosts, mutants of these genes can induce different phenotypes (see Bs:van and Chilton, s_upra). The phenotypes c~f tms and tmr are correlated with differences in ths: phytohormone levels present in the tumor. The differences in cytokinin:auxin ratios are similar to those which in culture induce shoot or root formation in. untransformed callus tissue (D. E. Akiyoshi et al (1983) Proc.. Natl. Acad. Sci. USA 80:407-411).
T-DNA containing a functional gene for either tms or tmr alone, but n.ot functional tml alone, can promote signif-icant tumor growth. Promotion of shoots and roots is respectively stimulated and inhibited by functional tml (L.W. Ream e.t al (1983) Proc. Natl. Acad. Sci. USA
80:1660-1664). Mutations in T-DNA genes do not seem to affect the insertion of T-DNA into the plant genome (J.
Leemans et al (1982) supra, L.W. Ream et al (1983) supra). The ocs gene encodes octopine synthetase, which has been sequenced by H. De Greve et al (1982) J. Mol.
Appl. Genet. _1:495-511. It does not contain introns (intervening' sequs:nces commonly found in eukaryotic genes which are posttranscriptionally spliced out of the messenger precursor during maturation of the mRNA). It 134071~~

does have sequences that resemble a eukaryotic tran-scriptional signal ("TATA box") and a polyadenylation site. As plant cells containing the enzyme octopine synthetase detoxify homo-arginine, the ocs gene may prove to be .a useful selectable marker for plant cells that have been transformed by foreign DNA (G.M.S. Van Slogteren et al (1982) Plant Mol. Biol. 1:133-142).
Nopalin~e Ti plasmids encode the nopaline synthetase gene (nos), 'which has been sequenced by A. Depicker et al (1982) J. Mol. Appl. Genet. _1:561-573. As was found with the ocs gene, nos is not interrupted by introns.
It has two putative polyadenylation sites and a potential "T:~TA box"'. In contrast to ocs, nos is pre-ceded by a sequence which may be a transcriptional signal known as a "CAT box". J.C. McPhersson et al (1980) Proc. Natl. Acad. Sci. USA 77:2666-2670, reported the in vitro translation of T-DNA encoded mRNAs from crown gall tissues.
Transcription .from hairy root T-DNA has also been detected (L. Willmitzer et al (1982) Mol. Gen. Genet.
186:16-22). Functionally, the hairy root syndrome appears to bra equivalent of a crown gall tumor incited by a Ti plasmid mutated in tmr (F. F. White and E.W.
Nester (198 0,) J. Bacteriol. 144:710-720.
In euka:ryotes, methylation (especially of cytosine residues) of DNA is correlated with transcriptional inactivation: genes that are relatively undermethylated are transcribed into mRNA. Gelvin et al (1983) Nucleic Acids Res. 1:159-174 have found that the T-DNA in crown gall tumors :is always present in at least one unmethyl-ated copy. '.that the same genome may contain numerous other copies of T-DNA which are methylated suggests that the copies o:E T-DNA in excess of one may be biologically inert. (See also G. Ooms et al (1982) Cell 30:589-597.) The Ti plasmid encodes other genes which are outside of tile T-DNA region and are necessary for the infection process. (See M. Holsters et al (1980) 8a Plasmid 3_:212-230 for nopaline plasmids, and H. De Greve et al (1981) Plasmid 6:235-248, D.J. Garfinkel and E.W. Nester (1980) J. Bacteriol. 144:732-743, and G.
Ooms (1980) .J. Bacteriol. 144:82-91 for octopine plas-mids. Most important are the one genes, which when mutated result in T:i plasmids incapable of oncogenicity.
(These loci .are also known as vir for virulence). The one genes function :in trans, being capable of causing the transformation of plant cells with T-DNA of a different plasmid type and physically located on another plasmid (J. l3ille et al (1982) Plasmid 7:107-118, H.J.
Klee et al (:1982) J. Bacteriol. 150:327-331, M.-D.
Chilton (18 ;January 1983) 15th Miami Winter Symp.
Nopaline Ti DNA has direct repeats of about 25 base pairs immediately adjacent to the left and right borders of the T-DNA which might be involved in either excision from the Ti plasmid or integration into the host genome (N.S. Yadav et al (1982) Proc. Natl. Acad. Sci. USA
79:6322-6326;x, and a homologous sequence has been observed adjacent to an octopine T-DNA border (R. B.
Simpson et a:l (1982) Cell 29:1005-1014). Opine catabolism i:a specified by the ocs and nos genes, respectively of octopine- and nopaline-type plasmids.
The Ti plasm:id also encodes functions ~. 3 ~ Q '~ 14 necessary for its own reproduction including an origin of replication. Ti plasmid transcripts have been detected in A. tumefaciens cells by S. B. Gelvin et al. (1981) Plasmid 6,:17-29, who found that T-DNA regions were weakly transcribed along with non-T-DNA sequences. Ti plasmid-determined characteristics have been reviewed by Merlo, supra (see especially Table II), and Ream and Gordon supra.
Agrobacterium-TIP Plasmid DNA
Different octopine-type Ti plasmids are nearly 100% homologous to each other when examined by DNA hybridization (T. C. Currier and E) W. Nester (1976) J. Bacteriol. l2Ei:157-165) or restriction enzyme analysis (D. Sciaky et al. (1978) Plasmid 1:238-253). Nopaline-type Ti plasmids have as little as 67% homology to each other (furrier and Nester, su ra . A survey revealed that different Ri plasmiids are very homologous to each other (P. Costantino _et al. (1981) Plasmid 5:17(1-182). N. H. Drummond and M.-D. Chilton (1978) J.
Bacteriol. 136:1178-118~~, showed that proportionally small sections of octopine and nopaline type Ti plasmids were homologous to each other. These homologies were mapped in detail by G. Engler _et _al. (1981) J. Mol. Biol.
152:183-208. They found. that three of the four homologous regions were subdivided into three (cw erlapping the T-DNA), four (containing some one genes), and nine (having one genes) homologous sequences. The uninterrupted homology contains at least one tra gene (for conjugal transfer of the Ti plasmid to other bacterial cells;), and genes involved in replication and incompatibility. This uninterrupted region has homology with a Stern plasmid (involved in symbiotic nitrogen fixation) from a species of Rhizobium, a different genus in the family Rhizobiaceae (R. K. Prakash _et _al. (1982) Plasmid 7:271-280). The order of the four regions is not conserved, though they are all oriented in the same direction. Part of the T-DNA sequence is very highly conserved between nopaline and octopine plasmids (M.-D. Chilton _et al. (1978) Nature 275:14'7-149, A. Depicker et _al. (1978) Nature 275:150-153). Ri plasmids have Keen shown to have extensive homology among themselves, and to both octopine (F. F. White and E. W. Nester (1980) J.
Bacteriol. 144:710-720) rind nopaline (G. Risulea- _et _al. (1982) Plasmid _7:45-51) Ti plasmids, primarily in regions encoding one genes. Ri T-DNA contains extensive though weak homologies to T-DNA from both types of Ti plasmid (L.
Willmitzer et al. (1982) Mol. Gen. Genet. 186:3193-3197). Plant DNA from uninfected Nicotiana glauca contains sequences, referred to as cT-DNA
-g_ ., (cellular T=DNA); that show homology to a portion of the Ri T-DNA (F. F. White et al. (1983) Nature 301.:348-350).
It has been shown that a portion of the Ti (M.-D. Chilton et _al. (1977) Cell 11:263-271) or Ri -(M.-D. Chilton (1982) Nature 295:432-434, F. F. White et al. (1982) Proc. Natl. Acad) S ci. USA 79:3193-3197, L. Willmitzer (1982) Mol. Gen. Genet. 186:16-22) plasmid is found in the DNA of tumorous plant cells. The transferred DNA is known as T-DNA. T-DNA is integrated into the host DNA (M. F. Thomashow et al. (1980) Proc. Natl. Acad. Sci. USA _77:6448-6452, N. S. Yadav et al. (1980) Nature 287:458-461) in the nucleus (M. P) Nuti et al. (1980) Plant Sci. Lett..18:1-6, L. Willmitzer _et _al. (1980) Nature 287:359-361, M.-D. Chilton et a_1. (1980) Proc. Natl. Acad) Sci. USA 77:4060-4064).
M. F. Thomashow et al. (19.80) Proc. Natl. Acad. Sci. USA _77:6448-6452, and M. F. Thomashow et al. (1980) Cell _19:729-739, found the T-DNA from octopine-type Ti plasmid~~ to have been integrated in two separate sections, TL-DNA and TR-DNA, left ~~nd right T-DNAs respectively. The copy numbers of TR
and TL can vary (D. J. Merlo et al. (1980) Molec. Gen. Genet. 177:637-643). A
core of T-DNA is highly homologo us to nopaline T-DNA (Chilton et _al. (1978) supra and Depicker et al" (1978;).su ra , is required for tumor maintenance, is found in TL, is generally present in one copy per cell, and codes for the genes tms, tmr, and tml. On the other hand TR can be totally dispensed with (M. De Beuckeleer et al. (1981) Molec. Gen. Genet. 183:283-288, G. Ooms _et _al.
(1982) Cell 30:589-597), though found in a high copy number (D. J. Merlo _et al. (1980) su ra . G. Oc~ns et dl. (1982) Plasmid _7:15-29, hypothesized that TR is involved in T-DNA integration, though they find that when TR is deleted from the Ti plasmid, A. _tumefaciens does retain some virulence. G. Ooms _et al. (1982) Cell 30:589-597, showed that though T-DNA is occasionally deleted after integration in the plant genome, it is generally stable and that tumors containing a mixture of cells that differ in T-DNA organization are the result of multiple transformation events. The ocs is found in TL but can be deleted from the plant genome without loss of phenotypes related to tumorous growth.
The left border of integrated TL has been observed to be composed of repeats of T-DNA sequences which .are in either direct or~inverted orientations (R. B.
Simpson et al. (1982) Cell 29:1005-1014).
In contrast to the situation in octopine-type tumors, nopaline T-DNA is integrated into the host ~Ienome in one continuous fragment (M. Lemmers et '~ -10-~34~'~1~

al. (1980) .J. Mol. Biol. 144:353-376, P. Zambryski et al (1980) Science 209:1385-1391). Direct tandem repeats were observed. T-DNA of plants regenerated from teratomas had minor modifications in the border frag-ments of the inserted DNA (Lemmers et al s. upra).
Sequence analysis of the junction between the right and left borders revealed a number of direct repeats and one inverted repeat. The latter spanned the junction (Zambryski et al (1980) supra). The left junction has been shown to vary by at least 70 base pairs while the right junction varies no more than a single nucleotide (P. Zambrysk:i et al (1982) J. Molec. Appl. Genet.
1:361-370). Left and right borders in junctions of tandem array; were separated by spacers which could be over 130 bp. The spacers were of unknown origin and contained some T-DNA sequences. T-DNA was found to be integrated into both repeated and low copy number host sequences.
N. S. Yadav et al (1982) Proc. Natl. Acad. Sci.
USA 79:6322-6326, have found a chi site, which in the bacteriophage lambda augments general recombination in the surrounding DN,A as far as 10 kilobases away, in a nopaline Ti plasmi~d just outside the left end of the T-DNA. R.B. Simpson et al (1982) Cell 29:1005-1014, have not obs<srved a chi sequence in an octopine Ti plasmid, though the possible range of action does not eliminate thE: possibility of one being necessary and present but outside of the region sequenced. The significance of the chi in the Ti plasmid is not known.
If the chi has a function, it is probably used in Actrobacteriurn_ cells and not in the plants, as chi is not found within the T-DNA.
Acrrobacterium-Maniyulations of the TIP Plasmids As detailed i:n the section on Shuttle Vectors, technology has been developed for the introduction of altered DNA sequences into desired locations on a TIP

plasmid. 'Transposons can be easily inserted using this technology (D. J. Garfinkel et al (1981) Cell 27:143-153). J.-P. Hernalsteen et al (1980) Nature 287:654-656, have shown that a DNA sequence (here a bacterial transposon) inserted into T-DNA in the Ti plasmid is transferred and integrated into the recipient ;plant's genome. Though insertion of foreign DNA has been done with a number of genes from different sources, tic date the genes have not been expressed under control of their own promoters. Sources of these genes include alcohol dehydrogenase (Adh) from yeast (K. A. Barton et al (1983) Cell, 32:1033-1043), AdhI
and zein from corn, interferon and globin from mammals, and the mammalian virus SV40. M. Holsters et al (1982) Mol. Gen. Genet. 185:283-289, have shown that a bacterial transposon (Tn7) inserted into T-DNA could be recovered :in a fully functional and seemingly unchanged form after integration into a plant genome.
Deletions can be generated in a TIP plasmid by several methods. Shuttle vectors can be used to introduce deletions constructed by standard recombinant DNA techniques (Cohen and Boyer U.S. Pat. 4,237,224).
Deletions with one predetermined end can be created by the improper excision of transposons (B.P. Koekman et al (1979) 1?lasmi~d 2:347-357, G. Ooms et al (1982) Plasmid 7::L5-29). J. Hille and R. Schilperoot (1981) Plasmid 6::L51-154, have demonstrated that deletions having both ends at predetermined positions can be generated by use of two transposons. The technique can also be usE:d to construct "recombinant DNA" molecules in vivo.
The nopaline synthetase gene has been used for insertion of DNA segments coding for drug resistance that can beg used to select for transformed plant cells.
M. Bevan (neport~ed by M.-D. Chilton et al (18 January 1340'14 12a 1983) 15th Miami Winter Symp., see also J.L. Marx (1983) Science 219:830) and R. Horsch et al (18 January 1983) 15th Miami Winter Symp., see Marx, supra, have inserted the kanamycin resistance gene (neomycin phosphotransferase) from Tn5 behind (under control of) the nopaline promoter. The construction was used to transform plant cells which in culture displayed resistance t~o kanamycin and its analogs such as 6418.
J. Schell et al (18 January 1983) 15th Miami Winter l0 Symp. (see also Marx, supra), reported a similar construction, in which the methotrexate resistance gene (dihydrofolate reductase) from Tn7 was placed behind the nopaline synthetase promoter. Transformed cells were resistant to methotrexate. As plant cells containing octopine synthetase are resistant to the toxic chemical homo-arginine, G.M.S. Van Slogteren et al (1982) Plant Mol. Biol. 1_:133-142, have proposed using that enzyme as a selectable marker.
M.-D. C:hilton. et al (1983) supra, reported that A.
De Framond has constructed a "mini-Ti plasmid". In the nopaline T-D:NA there is normally only one site cut by the restriction enzyme KunI. A mutant lacking the site was constructed anal a KpnI fragment, containing the entire nopaline T-DNA, was isolated. This fragment together with a ka.namycin resistance gene was inserted into pRK290, thereby resulting in a plasmid which could be maintained in A. tumefaciens and lacked almost all non-T-DNA Ti sequences. By itself, this plasmid was not able to transform plant cells. However when placed in an A. tumefaciens strain containing an octopine Ti plasmid; tumors were induced which synthesized both octopine and nopaline. This indicated that the missing nopaline Ti plasmid functions were complemented by the octopine Ti plasmid, and that the nopaline "mini-Ti" was functional in the transformation of plant cells. Chilton et al. (1983) supra also reported on the construction of a "micro-Ti" plasmid made by resectioning the mini-Ti with SmaI to delete essentially all of T-DNA but the nopaline synthetase gene and the left and right borders. The micro-Ti was inserted into a modified pRK290 plasmid that was missing its SmaI site, and employed in a manner similar to mini-Ti, with comparable results.
H. Lorz et al. (19132) in _~Plant Tissue Culture 1982, ed: A. Fujwara, pp. 511-512, reported v~the construction of a plasmid vector, apparently independent of the TIP ~~ystem for DNA uptake and maintenance, that used the nopaline synthetase gene as a marker.
Phaseolin and ene re ul'ation In general the genE~s of higher eukaryotes are highly regulated. A
multicellular organism, such as a plant, has a number of differentiated tissues, each with its own specialized functions, each of which requires specialized gene products. One such tissue is the cotyledon. In legumes, the cotyledons usually serves as the storage tissue for the seed, holding reserves of lipid, carbohydrate, minerals, and protein until the seed needs them during germination. In Phaseolus vulgaris L. (also known as the French bean, kidney bean, navy bean, green bean and other names), the maj or storage protein is known as phaseolin. This protein comprises a small number of molecular species that are extremely homologous and equivalent to one another.
Phaseolin contributes most of the nutrition value of dried beans, often comprising more than 10% of the weight of a dried bean.
Phaseolin is highly regulated during the life cycle of _P. vulgaris. The protein is made essentially only while seed is developing within the pod.
Levels rise from the limit of detection to as much as half the seed's protein content, following genetically .determined schedules for synthesis. At its peak, phaseolin synthesi~~ can a~~count for over 80% of a cotyledon cell's protein synthesis. At oi:her times and in other tissues, phaseolin synthesis is undetectable. The extreme nature of phaseolin's regulation, coupled with its worldwide nutritionall importance, has lead to much interest in the study of phaseolin, its properties, and its regulation.
__ -13-13~U~14 SUMMARY OF THE INVENTION
In accordance with one aspect of the present inention, there is provided a DNA vector comprising T-DNA having a plant structural gene inserted therein under control of a T-DNA promoter. The invention also includes a k>acterial strain containing and replicating the DNA
vector.
The novel C)NA vector of the present invention permits the provision of a plant comprising a genetically modified plant cell having a plant structural gene introduced and expressed therein under control of a T-DNA promoter. Further, the invention permits the provision of plant tissue comprising a plant cell whose genome includes T-DNA camprisin~g a plant structural gene inserted in such orientation and spacing with respect to a T-DNA promoter as to be expressible in the plant cell under control of the T-DNA promoter.
The experimental work disclosed herein is believed to be the first demonstration that plant structural genes are expressible in plant cells under control of a T-DNA promoter, after introduction via T-DNA, that is to say, by inserting the plant structural genes into T-DNA under control of a f-DNA promoter and introducing the T-DNA
containing the insert into a plant cell using known means. The disclosed experiments are also believed to provide the first demon-stration that plant structural genes containing introns are expressed in plant cells under control of a T-DNA promoter after introduction via T-DNA. These results are surprising in view of the fact that the genes previously ~~eported to be expressible in T-DNA under control of a T-DNA promoter, either endogenous T-DNA genes or inserted foreign genes, lacked introns. The results are unexpected also in view of the prior art fai:fure to demonstrate that a T-DNA promoter could function to control expression of a plant structural gene when the latter is introdu<:ed into T-DNA under the proper conditions. The invention is useful for genetically modifying plant tissues and whole plants by inserting useful plant structural genes from other plant species or strain~~. Such useful plant structural genes include, but are not limited to, genes coding for storage proteins, lectins, disease resistance factor's, herbicide resistance factors, insect resistance factors, environmental stress tolerance factors, specific flavor elements, an~d the like. The invention is exemplified by introduction and expression of a structural gene for phaseolin, t:he major seed storage protein of the bean 5 Phaseolus vul~aris, L., into sunflower and tobacco plant cells. Once plant cells expressing a plant structural gene under control of a T-DNA promoter are obtained, plant tissues and whole plants can be regenerated therefrom using methods and techniques well known in the 10 art. The re~~enerated plants are then reproduced by conventional means and the introduced genes can be transferred to other strains and cultivars by conven-tional plant breeding techniques. The introduction and expression of the structural gene for phaseolin, for 15 example, can be used to enhance the protein content and nutritional 'value of forage crops such as alfalfa.
Other uses of the invention, exploiting the properties of other structural genes introduced into other plant species will be readily apparent to those skilled in the art. The invention in principle applies to any introduction of a plant structural gene into any plant species into which 't-DNA can be introduced and in which T-DNA can remain stably replicated. In general these species include, but are not limited to, dicotyledenous plants, such as sunflower (family Compositeae), tobacco (family Sola:naceae), alfalfa, soybeans and other legumes (family Legu~minoseae) and most vegetables.
DET;I ED DESCRIPTION OF THE INVENTION
The following definitions are provided, in order to remove ambiguities to the intent or scope of their usage in the specification and claims.
T-DNA: A segment of DNA derived from the tumor-inducing principle (TIP) which becomes integrated in the plant genome. As used herein, the term includes DNA originally derived from any tumor-inducing strain of Aarobacterium including A.

tumefac:iens a=nd A. Rhizogenes, the inserted segment of the :Latter sometimes referred to in the prior art as R-DNA. In addition, as used herein the term T-DNA includes any alterations, modifications, mutations, insertions and deletions either natur-ally occ:urring or introduced by laboratory proce-dures, a principle structural requirement and limitat=ion to such modifications being that sufficiE:nt right and left ends of naturally-occurring T-DNAs be present to insure the expected function of stable integration in the transformed plant ce=ll genome which is characteristic of T-DNA.
In addit=ion, 'the T-DNA must contain at least one T-DNA promote=r in sufficiently complete form to control initiation of transcription and initiation of tran:elation of an inserted plant structural gene. ~~referably, an insertion site will be provided "downstream" in the direction of tran-scription and translation initiated by the promote:-, so :Lacated with respect to the promoter to enab7.e a plant structural gene inserted therein to be expressed under control of the promoter, either direct:ly or as a fusion protein.
Plant st=ructural gene: As used herein includes that pox-tion of a plant gene comprising a DNA
segment coding for a plant protein, polypeptide or portion thereof but lacking those functional element:c of a plant gene that regulate initiation of trans>cript:ian and inititation of translation, commonl~~ referred to as the promoter region. A
plant st=ructural gene may contain one or more introns or it may constitute an uninterrupted coding :>equence. A plant structural gene may be derived in whole or in part from plant genomic DNA, cDNA and chem=ically synthesized DNA. It is further contemplated that a plant structural gene could 13~0'~1~
include modifications in either the coding segments or the introns which could affect the chemical structure of the expression product, the rate of expression or the manner of expression control.
Such modifications could include, but are not limited to, mutations, insertions, deletions, and "silent" modifications that do not alter the chemical stru~,cture of the expression product but which affect intercellular localization, transport, excretion or stability of the expression product.
The structural gene may be a composite of segments derived from a plurality of sources, naturally occurring or synthetic, coding for a composite protein, the composite protein being in part a plant protein.
T-DNA t~:romote_r: Refers to any of the naturally occurring promoters commonly associated with integrated T-DNA. These include, but are not limited to, promoters of the octopine synthetase gene, nopaline synthetase gene, tms, tml and tmr genes, depending in part on the TIP source of the T-DNA. Expression under control of a T-DNA
promoter may take the form of direct expression in which the structural gene normally controlled by the promoter is removed and replaced by the in-serted plant structural gene, a start codon being provided either as a remnant of the T-DNA struc-tural gene or as part of the inserted plant structural gene, or by fusion protein expression in which part or all of the plant structural gene is inserted in correct reading frame phase within the existing T-DN;~1 structural gene. In the latter case, the expression product is referred to as a fusion protein.

i3 40'~ 14 Plant tissue: Includes differentiated and undif-ferentiated tissues of plants including roots, shoots, pollen, seeds, tumor tissue, such as crown galls, and various forms of aggregations of plant cells in culture, such as embryos and calluses.
Plant ce~~: As used herein includes plant cells in plants and plant cells and protoplasts in culture.
Production of a genetically modified plant expres-sing a plant structual gene introduced via T-DNA com-bines the spE:cific teachings of the present disclosure with a variei:y of 'techniques and expedients known in the art. In moss: instances, alternative expedients exist for each stage of 'the overall process. The choice of expedients dE~pends on variables such as the choice of the basic TI1?, the plant species to be modified and the desired regeneration strategy, all of which present alternative process steps which those of ordinary skill are able to :select and use to achieve a desired result.
The fundameni:al aspects of the invention are the nature and structure= of t:he plant structural gene and its means of insertion into 'r-DNA. The remaining steps to obtaining a genetically modified plant include trans-ferring the modified T-DNA to a plant cell wherein the modified T-DNA becomes stably integrated as part of the plant cell genome, techniques for in vitro culture and eventual regcaneration into whole plants, which may include step:a for selecting and detecting transformed plant cells and steps of transferring the introduced gene from the originally transformed strain into commercially acceptable cultivars.
A principal feature of the present invention is the construction of T-DNA having an inserted plant struc-tural gene wader control of a T-DNA promoter, as these terms have been defined, supra. The plant structural gene must be inserted in correct position and orienta-tion with respect to the T-DNA promoter. Position has 1340'~1~

two aspects. The first relates to on which side of the promoter the structural gene is inserted. It is known that the majority ~of promoters control initiation of transcription and 'translation in one direction only along the DNA. The region of DNA lying under promoter control is s<iid to lie "downstream" or alternatively "behind" the promoter. Therefore, to be controlled by the promoter,, the correct position of plant structural gene insertion must be "downstream" from the promoter.
(It is recognized that a few known promoters exert bi-directional ~~ontrol, in which case -either side of the promoter cou:Ld be considered to be "downstream" there-from). The ;second aspect of position refers to the distance, in base pairs, between known functional elements of ithe promoter, for example the transcription initiation sate, and the translational start site of the structural gene. Substantial variation appears to exist with regard to this distance, from promoter to promoter.
Therefore, tine structural requirements in this regard are best described :in functional terms. As a first approximation, reasonable operability can be obtained when the distance between the promoter and the inserted structural gene is similar to the distance between the promoter and the T-DNA gene it normally controls.
Orientation :refers to the directionality of the struc-tural gene. By convention, that portion of a structural gene which ultimately codes for the amino terminus of the plant protein is termed the 5' end of the structural gene, while that e.nd which codes for amino acids near the carboxyl end o~f the protein is termed the 3' end of the structural gene. Correct orientation of the plant structural gene is. with the 5' end thereof proximal to the T-DNA promoter. An additional requirement in the case of constructions leading to fusion protein expres-sion is that the insertion of the plant structural gene into the T-DNA structural gene sequence must be such 13~071~
that the coding sequences of the two genes are in the same reading frame phase, a structural requirement which is well understood in the art. An exception to this requirement, of relevance to the present invention, 5 exists in the case where an intron separates the T-DNA
gene from the first coding segment of the plant structural gene. In that case, the intron splice sites must be so positioned that the correct reading frame for the T-DNA gene and the plant structural gene are 10 restored in phase after the intron is removed by post-transcr:iptional processing. The source of T-DNA
may be any o:E the 'TIP plasmids. The plant structural gene is inserted by standard techniques well known to those skilled in t:he art. Differences in rates of 15 expression may be observed when a given plant structural gene is inserted under control of different T-DNA
promoters. Different properties, including such prop-erties as stability" inter-cellular localization, excretion, antigenicity and other functional properties 20 of the expre:used protein itself may be observed in the case of fusion proteins depending upon the insertion site, the length and properties of the segment of T-DNA
protein included within the fusion protein and mutual interactions between the components of the fusion protein that effect folded configuration thereof, all of which preseni~ numerous opportunities to manipulate and control the :Euncti~onal properties of the expression product, depending upon the desired end use. Expression of the phaseolin structural gene has been observed when that gene wa:~ inserted under control of the nopaline synthetase promoter from an octopine plasmid of A.
tumefaciens (see Example 1).
A convenient :means for inserting a plant structural gene into T-1~NA involves the use of a shuttle vector, as described supra, having a segment of T-DNA (that segment into which insertion is desired) incorporated into a 1~~0'~1~~

plasmid capable of replicating in ~. coli The T-DNA
segment contains a. restriction site, preferably one which is unique to the shuttle vector. The plant structural gene ca.n be inserted at the unique site in the T-DNA segment and the shuttle vector is transferred into cells of the appropriate Agrobacterium strain, preferably one whose T-DNA is homologous with the T-DNA
segment of the shuttle vector. The transformed Agrobacterium_ strain is grown under conditions which permit selection of a double-homologous recombination event which results in replacement of a pre-existing segment of the Ti plasmid with a segment of T-DNA of the shuttle vector.
Following the: strategy just described, the modified T-DNA can be transferred to plant cells by any technique known in the art. For example, this transfer is most conveniently accomplished either by direct infection of plants with the novel Ag~robacterium strain containing a plant structural grease incorporated within its T-DNA, or by co-cultivation of the Actrobacterium strain with plant cells. The former technique, direct infection, results in due course in the appearance of a tumor mass or crown gall at the site of infection. Crown gall cells can be subsequently grown in culture and, under appropriate circumstances known to those of ordinary skill in the art, regenerated into whole plants that contain the inserted T-DNA segment. Using the method of co-cultivation, a certain proportion of the plant cells are transformed, that is to say have T-DNA transferred therein and inserted in the plant cell genome. In either case, the transformed cells must be selected or screened to distinguish them from untransformed cells.
Selection is most readily accomplished by providing a selectable marker incorporated into the T-DNA in addi-tion to the plant structural gene. Examples include either dihydrofola:te reductase or neomycin phosphotrans-~3~~'~14 ferase expressed under control of a nopaline synthetase promoter. These markers are selected by growth in medium containing methotrexate or kanamycin, respec-tively or their analogs. In addition, the T-DNA
provides endogenous markers such as the gene or genes controlling hormone-independent growth of Ti-induced tumors in cu:Lture, the gene or genes controlling abnormal morphology of Ri-induced tumor roots, and genes that control resistance to toxic compounds such as amino acid analogs,, such resistance being provided by an opine synthetase. Screening methods well known to those skilled in the art include assays for opine production, specific hybridization to characteristic RNA or T-DNA
sequences, or immunological assays for specific proteins, inc:ludin~g ELISA (acronym for "enzyme linked immunosorbani~ _assay"), radioimmune assays and "western"
blots.
An alternative to the shuttle vector strategy involves the use of plasmids comprising T-DNA or modified T-D1JA, into which a plant structural gene is inserted, sa:Ld plasmids being capable of independent replication :in an ;Acrrobacterium strain. Recent evidence ind:LCates that the T-DNA of such plasmids can be transferrcad from an Aqrobacterium strain to a plant cell provided the ;Actrobacterium strain contains certain trans-acting genes whose function is to promote the transfer of '.C-DNA to a plant cell. Plasmids that contain T-DNA and are able to replicate independently in an Aqrobacter'um strain are herein termed "sub-TIP"
plasmids. A spectrum of variations is possible in which the sub-TIP ;plasmids differ in the amount of T-DNA
they contain.. One end of the spectrum retains all of the T-DNA from the TIP plasmid, and is sometimes termed a "mini-TIP" plasmid. At the other end of the spectrum, all but the minimum amount of DNA surrounding the T-DNA
border is de:Leted, the remaining portions being the 1340'14 minimum necessary to be transferrable and integratable in the host cell. Such plasmids are termed "micro-TIP".
Sub-TIP plasmids a:re advantageous in that they are small and relatively easy to manipulate directly. After the desired structural. gene has been inserted, they can easily be introduced directly into an Aarobacterium containing the traps-acting genes that promote T-DNA
transfer. Introduction into an Agrobacterium strain is conveniently accomplished either by transformation of the Agrobacterium strain or by conjugal transfer from a donor bacterial cell, the techniques for which are well known to those of ordinary skill.
Regeneration is accomplished by resort to known techniques. An object of the regeneration step is to obtain a whole plant that grows and reproduces normally but which retains integrated T-DNA. The techniques of regeneration vary somewhat according to principles known in the art, depending upon the origin of the T-DNA, the nature of any modifications thereto and the species of the transformed plant. Plant cells transformed by an Ri-type T-DN,A are readily regenerated, using techniques well known to those of ordinary skill, without undue experimentation. Plant cells transformed by Ti-type T-DNA can be regenerated, in some instances, by the proper manipulation of hormone levels in culture.
Preferably, :however, the Ti-transformed tissue is most easily regenerated if the T-DNA has been mutated in one or both of t:he tmr; and tms genes. Inactivation of these genes return's the hormone balance in the transformed tissue towards normal and greatly expands the ease and manipulation of the tissue's hormone levels in culture, leading to a plant with a more normal hormone physiology that is readily regenerated. In some instances, tumor cells are able to regenerate shoots which carry integra-ted T- DNA a;nd express T-DNA genes, such as nopaline synthetase, ,and which also express an inserted plant 13~0~14 structural gene. The shoots can be maintained vege-tatively by grafting to rooted plants and can develop fertile flowers. The shoots thus serve as parental plant materiel for normal progeny plants carrying T-DNA
and expressing the plant structural gene inserted therein.
Examples The following Examples utilize many techniques well known and accessible to those skilled in the arts of molecular biology and manipulation of TIPs and Ag~robacterium; such methods are not always described in detail. Enz,Ymes are obtained from commercial sources and are used according to the vendor's recommendations or other variations known to the art. Reagents, buffers and culture ~~onditions are also known to those in the art. Reference works containing such standard techniques include the following: R. Wu, ed. (1979) Meth. Enzymol. 68: J.H. Miller (1972) Experiments in Molecular Genetics; R. Davis et al (1980) Advanced Bacterial Ge:netics,; and R.F. Schleif and P.C. Wnesink (1982) Practical Methods in Molecular Biologv.
In the :Examples, special symbols are used to clarify sequences. Sequences that do or could code for proteins are underlined, and codons are separated with slashes (/). The positions of cuts or gaps in each strand caused by restriction endonucleases or otherwise are indicated by the placement of asterisks (*). (In Example 4 a double-stranded DNA molecule is represented by a single line flanked by asterisks at the sites of restriction enzyme. cuts; the approximate position of a gene is there indicated by underlined "X"'s under the single line). With the exception of the plasmid IIc, plasmids and only plasmids are prefaced with a "p", e.g.
p3.8 or pKS4. Cells containing plasmids are indicated by identifying the: cell and parenthetically indicating 13~0'~~.4 _ the plasmid, e.g., A. tumefaciens(pTi15955) or K802 (pKS4-:KB) .
In the Examples, reference is made to the 5 accompanying drawings:
Figure 1 depicts the T-DNA region of pTi15955;
Figure 2 contains the nucleotide and derived amino acid sequences of the octopine synthase gene:
Figure 3 contains the nucleotide sequence for a 10 phaseolin gene and the nucleotide and derived amino acid sequences of a cDNA:
Figure 4 contains the nucleotide and derived amino acid sequences of nopaline synthase;
Figura_ 5 shows the restriction sites for plasmid 15 pKS-nop IV;
Figure 6 shows the steps of formation pKS Nop IV
KB 3.8 from pTic58;
Figur~ss 7 and 8 show the restriction sites for plasmids plKS4-KB and pNNNl;
20 Figure 9 shows formation of plasmid pNNN2;
Figure 10 contains the nucleotide sequence for the DNA from the HindIII site of plasmid p401 past the ClaI
site to it;~ right,;
Figure 11 shows the mapping of a 1450bp mRNA;
25 Figure 12 shows the formation of plasmid pKS-ProI;
Figure. 13 shows the restriction sites of p7.2;
Figure 14 shows the restriction sites of pKS-PRI
I-KB:
Figure 15 shows the structure of the phaseolin storage protein gene;
Figures 16, .L7, 18 and 19 show the restriction sites for ~?lasmids p 3.8, pBR 322, pKS-4 and pKS-KB 3.8 respectively;
Figure 20 shows the formation of plasmid pKS4-KB
2,4:
Figure 21 shows the restriction map for plasmid pKS 4 -KB 2 . ~~

13 4 0'~ 1'~

Figure 22 shows the cloning of phaseolin cDNA into phaseolin genomic environment:
Figure 23 chows the formation of plasmid pl-B:
Figure 24 shows the restriction map for plasmid pKS-proI A:
Figures 25, 26 and 27 show the formation of plasmids pKS-5 .arid pKS-oct.Cam203:
Figu~.~es 28, 29 and 30 show the restriction maps 10 for plasmids pK;S-oct.del. II, pKS-oct.del. I and pRK290 respectivEaly Figures 31, 32, 33, 34, 35, 36 and 37 show the formation of pl~asmids p2f, pie, pKS-oct.del. III, pKS-6, p2, pKf>-oct.del. IIIa and p203 with inserted BalII
site, respectimely:
Figure 38 chows the restriction sites for plasmid pKS-oct . trnr .
Figure 39 captains a comparison of the restriction sites for constructions described in Examples 11, 12 and 14;
Figure 40 :is a Table containing the genetic code;
Figure 41 .is a nucleotide sequence of a "large tumor" gene;
Figures 42 and 43 are the restriction maps for plasmids containing a Bam 17 T-DNA fragment and pKS-B17-KB3.0 respectively.
Table: I provides an index useful for identifying the plasmids their formation and their interrelat:ionsh:ip with respect to the various Examples.
30 Of the drawings not specifically identified in Table I, Figure 3 illustrates the structural gene for the bean :need storage protein phaseolin, Figure 4 illustratsa the structural gene for nopaline synthetase. Figure l0 illustrates the structural gene 35 for the portion of the construct of Figure 1 from the HindIII site of p401 past the ClaI site to its right, and Figure: 37 i:ll.ustrates conversion of the HpaI site in p203 to a Bg:III site.
Tabls: 2 provides an index of deposited strains.

13~U714 Fig. 39 provides a useful comparison of the constructions described in Examples 11, 12, and 14.
Fig. 40 se=is forth the genetic code and is useful forinterpreting sequences. The nucleotide sequence of an important T-1DNA gene, tml, though not used in these Examples, is set forth in Fig. 41; it is useful in designing constructions not described herein.
Example 1 A fusion protein gene was constructed consisting of the ocl:opine synthetase promoter, the amino terminal 90 amino acids of the structural gene for octopine synthetasE: a 3 amino acid overlap between the two genes, and all of phaseolin except for codons encoding its first 11 amino acids. Prior to the start of construct~Lon, a clone of pTi15955 T-DNA, p233, (the sequences defined by p203 and p303, in pBR322, see Fig.
1) was sec;uenced from the BamHI site to the PvuII site.
This includes a:Ll of the octopine synthetase gene (Fig.
2). The octopine synthetase sequence and reading frame ...
were found to be as follows near a site cut by the restriction enzyme EcoRI:
EcoRI
5'...AT(J/GGC CAG/CAA/GG*A,/ATT/CTT...3' 3'...TAC CCG GTC GTT CC T TAA*GAA...5' ...Mei~ Gly Gln Gln Gly Ile Leu...
84 85 8fi 87 88 89 90 Cleavage with EcoRI yields a fragment with the following end:
...ATG GGC/CA~~CAA GG 3' ...TAC CCG GTC GTT CCTT 5' The structural gene for the bean seed storage protein phaseolin (previously sequenced, Fig. 3) contains an EcoRI site near its 5' (amino terminal) end as follows:
ECORI
...CTG '.CTG CT~~GG*A/ATT/CTT/TTC...
...GAC AAC GAC CC T TAA*GAA AAG...
. . . Leu l~eu Leu Gly Ile Leu Phe. . .
9 :LO 11 12 13 14 15 Cleavage with EcoRI yields a fragment with an end as follows:
5' ~3TT CT'r TTC . . .
3' GAA AAG...
These two fragments, after ligation, form the following structure:
EcoRI
...ATG/GGCJCAGJCAA./GG*~AT T/CTT/TTC...
...TAC CCG GTC GTT CC T TA*A GAA AAG...
...Met Gly G~Ln Gln Gly Ile Leu Phe...
84 85 8(i 87 88 89 90 octopine synthetase 12 13 14 15 phaseolin Not only are the same reading frames preserved, but there are no intervening stop signals generated.
So in short, 'the EcoRI/BamHI restriction endo-nuclease fragment ~of the Phaseolin gene was ligated at the EcoRI sii:e to 'the octopine synthase gene of the T-DNA of pTi:L5955. This fusion gene contains the ocs promoter, thEa first 90 amino acids of octopine synthe-tase, the phaseoli:n gene minus its promoter and its first 11 amino acids, and a three amino acid junction identical to sequences present in both parent proteins.

1340'14 _1~ Removal of the EcoRI site from pBR322 The EcoRI site in pBR322 was removed by digesting with EcoRI, filling in with T-4 DNA polymerase, blunt end ligation and transformation into ~. coli strain HB101. Selecaion of transformants was made with ampicillin and colonies were screened by isolating small amounts of pl.asmid DNA (D. Ish-Horowicz (1982) in Molecular Clon_ incr, c:.S.H.) and selecting a clone without an EcoRI site' callead pBR322-R.
~ Clonina of the BamHI T-DNA fractment into pBR322-R
p203 (Fig. 42;1 was isolated and digested with BamHI. The 9a.7kbp fragment of T-DNA was isolated by agarose gel electrophoresis and ligated into the BamHI
site of pBR3~:2-R. 'This plasmid was transformed into E.
co ' strain HB101 and selected for using ampicillin resistance and tetracycline sensitivity. A positive clone was selected and called pKS169.
1.3 Removal of the EcoRI sites and fragments from the octopine: synthetase gene pKS169 Gras isolated and digested with EcoRI. An 8.6kbp fragment wa:~ isolated by agarose gel electro-phoresis and purified. This fragment had the 2 small (0.36kbp and 0.2kbp) fragments in the ocs gene removed.
1-44 Isolation of the EcoRI fragment containing' the phaseolin gene. DNA fragment and the kanamycin resistance qen~
pKS4-KB (Fig. 7) was purified and digested with EcoRI. A 4.8kbp fragment was isolated using 3.Okbp EcoRI/BamHI F~haseo:lin gene fragment ligated at the BamHI
site to a l.EtSkbp DNA fragment containing the kanamycin resistance gene encoding neomycin phosphotransferase II
(NPTII).
1.5 Ligation of the phaseolin gene to the octopine synthase~
The pha:~eolin,iNPTII fragment was then ligated at the EcoRI sites to the EcoRI fragment described in Example 1.3. The :Ligated DNA was transformed into HB101 and colonies were :elected on ampicillin and kanamycin.

~3~~71~
A colony named pKS-B17-KB3.0 (Fig. 43) was selected that contained a plasmid that had the correct orientation (i.e., the phaseolin gene ligated to the ocs gene in the correct direction and reading frame). This 5 was ascertained by the restriction mapping of plasmids from a small number of colonies. DNA sequence of the appropriate region was determined to verify the construction.
1.6 Transfer of the T-DNA fragment containing the 10 NPTII, aseolin and ocs DNA into pRK290 pRK290, a broad host range plasmid, was digested with BalII a:nd ligated to a 9.lkbp BamHI fragment containing t:he T-DNA, the NPTII gene, and the phaseolin DNA from pKS-B17-KB3Ø This was accomplished by 15 partially digesting pKS-B17-KB3.0 with BamHI and isolating a 9.lkbp fragment from 6 other bands from an agarose gel .electrophoresis. After ligation and transformati~~n into E. coli strain K802, colonies were selected on :kanamycin and tetracycline. A colony was 20 selected that had the desired restriction pattern and was labeled ;pKS-OS-KB3Ø
1.7 Replacement of octopine synthetase on pTi15955 with the octouine ~nthetase phaseolin fusion protein gene 25 Using triparental mating of A. tumefaciens (streptomyci:n resistant), E_. coli(pKS-OSI-KB3.0), and E.
coli(pRK2013), we selected for colonies resistant to streptomycin, kanamycin, and tetracycline. One colony was mated with E_. coli (pPHlJ1). A colony was selected that is resistant to kanamycin and gentamycin. This was shown to be ;A. tum.efaciens with p15955-12A, a pTi15955 that has the phaseo:lin gene and kanamycin resistance gene engineered into the EcoRI site of the ocs gene by restriction enzyme mapping, and filter hybridization of electrophore~tically separated restriction fragments (Example 19). An analogous triparental mating is done with A_. tumefaciens(pTiA66). Shoots transformed by the ~340'~14 _. 25; ~ _,..
resulting pl~~smid, pA66-12A, are shown to contain phaseolin as described above.
1-88 Crown crall foformation and expression Sunflower plants were inoculated with the engineered T:i plas~mid. Crown galls were established in tissue culture. Expression was tested by running ELISAs and by filter hybridization to electrophoretically separated mRIJA ("Northern blots", Example 19). RNA of the expected size 'was detected with hybridization probes to both the ~?haseolin and octopine synthetase genes, and comprised about 0.5~ of total poly(A)5+4 RNA. Poly (A)5+4 RNA isolated from galls directed the in vitro synthesis of a protein of the expected size which was precipitatab:le by antibodies raised against phaseolin.
Example 2 A fusion protein gene similar to that taught in Example 1 wars constructed from phaseolin and nopaline synthetase, under control of the latter gene's promoter.
It contained the nopaline synthetase promoter, and encoded the :first 59 amino acids of nopaline synthetase (of which the last residue was synthetically added); a one amino acid junction; and all of the phaseolin structural gene except for its first 12 amino acids. Prior to the start of construction, a clone of pTiC58 T-DNA (pCF44A; Fig. 6) was sequenced from the ,VIII site on the extreme left through the middle HindIII site, which is x outside of the T-DNA re~~ion. 'This included all of the nos gene (Fig. 4).
The nopaline synthetase sequence and reading frame were found to be as follows near a site cut by the restriction enzyme CIaI:
CIaI
5'...CCA/GGA/T~/ATC/TCA...3' 3'...GGT CCT A GC*TAG A(;T...S' ...Pro Gly Ser Ile Ser...

Cleavage with CIaI yields a fragment with the following end:
...CCA GGA T 3' ...GGT CCT AGC 5' As stated in Example 1, the following phaseolin EcoRI site:
EcoRI
...CTG/GG*7CTCTT/CTT/TTC...
...GAC CC T TAA*GAA AAG...
...Leu Gly Ile Leu Phe...

can be cleaved to following structure:
5' A/ATT CTT TTC...
3' G G...
The following two linkers a) 5' CGATCCC 3' b) 5' AATTGGGAT 3' can be annealed to form the following structure 5' CGATCCC 3' (a 3' TAGGGTTAA 5' (b .. 4340714 Which can link together the DNA fragments to form the following structure:
New Linker ...CCA/GGA/T*CG ATC/CC*A/ATT/CTT/TTC...
...GGT CCT A GC*TAG GG T TAA*GAA AAG...
...Pro Gly S~sr Ile Pro Ile Leu Phe...
56 57 5.3 59 nopaline synthetase 13 14 15 phaseolin Note that the linker serves several functions: a new amino acid is introduced; part of the deleted sequence of nopaline synthetase is reconstructed; two incompatible restriction sites are made compatible, and an open reading frame is preserved.
So in slaort, the EcoRI/BamHI restriction fragment of the phaseolin gene was ligated to the ClaI site of the nopaline synthase gene after a linker converted the EcoRI site to a Cla_I site. The fusion gene contained the nopaline synthetase promoter, the first 58 amino acids of nopaline synthetase, a linker which reconstruc-ted some of 'the nopaline synthetase sequence and inserted a newel amino acid, and all of phaseolin except for the first twelve amino acid residues.
2.1 Synthesis of Linkers The following two linkers were synthesized:
a ) 5' CG~~TCCC 3 ' b ) 5' AA'rTGGGAT 3 ' These were s:Ynthesized by the methods of Example 17.
The oligonuc:leotide a) and b) were annealed together to form the structure 5' CGATCCC 3' (a 3' TAGGGTTAA 5' (b 2.2 Preparation of the shuttle vector pKS-nop:IV, whose construction wa.s described in Fig.
6, is pRK290 with nopaline T-DNA cloned into its BalII
site. Its n~opaline T-DNA contains a single ClaI site resulting fr~~m deletion between the ClaI site in nos and the C7~I site downstream outside the nos gene (Figs. 5 27a ~3~071~
and 6). We purified pKS4-KB (Fig. 7) and digested with EcoRI. The ~E.8kbp kan bean resistance fragment --- ~3~0'~1~
was purified by gel electrophoresis. This fragment contains the EcoRI/BamHI
phaseolin DNA fragment (referred to as bean in the label kan bean) ligated at the BamHI site to the BamHI/EcaRI fragment of the kanamycin resistance gene (kan of TnS.
We ligated CIaI linearized pKS-nopIN with purifed kan/bean fragment and the linkers from Example 2.1. E. coli K802 was transformed and selected for kanamycin and tetracycline resistant colonies. Two orientations were present, one with phaseolin DNA ligated to nopaline synthetase gene and the other with kanamycin resistance gene ligated next to nopaline synthetase gene.
Restriction site mapping was used to determine which cells contained a plasmid, pNNNl, having the desired orientation as shown in Fig. 8.
2.3 Replacement of the nopaline synthetase ene '. Tp iC58 with the modified phaseolin A triparental matin!~ (see Background-Shuttle Vectors) with _A.
tumefaciens-strR C58, E. coli(pRK2013), and E. coli(pNNNly was used to insert the construction into a '~i plasmid. We selected for A. tumefaciens cells resistant to streptomycin, kanamycin and tetracycline. The selected transformants were mated with E. coli(pPHlJ1) and colonies resistant to kanamycin and gentamycin were selected.
2.4 Crown Gall Formation and Expression Sunflowers were inoculated and crown galls established in tissue culture. Expression was tested by ELISA and hybridization to mRNA as described in Examples 17 and 20"
Example 3 The aim of this example is to reconstruct the complete phaseolin gene coding sequence from the ATG translational start signal to the EcoRI site which can then be ligatedl to the remainder of the structural gene. A CIaI
site will be constructed at the 5' end so the gene can be easily recovered.
The following two oligonu~cleotide sequences will be synthesized:

~~4~'~1~
a) 5' AATTCCCAGCAACAGGAGTGGAACCCTTGCTCTCATCAT 3' b) 5' CGATGATGAGAGCAAGGGTTCCACTCCTGTTGCTGGG 3' These can be rennealed to form the following structure:
CIaI EcoRI
5' CG/ATG/ATG/AGA/GCA/AGG/GTT/CCA/CTC/CTG/TTG/CTG/GG 3' (a TAC TAC TCT CGT TCC CAA GGT GAG GAC AAC GAC CCT TAA 5' (b Met Met Arg Ala Arg Hal Pro Leu Leu Leu Leu Gly Ile As stated in Example 1, the following phaseolin EcoRI site:
...CTG/GG*A/ATT/CTT/TTC...
...GAC CC T TAA*GAA AAG...
Leu Gly Ile Leu Phe can be cleaved to following structure:
5' A/ATT/CTT TTC...
3' GAA G...
Ligation of this end to the synthetic double-stranded oligonucleotide described above results in a structural gene encoding a complete phaseolin polypeptide, with CIaI sticky-ends immediately ahead of the start of the coding sequence.
3.1 Synthesis of linker, The following two linkers were synthesized by the method of Example 17:
a) 5' AATTCCCAGCAACAGGAGTGGAACCCTTGCTCTCATCAT 3' b) 5' CGATGATGAGAGCAAGGGTTCCACTCCTGTTGCTGGG 3' They were annealed i;o form the following structure:
5' CGATGATGAGAGCAAGGGTTCCACTCCTGTTGCTGGG 3' (b 3' TACTACTCTCGTTCCCAAGGTGAGGACAACGACCCTTAA 5' (a 3.2 Construction of complete phaseolin ene and kanamycin resistance ene cloned in pKS-nopIV

CIaI linearized pKS-nopIV is ligated with reannealed linker from Example 3.1 and purified kan/bean EcoRI fragment from KS4-KB (see Example 2.2). E.
coli K802 is transformed and selected for tetracycline and kanamycin resistant colonies. Again; though two orientations are possible, only one is phaseolin gene ligated next to the nopaline synthetase gene. The correct orientation is selected after endonuclease mapping the clones.
3-33 Crown gall formation and expression The homologous recornbination and crown gall tissue culture isolation is performed as outlined in Example 21, and the testing of crown gall tissues for phaseolin gene expression is as in Examples 19 and 20.
Exam le 4 The purpose of this construction is to teach how to construct a Shuttle Vector to be used in pTi system for expressing foreign genes in crown gall cells, the foreign gene being under control of the nos promoter, part of which is chemically synthesized, and 'is missing codons for the nopaline synthetase gene. Prior to the start: of construction, a clone of pTiC58 T-DNA (pCF44A) was sequenced to discover the nns promoter (Fig. 4).
4.1 Isolation of the 5' op rtion of the nos promoter pCF44A is cut with x;hoI, religated, and labeled pCF44B, which has the following structure:
BgIII CIaI C1'aI SstII SstII SstII BgIII
...* 1160bp * 1300 * 355 * 620 * 420 * 1155b~ *...
3' nopaline 5' synthetase This new plasmid is of the SstII fragments. The resulting plasmid, pCF44C
~gIII CIaI CIaI SstII BgIII
... 1160 ~ 1300 ~ 355 * 1155 *...
* * XXXXXXXXXXXXXXX*XXXXXXXXX*
3' nopaline 5' synthetase is digested with Bc~III, and a 3.6kbp fragment is inserted into the Bc~III
site of pRK290. A colony selected for hybridization to T-DNA in a Grunstein-._.
Hogness assay is labeledl pKS-napV; digested with CIaI; and relegated; forming pKS-nopVI.
B~,1TII CIaI SstII B~III
...* 1160bp * 355 * 1155bp **...
* *XXXXXXXXX*
--This is digested with CIaI and SstII giving a 22kbp linearized vehicle and a 355bp fragment. These are easily separated by centrifugation through a salt gradient. After the small fragment is digested with HinfI the 149bp SstII/HinfI and the 208bp CIaI/HinfI fragments are isolated by gel electrophoresis.
4.2 Synthesis of linkers The following two linkers were synthesized by the method of Example 17:
a) 5' AGTCTCATACTCACTCTCAATCCAAATAATCTGCCATGGAT 3' b) 5' CGATCCATGGCAGATTATTTGGATTGAGAGTGAGTATGAG 3' They were annealed together to form the following structure:
5' AGTCTCATACTCACTCTCAATCCAAATAATCTGC_CATGGAT 3' (a 3' GAGTATGAGTGAGAGTTAGGTTTATTAGACGGTACCTAGC 5' (b This sequence has a HinfI site on the left, and NcoI and CIaI sites on the right. An alternate sequence will have a BcII site between the NcoI and CIaI
sites. The sequence is identical to that found in T-DNA except for the underlined bases which replace an A-T base pair with a C-G base pair.
4.3. Assembly of Np NN2 The 22kbp CIaI/SstIt vehicle is legated as shown in Fig. 9 with the 149bp SstII/HinfI fragment and the synthetic linker, forming the following structure:
HinfI synthetic linker NcoI CIaI
5'...149bp...TAG*AGT CTCATACTCACTCTCAATCCAAATAATCTGC*CATG GAT*CG
AT...1160bp...3' 3'...T-DNA...ATC TCA*GAGTATGAGTGAGAGTTAGGTTTATTAGACG GTAC*CTA GC*TA...T-DNA....5' I3~07I~

4.4 Insertion and expression of a phaseolin gene pNNN2, t:he plasmid constructed in Example 4.3 (Fig.
9) is cut with Cla:I, mixed with the ClaI/EcoRI linker synthesized in Example 4.2 and electrophoretically purified EcoRI/Cla:I kan bean fragment from pKS4-KB, ligated, transformed, isolated, and restriction mapped.
The appropriate plasmid, pNNN4, is transferred and tested for expression as described in Examples 21, 19 and 20.
4.5 Insertion and expression of a phaseolin gene lacking intro:ns The procedure outlined in Example 4.4 is repeated with the sub:~titution for pKS4-KB of a pcDNA31 or pMC6-cDNA-derived .analog of pKS4-KB. (see Example 9).
4.6 Insertion and expression of a phaseolin cDNA
This construction is analogous to Example 10 in its use of cDNA, a single stranded PstI linker, and the PstI
kan fragment,, and is analogous to Examples 4.1, 4.2 and 4.3 in the u:ae of the semisynthetic nos promoter. Char-acterization,, transfer and testing of expression is as described in Example 4.4.
pNNN2, ithe plasmid constructed in Example 4.3 (Fig.
9) is cut wiith ClaI, mixed with and ligated to the ClaI/EcoRI linker synthesized in Example 4.2, the electrophoretically purified l.7kbp EcoRI/PstI bean fragment iso:Lated from pKS4-KB~, the electrophoretically purified 0.9:3 kbp PstI Tn5 kan fragment, and the single-strancied Cla_I/PstI linker 5'CGAATT3', previously synthesized by the method of Example 17.
Example 5 The purpose of this construction is to ligate the phaseolin gene from the EcoRI site to BamHI site, into the active T~-DNA gene that lies across the HindIII sites on p403. This mRNA of this T-DNA gene is labeled 1.6 on the map, shown in Fig. 1, and 1450 by and ProI in the map shown in Figure 11. This T-DNA gene is referred to a340'~1~

herein as the "1.6 transcript gene". The sequence (see Fig. 10) was determined from the HindIII site of p401 past the Cla:C site to its right (see Fig. 11). There is an open reading frame that starts between the HindIII
and ClaI site going toward the HindIII site (see the 1450bp mRNA mapped in Fig. 11). The ClaI site is in the untranslated leader of the mRNA of the gene spanning the HindIII rites. We create a promoter vehicle by cutting out i~he Clal fragment in the middle of p403.
This is possible because the internal ClaI sites are not methylated in some E. coli strains, whereas the ClaI
site next to the EcoRI site is methylated.
The phaaeolin gene is now ligated into the ClaI
site bringing with it an ATG. This can be acaomplished by using pKS~~-3.OKB. The base sequence from the ClaI
site of pBR3:Z2 through the EcoRI site of phaseolin is as follows:
ClaI FmRI
5' . . .AT*C,/G AT/GATE',~CIGJ~/AAC/A~/AG*~,/ATr~~. . . 3' 3'...TA G C*TA CIA TfG GAC GAC AGT TIG TAC TC T TAA*GAA AAC...S' Met Arg/Ile Leu Phe...

...derived frcan pBR322/phaseolin...
Note the open reading frame and the ATG. There are l8bp between the ClaI site and the translational start signal (ATG). This compares to l2bp from the ClaI site to the start of the T-DNA gene:
_Cla_I
5'...AT'*CG ALTGG/ACA,/TGC/TGT/ATG...3' 3'...TA GC*T ACC TGT ACG ACA TAC...5' Met...
Again, ;note the open reading frame and the ATG.
Thus, ligati~on into the ClaI site of the promoter clone should create an active phaseolin gene in T-DNA. The phaseolin gene has a substitution of 2 amino acids for the naturally occur.ing amino terminal 12 residues.
5.1 Construction of a Promoter vehicle pKSIII, which is a pRK290 clone corresponding to the T-DNA clone p403 (see Fig. 1), is digested with ClaI

134Q'~14 and then reli.gated. The ligation mix is transformed into K802 andl selecaed for kanamycin resistance.
Plasmids are isolated by doing "minipreps" (plasmid preparations from :small volume cell cultures) and restriction maps are obtained to prove the structure.
The new vehicle, pKS-proI, is not able to be digested by HindIII but can be linearized by ClaI (Fig. 12).
pKS-proI is purified and linear molecules are produced by digestion with <:laI.
5.2 Ligation of a partial phaseolin crene to a kanamyci.n resistance crepe A 3.Okbp fragment containing extensive 3' flanking sequences andl all but the extreme 5' coding sequences of the phaseolin gene was obtained by elution from an agarose gel after electrophoresis of an HindIII and BamHI digest of p7,.2 (Fig 13), a pBR322 subclone of the phaseolin genomic clone 177.4 whose construction is described in Examp.'Le 6.1. This was mixed with and ligated to a 3.Okbp kanamycin resistance HindIII/BamHI
fragment similarly isolated from pKS4 (Fig. 18), and HindIII-linearized pBR322. After restriction mapping of plasmids isolated from ampicillin resistant tranwsfor-mants, a plascmid having the structure shown in Fig. 7 was labeled ~>KS4-KB.
5.3 Purifica 'on of the kan/bean fragment from pKS4-3 . C)KB
pKS4-KB (Fig. 7) is digested with ClaI and the 4.9kbp fragment purified by agarose gel electrophoresis.
5.4 Lig~atior~ of C:LaI kan/bean resistance gene into ClaI
digested pKS-ProI
pKS-pro7: is l:inearized by digestion with ClaI and the kanamycir~ resi:atance gene/bean fragment from Example 5.3 are ligat:ed together and transformed into K802.
Kanamycin re:~istanit transformants are selected and plasmids isolated by "minipreps" are restriction mapped to detect onE: having the proper orientation. The plasmid is labeled pKSProI-KB (Fig. 14).
5.5 Transfo~:~mation and expression Cells containing pKS-proI-KB are mated with 5 Aarobacteriurn_ cells containing pTi15955 or pTiA6 or other appropriate 'TIP plasmids. After selection of recombinants with :kanamycin, plants are inoculated and crown galls are established in tissue culture. Testing for the synthesis ~of phaseolin is as described in 10 Examples 19 and 20.
Example 6 This example teaches manipulations of a gene for phaseolin, the major seed storage protein of the bean Phaseolus vul~aris L., preparatory to further manipula-15 tions which :insert the phaseolin gene into vectors described in various other examples.
6.1 Subclon:ing of a phaseolin gene A genom:ic clone of phaseolin in a Charon 24A
AG-PVPh177.4 (or 177.4; S.M. Sun et al (1981) Nature 20 289:37-41, J.L. Slightom et al (1983) Proc. Natl. Acad.
Sci. USA 80 l?ig. 15) was digested with BalII and BamHI.
The 3.8kbp fragment carrying the phaseolin gene and its flanking sequences, isolated by agarose gel electrophor-esis was mixed with and ligated to BamHI-linearized 25 p8R322. The mixture was transformed into HB101, and colonies resistant to ampicillin and sensitive to tetracycline were selected. Plasmid isolated from these clones was restriction mapped. A plasmid having the structure shown in Fig. 16 was selected and labeled 30 AG-pPVPh3.8 (or alternatively, p3.8). The ligation of BalII and Bam_HI sites with each other inactivates both sites.
Another subclone of 177.4 was constructed by digestion with EcoRI, isolation of a 7.2kbp fragment 35 containing e:Ktensive 3' flanking sequences and all but the extreme !~' end of the phaseolin gene, and isolated ~340'~1~
35a after ampicillin selection of HB101 transformants were restriction mapped. A plasmid having the insert oriented so that tree HindIII site of pBR322 was adjacent to the 5' end. of the phaseolin gene and distal to the 3' untranslated region was labeled AG-pPVPh7.2 (or p7.2;
Fig. 13; Sun et al and Slightom et al, supra).
6.2 Cloninct and isolation of a kanamycin resistance gene pRZ102 (R. A. Jrorgenson et al (1979) Molec. Gen.
Genet. 177:65-72), a ColEI plasmid carrying a copy of the transposo~n TnS) was digested with BamHI and HindIII, mixed with pE~R322 (Fig. 17) previously linearized with the same two enzymea, ligated, and transformed into K802. Plasmi.ds, i:~olated from transformants selected for resistance to both ampicillin and kanamycin were restriction mapped and one having the structure shown in Fig. 18 was labeled pKS-4.
6.3 Linkage of then phaseolin Qene with a kanamycin resistance gent p3.8 was. digested with ClaI and BamHI, and a 4.2kbp fragment cont.aininc3 the phaseolin gene and some pBR322 sequences was. isolated by agarose gel electrophoresis.
This was mixed with a ClaI/BamHI fragment of Tn5 carrying a ka.namycin resistance (neomycin phosphotransferase LI) gene from pKS4 (Fig. 18) and pBR322 (Fig. 17) which had been linearized with ClaI.
The mixture was ligated and transformed into K802.
After selection of colonies resistant to ampicillin and kanamycin, pl.asmids were isolated and restriction mapped. A colony having the structure shown in Fig. 19 was labeled pKS-KB:3.8.
The cons~truct:Lon of another useful plasmid, pKS4-KB, is described in Example 5.2.

~~~o~~~
Example 7 This example is analogous to the construction described in Example 5, except for the substitution of a cDNA clone for the genomic clone of phaseolin. This construction will result in a gene lacking introns.

7.1 Construction of ~~4-KB2.4 analo ous to KS4-KB
After pMC6 (Fig. 20~) is digested with EcoRI and BamHI, a 2.4kbp phaseolin cDNA fragment is isolats~d by centrifugation through a salt gradient or gel electrophoresis. A 1.9k.bp fragment containing a gene for kanamycin resistance is purified from a EcoRI and BamHI digest of pKS4 (Fig. 18); mixed with the cDNA fragment and EcoRI-linearized pBR322, ligated, and transformed into K802. Colonies are selected for kanamycin resistance, and after plasmid isolation and restriction mapping, a plasmid as shown in Fig. 21 is labeled pKS4-KB2.4.
7.2 Ligation of the CIaI kan bean DNA into CIaI digested S-ProI
pKS4-KB2.4 is digested with CIaI and ligated with CIaI-linearized pKS-proI (Fig. 12). After transformation, selection, plasmid isolation and characterization, the desired construction, having the phaseolin sequences adjacent to the T-DNA promoter, is transferred to a Ti plasmid. Inoculation and testing is as described in Examples 21, 19, and 20.
Exampl a 8 This example teaches a method of removing the introns from a gene. This is the same as placing a cDNA in a genomic environment. Restriction enzyme sites are found, or created by site specific mutagenesis, in exons on both the 5' and 3' extremities of the unprocessed transcript. These sites exist in both the genomic clones and cDNA. The intervening intron-containing DNA can be removed from the genanic clone and be replaced with the corresponding intronless cDNA clone fragment spanning the two sites. The reverse operation is also possible: intron-containing genomic sequences can be placed in a cDNA
environment. One inserts an internal fragment of the genomic clone into a corresponding gap cut oui: of a cDNA clone. This latter strategy is analogous, though often technically more difficult as the introns may contain sites susceptible to the enzymes chosen to create the exchanged fragment. This difficulty can be overcane by careful selection of conditions of partial digestion and by purification of the desired fragment by agarose gel electrophoresis. Further elaborations of this strategy include the manipulation of individual introns within a gene while leaving other introns and exons unaffecaed, and the stepwise exchange of sequences when inconvenient :intervening restriction sites are present within intr0ns as discussed above.

8.1 Replacement o:f a fragment containing phaseolin's introns with cDNA
p3.8, a plasm:id clone of the phaseolin gene and its flanking sequences, was digested respectively partially and to complexion with EcoRI and SacI, and a 6.4kbp fragment, containing the pBR322 vector and both the 5' and 3' ends of the gene, was isolated by agarose gel electrophore:ais. pcDNA3l, a pBR322 plasmid clone of cDNA made from pha;seolin mRNA, was digested respectively partially and to completion with SacI and EcoRI, and a 1.33kbp fragnnent, containing the entire phaseolin cDNA
except for sE:quences at the extreme 5' and 3' ends, was isolated by agarose gel electrophoresis. These two fragments were ligated together and transformed into HB101. After selection of colonies, growth of cells, and plasmid isolat.ian, restriction mapping identified a plasmid having the desired structure. This plasmid was labeled p3.8--cDNA (Fig. 22).
8.2 Use of x~3.8~cDNA
Note that p3.~B--cDNA can substitute for the genomic DNA source, E~.g., p3.8, used in other Examples and that when so used will :result in analogous constructions differing in that 'they are lacking introns. Alterna-tively, this strategy can be used to remove introns from construction: already made.
Example 9 This example 'teaches the expression of an intron-less gene. '.Che phaseolin cDNA is prepared as described ~~4om4 in Example 8, but a gene that naturally lacks introns could also be: used,.
An analogous c:onstruction to those taught in Examples 7 and 8 i:a used. pKS4-KB and pMC6 are digested with EcoRI arid Sac7C as taught in Example 8 and as described thE:re, the cDNA insert is ligated into the pKS4-KB fragment containing the vector and the 5' and 3' extremities of the phaseolin gene. The new plasmid, pKS4-KBc, is used in constructions in an analogous manner to pKS:4-KB.
Example 10 The purpose of this example is to teach the place-ment within T-DNA of the cDNA for a Phaseolus vulgaris lectin under the control of a T-DNA gene promoter, the transfer of this construction to a plant cell, and the detection of this c:onstruction's expression within plant tissue.
This cor~struci~ion utilizes a single-stranded linker to connect the sticky-ends resulting from digestion with the restriction en~.ymes PstI and HindIII. When PstIII
and HindIII sites Pst7: HindIII
5'...C TCCA~'G...3'' S'...A*AGCT T...3' 3'...G*ACGT C...5'' 3'...T TCGA*A...S' are cleaved t:o form the following ends:
PstI HindIII
5'...CTGCA AGCTT...3' 3'...G A...5' and are mixed together in the presence of a linker of 3 0 appropriate :sequence 5' . . . CT~rCA AGCTT . . . 3' 3'...G A...5' 3' ACGTTCGAS' they can be l.igated together to form the following suture:

13~071~

_HindIII
5' ..C '.fGCA*AGCT T...3' 3'...G*ACGT TOGA*A...5' Note that a HindIII site is reconstructed.
The lecmin cD:HA is obtained from a plasmid clone, pPVL134, ATCC39181, that was constructed by poly C-tailing double-stranded cDNA followed by insertion into PstI cut, G-mailed pBR322. This clone was described by L. Hoffman eit al (1982) Nucleic Acids Res.10:7819-7828.
10.1 Svnthe:ais of the linker The linlter 5'.AGCTTGCA3' is synthesized by the method of Example 1'l.
10.2 Construction of a clone containing lectin cDNA and a kanam~,rcin resistance ciene pPVL134 is digested with Bcll and PstI, and the intermediate-sized fragment containing the lectin coding sequence, 3' untranslated region, and a C/G tail is isolated by elution from an agarose gel after separation by electrophoresis. pBR325 is digested with BclI and HindIII and 'the largest fragment is isolated after sedimentation through a salt gradient. The BclI/HindIII
pBR325 vector is mixed with and ligated to the BclI/PstI
lectin fragment and the PstI/HindIII linker prepared in Example 10.1. E. coli K802 is transformed, selected for drug resistance and presence of lectin sequences, and the plasmid isolated from such cells is labeled IIc.
The largest fragment resulting from HindIII and BamHI
digestion of IIc is mixed with and ligated to the kanamycin resistance gene-carrying HindIII/BamHI frag-ment of pKS-4 which is previously isolated by agarose gel electrophoresis (Fig. 23). K802 is transformed, and colonies are selected for kanamycin resistance, plasmics are isolated and characterized by restriction mapping.
The desired plasmid is labeled pL-B.
10.3 Chance of a ClaI site to GamHI site in pKS-proI
pKS-proI, whose construction was described in Example 5.1 (see F'ig. 12) is digested with ClaI. This 13~071~
39a cut is located between the promoter and ATG translation start signal of the l.6kbp transcript (see Fig. 1). The sticky-ends are converted to blunt-ends by filling in by DNA polymerase I. BamHI linkers are ligated into the gap, trimmed to expose BamHI sticky-ends, ligated, and transformed into K802. Colonies harboring the desired plasmid, pKS-proIA (Fig. 24), are selected after "miniprep" plasmid isolations and a characterization by restriction enzyme mapping.
10.4 Insertion of lectin and kanamycin resistance enes into pKS-proI~~
pL-B (Ex:ample 10.2) is digested with BclI and BamHI, and th.e fragment carrying the kanamycin resistance gene and lectin sequences is eluted from an agarose gel after e~lectrophoretic separation. This fragment is mixed with and ligated to BamHI linearized pKS-proIA. T'he lic~ation mixture is transformed into K802. Plasmids are: isolated from kanamycin resistant colonies, characterized by restriction mapping, and the desired construction labeled pLK-proIA (Fig. 25).

h ~-_ ~3~0'~1~
10.5 Expression i_n plants pLK-proIA is transferred to a Ti plasmid by a triparental mating (Example 21) of K802(pLK-proIA);.E-.. coli.(2013),and A. tumefaciens (pTi15955) (streptomycin resistant). After additional conjugational transfer of pPHlJ1 into the Agrobacterium, double-homologous recombinants are selected by growing cells on kanamycin, streptomycin, and gentamycin. Lectin is detected by ELISA
with the appropriate antibodies.
Example 11 The purpose of this example is to generate a Ti plasmid with a deletion from the tms ("shooting" locus) through the tmr ("rooting" locus) of pTi15955 and other octopine Ti pl,asmids. This derivative is useful because cells transformed by it are easier to regenerate to whole plants than cells transformed by pTi15955 with intact tms and tmr genes.
The tms-tmr deleted pTi15955 is ultimately changed in two ways: the in activation of tms-tmr and the insertion of a foreign gene. Should these two changes be located at different points of the T-DNA, each change is inserted independently by different shuttle vectors. Each shuttle vector dependent change is selected independently which will necessitate use of at least two markers selectable in Agi~obacterium. In addition to the usual kanamycin resistance, this example utilized a chloramphenicol resistance derived from pBR325.
11.1 Construction of a c:hloramphenicol resistance ene clone pBR325 is digested with HincII and blunt end legated with HindIII
linkers. The resultant preparation is digested with HindIII, relegated, selected for chlorampheniicol resistance (cam), and labeled pKS-5 which will serve as a source of the HindIII/BcII fragment which contains the cam ~ gene (Fig. 26).
11.2 Construction of a pBR322 clone of T-DNA with a deletion and a cam ene A 9.2kbp linear DNA fragment is isolated from a complete HindIII and partial BamHI digest of E>203. lfhe fragment carrying the cam gene is isolated from pKS-5, mixed with the 9.2kbp linear fragment, legated, transformed into E. cola) selected for chloramphenicol resistance, and labeled pKS-Oct.Cam203 (Fig. 27).

13~~711~
pKS-oct.Cam203 is a plasmid clone that can now be used to construct a number of deletion n mutants of pTi15955. It contains the right hand arm of TL and a resistance gene to the left of the right arm. We can attach various left-hand arms of TL to the left of the cam gene (HindIII site). For instance, if p102 is attached the deletion is 5.2kbp long and includes all of tms and tmr. If p103 is attached the deletion is 3.2kbp long and includes part of tms and all of t~,mr. See Fig. 1.
pKS-oct.Cam203 is digested with HindIII. p102 or p103 is digested with HindIII and the 2.2kbp or 2.Okbp T-DNA fragment is isolated and ligated with the linearized pKS-oct.Cam203, transformed, isolated yielding pKS-oct.delII
(Fig. 28) or pKS-oct.delI (Fig) 29), respectively. These constructions are moved into A. tumefaciens by mating, homologous recombinations, and selection for chloramphenicol resistance. Alternatively, one moves the constructions into pRK290 by use of established methods by linearizing the construction carrying plasmids with B~amHI and ligating into the B~,1_II site of pRK290 (Fig.
30).
Example 12 The Ti plasmid is mutated in this example by deleting the T-DNA between the HpaI site in tmr to vthe SmaI site in tml. The Ti plasmids that can be modifed include pTi15955) pTiB6, pTiA66 and others. This construction is diagrammed in Fig. 31.
12.1 Isolation of the cam ene pKS-5 (Fig. 26) is digested with HindIII and BcII. The smallest fragment is isolated after separala on on an agarose gel, as taught in Example 11.
12.2 Construction of a pBR322 clone of T-DNA with a deletion The right hand arm of the 'f-DNA deletion is constructed by insertion of Bc~III sites into the Smal~ sites of p203 (see Fig. 1). p203 is digest, by SmaI, ligated with BqIII linkers, digested with Bc~III, religated, and transformed into K80i!. In an alternative construction, BamHI linkers may be substituted for Bc~III linkers and the appropriate BamHI partial digest products are isolated.) fhe resultant plasmid is labeled p203-BgIII, and is digested with B~,1_II and HindIII. The large BqIII/HindIII vector containing fragment is ligated with the chloramphenicol resistance fragment whose ... -41-isolation was described in Example 12.1. Chloramphenicol resistance is selected for after transformation into K802. The resultant plasmid is labeled p2f (Fig. 31).
12.3 Construction of left-hand arm of T-DNA deletion clone HindIII sites are inserted into the H~aI site of p202 by digestion with H~aI and ligation with HindIII linkers. After unmasking of the HindIII sticky ends by digestion with tlhat restriction enzyme, the 2kbp HMI fragment which now bears HindIII ends i~s isolated. HindIII digested HindIII-ended H~aI
fragment and transformed into K~B02. After a colony containing the desired construction is isolated; and characterized, the plasmid is labeled pie (Fig.
32).
12.4 Construction of the T-DNA deletion clone The left-hand arm of the clone is obtained by purifying a 2kbp fragment of a HindIII digest of p:3e by elution from an agarose gel after electrophoresis. p2f is cut by HindIII, treated~~with alkaline phosphatase, mixed with the 2kbp fragrnent, ligated, transformed into K802; and selected for chloramphenicol resistance) Plasmids are isolated from individual colonies and characterized by resi:riction mapping. A plasmid having the two arms in the desired tandem orientation is chosen and labeled pKS-Oct.delIII (Fig. 33).
pKS-oct.delIII is moved into A. tumefaciens by mating, and homologous recombinants are selected with chloramphenicol. Sunflower and tobacco roots and shoots are inoculated as described in other Examples and the tumors generated are tested for opines.
Exam le 13 This example teaches a construction deleting tmr and tml that provides an alternative to that taught in E;Kample 12.
13.1 Construction of a c;hloramphenicol resistant fragment with a BgIII site pBR325 is digested with HincII, blunt-end ligated with BgI,II
linkers, digested with BdIII, and religated (Fig. 34). Chloramphenicol resistance is selected for after transformation of either K802 or GM33. The resultant plasmid, pKS-6 serves as a source of the BgIII/BcII fragment carrying the cam gene.

I3~0'~14 13.2 Construction of the tmr; tml deletion clone p203 is digested with H~aI and SmaI. After blunt end ligation with Bc~II
linkers, it is digested with B~III to expose the BgIII sticky-ends, religated, and transformed into K802. The desired construction is identified and labeled p2 (Fig. 35).
13.3 Construction of the T-DNA deletion clone (pKS-oct.delIIIa) The BgIII fragment carrying the cam gene is isolated from pKS-6 and ligated into BgIII-cut p~2. Chloramphenicol resistance is selected for after transformation of K802. The resultant plasmid is labeled pKS-oct.delIIIa (Fig. 36), and is tested as described in Example 12.4.
Example 14 The purpose of this construction is to provide an example of the mutation of the tmr locus only at the H~aI site by insertion of the chloramphenicol resistance gene. This gene is isolated as the Bc~III/BcII fragment from pKS-6, and is ligated into the,H~aI site of p203 after that site is changed to a _B~III site.
14.1 Conversion of the HpaI site to a BgIII site p203 is digested with H~aI, ligated to BgIII linkers, trimmed with BgIII
and relegated. After transformation of K802, colonies are selected and screened by restriction mapping for insertion of B~III sites (Fig. 37).
14.2 Isolation of the cam gene, pKS-6 is digested with BgIII and BcII. The smallest fragment is isolated by agarose gel electrophoresis.
14.3 Construction of the mutated T-DNA clone The modified p203 from Example 14.1 is digested with BgIII, legated with the purified cam gene from Example 14.2 and transformed into K802.
Chloramphenicol resistance is selected for, and after isolation from the resistant transformants and characterization by restriction enzyme mapping, the plasmid is labeled pi~CS-oct.tmr (Fig. 38).

1340~1~

Example 15 Regeneration in this example involves carrot tumors incited by an Ri-based TIP plasmid and is effected essentially ~~s described by M. D. Chilton et al (1982) Nature 295:4:32-434.
15.1 Infection with hairy root Carrot disks axe inoculated with about 10594 bacteria in 0.1 ml of water. One to 1.5 cm segments of the ends of 'the roots obtained are cut off, placed on solid (1-1.5~~ agar) Monier medium lacking hormones (D. A.
Tepfer and J.C. Tempe (1981) C.R. Hebd. Seanc. Acad.
Sci., Paris 295:153-156), and grown at 25°C to 27°C in the dark. Cultures uncontaminated by bacteria are transferred ~svery 2 to 3 weeks and are subcultured in Monier medium lacking hormones and agar.
15.2 Regeneration of roots to plants The cultured root tissue described in Example 15.1 is placed on solidified (0.8% agar) Monier medium supplemented with 0.36uM 2,4-D and 0.72uM kinetin.
After 4 week,, the resulting callus tissue is placed in liquid Monie:r medium lacking hormones. During incuba-tion at 22 t~o 25°C on a shaker (150 r.p.m.) for one month, the callus disassociates into a suspension culture from which embryos differentiate, which, when placed in Petri dishes containing Monier medium lacking hormone, develop into plantlets. These plantlets are grown in culture, and after "hardening" by exposure to atmospheres of progressively decreasing humidity, are transferred to soil in either a greenhouse or field plot.
15.3 Use of non-ha.irv root vectors Ti-based vectors which do not have functional tmr genes are used instead of the Ri-based vectors as described in Examples 15.1 and 15.2. Construction of suitable deletions. is described in Examples 12, 13, and 14.

Examt~le 16 Regeneratic>n in this example involves tobacco tumors incited x>y a Ti-based TIP plasmid and is 5 effected essentially as described by K.A. Barton et al (1983) Cell.
16.1 Infection with crown .gall Tobacco tissue is transformed using an approach utilizing invert:ed stem segments first described by 10 A.C. Braun (195E~) Canc. Res. 16:53-56. Stems are surface sterilized with a solution that was 7%
commercial Chlot-ox* and 80% ethanol, rinsed with sterile distills:d water, cut into 1 cm segments, and placed basal end up in Petri dishes containing 15 agar-solidified MS medium (T. Murashige and F. Skoog (1962) Physiol. Plant. 15:473-479) lacking hormones.
Inoculation is effected by puncturing the cut basal surface of the :aem with a syringe needle and injecting bacteria. Stem:: are cultured at 25°C with 16 hours of 20 light per day. The calli which develop are removed from the upper surface of the stem segments, are placed on solidified M~~ medium containing 0.2 mg/ml carbenicillin and lacking hormones, are transferred to fresh MS-c:arbenicillin medium three times at intervals 25 of about a. month, and are tested to ascertain whether the cultures had been ridden of bacteria. The axenic tissues are maintained on solidified MS media lacking supplements unde=r the culture conditions (25°C; 16 hr.:8 hr. light:: dark) described above.
30 16.2 Culture of transformed tissue Clonea are obtained from the transformed axenic tissues as'. described by A. Binns and F. Meins (1979) Planta 145:365-:369. Calli are converted into suspensions of c=ells by culturing in liquid MS having 35 0.02 mg/1 naphthalene acetic acid (NAA) at 25°C for 2 or 3 days while being shaken at 135 r.p.m.,and filter-ing in turn through 543 and 213~,m stainless steel 1340!714 meshes. The passed filtrate is concentrated, plated in 5m1 of MS
* Trade-mark medium containing 0.5% melted agar, 2.0 mg/1 NAA, 0.3 mg/1 kinetin and 0,.4 g/1 Difco yeast extract at a density of ax~out 8 x 10534 cells/ml. Colonies reaching a diameter of aboui~ 1 mm are picked by scalpel point, placed onto a:nd grown on solidified MS medium having 2.0 mg/1 NAA and 0.3 mg/1 kinetin. The resulting calli are split into pieces and tested for transformed phenotypes.
16.3 Regeneration of plants Transformed c7Lones are placed onto solidified MS
medium having 0.3 mg/1 kinetin, and cultured as des-cribed in Example 16.1. The shoots which form are rooted by putaing i:hem on a solid (1.0% agar) medium containing 1/'10 strength MS medium salts, 0.4 mg/1 thiamine, lacking :sucrose and hormones, and having a pH of 7Ø Footed plantlets are grown in culture, hardened as described in Example 15.2, and are transferred t:o soi7L in either a greenhouse or field plot.
16.4 Vectors used The methods dEascribed in Examples 16.1, 16.2 and 16.3 are suitable Ti.-based vectors lacking functional tmr genes. ~'.onstruction of suitable deletions is described in Examp7Les 12, 13, and 14. These methods are also effective when used with Ri-based vectors. The method described in Example 16.1 for infection of inverted stem segments is often useful for the establishment: of T7CP transformed plant cell lines.
Example 17 The techniques for chemical synthesis of DNA
fragments used in i~hese Examples utilize a number of techniques well known to those skilled in the art of DNA
synthesis. Z'he modification of nucleosides is described by H. Schalle:r et al. (1963) J. Amer. Chem. Soc. 85:
3821-3827. The prs:paration of deoxynucleoside phosphor-amidites is described by S.L. Beaucage and M.H.
Caruthers (1981) TEarahedron Lett. 22:1859. Preparation of solid phase resin is described by S.P. Adams et al (1983) J. Ame:r. ChEam. Soc. Hybridization procedures useful for tree formation of double-stranded synthetic linkers are olescribed by J.J. Rossi et al (1982) J.
Biol. Chem. 2.57:92:'6-9229.
Example 18 Phaseoli.n is 1_he most abundant storage protein (approximatel.y 50% of the total seed protein) of Phaseolis vul.Qaris. Transfer of the functional phaseolin gene to alfalfa plants and translation of the phaseolin m-F;NA ini:o stored phaseolin is of significant economic value since it introduces storage protein into leaf material. to be used as fodder. Alfalfa is a valuable plant for the transfer and expression of the phaseolin genie bec<iuse of its acceptance as cattle fodder, its rapid growth, its ability to fix nitrogen through Rhizobial :symbiosis, its susceptibility to crown gall infection and the ability to regenerate alfalfa plants from ~~ingle cells or protoplasts. This example teaches the introduction of an expressible phaseolin gene into intact alfalfa plants.
18.1 Construcaion of shuttle vector Alfalfa plant:a are regenerated from crown gall tissue containing genetically engineered Ag~robacterium plasmids as described hereafter. In the first step we construct a "'shutt:Le vector" containing a tmr5-4 and a tms5-T-DNA mutant :Linked to a phaseolin structural gene under control. of a T-DNA promoter. This construction is, in turn, linked to a nopaline synthetase promoter which has a functional neomycin phosphotransferase (NPTII) strucaural gene (kanamycin resistance) down-stream (reported b~~ M.D. Chilton et al (18 January 1983) 15th Miami Winter Symposium; see also J.L. Marx (1983) Science 219:830 and R. Horsch et al (18 January 1983) 15th Miami Winter Symposium). A phaseolin structural gs:ne under control of a T-DNA promoter is illustrated in Example 1.
18.2 Transfer to Ac~robacterium and plant cells The "shuttle vector" is then transformed by con-s ventional tec;hniques (Example 21) into a strain of Aqrobacterium_ cont<~ining a Ti plasmid such as pTi15955.
Bacteria containing recombinant plasmids are selected and co-cultivated with alfalfa protoplasts which are regenerated c;ell w<~lls (Marton et al (1979) Nature 277:129-131: G. J. Wullems et al (1981) Proc. Natl Acad. Sci. (Lf.S.A.) 78:4344-4348; and R.B. Horsch and R.T. Fraley (18 January 1983) 15th Miami Winter Symposium).
Cells are grown in culture and the resulting callus tissue is tee>ted for the presence of the appropriate mRNA by Northern blotting (Example 19) and for the presence of t:he appropriate proteins by ELISA tests (Example 20) (see ~T.L. Marx (1983) Science 219:830: R.B.
Horsch and R.T. Fraley (18 January 1983) 15th Miami Winter Sympo~~ium) .
18.3 Plant re:aeneration Alfalfa plant:a are then regenerated from callus tissue by methods :similar to those previously used by A.V.P. Dos Santos eat al (1980) Z. Pflanzenphysiol.
99:261-270, T.J. McCoy and E.T. Bingham (1977) Plant Sci. Letters 10:59-66 and K.A. Walker et al (1979) Plant Sci. Leaters 16:23-30. These regenerated plants are then propagated by conventional plant breeding techniques forming the basis for new commercial varieties.
Example 19 In all Examples, RNA was extracted, fractionated, and detected by the following procedures.
19.1 RNA extractioy This procedure was a modification of Silflow et al (1981) Biochemistry 13:2725-2731. Substitution of LiCl 48a precipitation for CsCl centrifugation was described by Murray et al (1981;) J. Mol. Evol 17:31-42. Use of 2 M
LiCl plus 2M urea to precipitate was taken from Rhodes (1975) J. Biol. Chem. 25:8088-8097.
Tissue was homogenized using a polytron or ground glass homogenizer :in 4-5 volumes of cold 50 mM Tris-HC1 (pH 8.0) containing 4% p-amino salicylic acid, 1%
tri-isopropyl. naptlzalene sulfonic acid, to mM
dithiothreitol (fr~ashly made) and 10 mM Na-metabisulfite (freshly madE:) . N~-octanol was used as needed to control foaming. An equal volume of Tris-saturated phenol containing :1% 8-hydroxyquinoline was added to the homogenate which was then shaken to emulsify and centrifuged at 20,000-30,000 g for 15 minutes at 4°C.
The aqueous upper phase was extracted once with chloroform/ocaanol (24:1) and centrifuged as above.
Concentrated LiCl-urea solution was then added to a final concentration of 2 M each and the mixture was left to stand at :>.0°C for. several hours. The RNA precipitate was then centrifuged down and washed with 2 M LiCl to disperse the pellet. The precipitate was then washed with 70% ethanol-0.3M Na-acetate and dissolved in sufficient sterile water to give a clear solution. One half volume of ethanol was added and the mixture put on ice for 1 hour, after which it was centrifuged to remove miscellaneous polysaccharides. The RNA precipitate was then recoverE:d and re-dissolved in water or in sterile no salt poly I;U) bu:Efer.
19.2 Poly(U)/Sephadex chromatocrraphy Two poly(U) Sephadex (trademark: Pharmacia, Inc., Uppsala, Sweden) buffers were used: the first with no salt contain~Lng 20 mM Tris, 1 mM EDTA and 0.1% SDS, and the second with 0.:1 M NaCl added to the first. In order to obtain a good match at A42605, a 2x stock buffer should be made and the salt added to a portion. After adjusting then final concentrations, the buffers were autoclaved.

~~~:o~~~
Poly(U) Sephadex ways obtained from 8ethesda Research Laboratories and lgm poly(U) Sephadex was use, per 100ug expected poly(A)RNA. The poly(U) Sephadex was hydrated in no salt ipoly-U buffer and poured into a jacketed column. The temperature was raised to 60°C and the column was washed with no salt buffer until the baseline at 260mm was flat. Finally the column was equilibrated with the salt containing poly(U) buffer at 40°C. The RNA at a concentration of less than 500ug/ml was then 'heated in no salt buffer at 65°C for 5 minutes, after which it was cooled on ice and NaCI added to a concentration of O.1M.
The RNA was then placed on the column which should be run at no more than lml/min until the optica~i density has fallen to a steady baseline. The column temperature was then rai~~ed to 60°C and the RNA was eluted with no salt poly(U) buffer. The RNA will usually wash off in three column volumes. The eluted RNA was then concentrated with secondary butanol to a convenient volume after addition of NaCI to lOmM, and precipitated with 2 volumes ethanol. The ethanol precipitate was dissolved in water and NH445-acetate added to O.1M, followed by re-precipitation with ethanol. Finally the RNA was redissolved in sterile water and stored at -70'°C.
19.3 Formaldehyde RNA gels The method used followed that of Thomas (1980) Proc. Nat'1. Acad. Sci.
(U.S.A) 77:5201 and Hoffman, et al. (1981) J. Biol. Chem. 256:2597.
0.75-1.5% agarose gels containing 20mM Na-phosphate (pH 6.8-7.0) were cast. If high molecular weight aggregate bands appeared, then the experiments were repeated with the addition of 6% or 2.2M formaldehyde (use stock solution of 36%) to the gels. The formaldehyde was added to the agarose ater cooling to 65°C. Addition of fonnaldehyde caused visualization with ethidium bromide to be very difficult. The running buffer was lOmM Na-phosphate (pH 6.8-7.0).
Prior to electrophoresis, the RNA was treated with a denaturing buffer having final concentrations of 6% formaldehyde, 50% formamide) 20mM Na-phosphate buffer and 5mM EDTA. The RNA was incubated in the buffer at 60°C
for 10-20 minutes. The iincubation was terminated by addition of stop buffer. For a 20u1 sample, 4u1 50% glycerol, lOmM EDTA, 5mM Na-phosphate and bromphenol blue were added.
Submerged electrophoresis was used. The RNA was loaded before the gel was submerged, and run into the gel at 125mA for 5 minutes. The gels were then submerged and the current reduced to 30mA (overnight) or 50mA (6-8 .r hours). The buffer was recirculated and the electrophoresis was done in a cold room.
19.4 "Northern" blots If the gel was to b~e blotted to detect a specific RNA, it was not stained; but a separate marker lane was used for staining. Staining was with 5ug/ml ethidium bromide in O.1M Na-acetate and destaining was for several hours in O.1M Na-acetate. Treatment in water at 60-70°C for 5-10 minutes prior to staining helped visualization.
A gel to be blotted was soaked for 15 minutes in lOx standard saline citrate (SSC)-3% formaldehyde. If large RNA molecules were not eluting from the gel then a prior treatment in 50mM NaOH for 10-30 minutes helped to nick the RNA. If base treatment was used, the gel should be neutralized and soaked in SSC-formaldehyd a before blotting. Transfer of the RNA to nitrocellulose was done by standard methods.
Prehybridization was done at 42°C for a minimtan of 4 hours in 50%
formamide, 10% dextran sulfate, 5x SSC, 5x Denhardt's, 100ug/ml denatured carrier DNA, 20ug/ml pol;~(A), 40mM Na-phosphate (pH 6.8-7.0) and 0.2% SDS.
Hybridization was done b~~ addition of the probe to the same buffer with overnight incubation. The probe was not be used at more than approximately 5 x 10554 c.p.m./ml.
After hybridization, the nitrocellulose was washed a number of times at 42°C with 2x SSC, 25mM Na-phosphate, 5mM EDTA and 2mM Na-pyrophosphate followed by a final wash for 20 minutes at 64°C in lx SSC. Best results were obtained if the filter was not dried prior to autoradiography and the probe could be removed by extensive washing in 1mM EDTA at 64°C.
Exa_ mple 20 "Western" blots, to detect anitgens after SDS-polyacryamide gel electrophorsis, were done essentially as described by R. P. Legocki and D. P.
S. Derma (1981) Analyt. f3iochem. 111:385-392.
Micro-ELISA (enzyme--linked inmuno-sorbant assay) assays were done using Imnulon-2 type plai:es with 96 wells by the following steps:

~3407~~
20.1 Bindin antibody to lp ates On Day 1; thE~ wells were coated with 1:1000 dilution of antibody (rabbit antiphaseolin IgG) in coating buffer. 200u1/well incubated at 37°C for hours. The plates. were covered with Saran Wrap: Then the plates were rinsed three times with phosphate buffered saline-Tween *(PBS-Tween) allowing a 5 minute waiting period between each rinse step. Then 1% bovine serum albumin (BSA) was added to rinse and, after addition to the well, left to sit for 20 minutes before discarding. Rinsing was repeated five times more with PBS-Tween.
20.2 Tissue homogenization The tissue was sliced up into small pieces and then homogenized with a poly~tron using lgm of tissue/ml phosphate buffered saline-Tween-2% polyvinyl pyrollidone-40 (PEIS-Tween-2% PVP-40). All samples were kept on ice before and after grinding and standard phaseolin curves were obtained. One standard curve was done in tissue homogenates and one standard curve was also done in buffer to check tree recovery of phaseolin when ground in tissue. Following centrifugation of the homogenized samples, 100u1 of each sample were placed in a well and left overnight at 4°C. To avoid errors, duplicates of each sample were done. The plates were sealed during incubation.
20.3 Bindin enmnne After the ovE~rnight incubation, the antigen was discarded and the wells were washed five tames with PBS-Tween allowing 5 minutes between each rinse.
A conjugate (,rabbit anti-phaseolin IgG alkaline phosphatase-linked) was the diluted 1:30011 in PBS-Tween-2% PVP containing 0.2%BSA and 150u1 was added to each well; followed by incubation for 3-6 hours at 37°C. After the incubation, the conjugate was discarded and the wells were rinsed five times with PBS-Tween) allowing five minutes between each rinse as before.
20.4 Assay Irtmediately before running the assay) a 5mg tablet of p-nitrophenyl phosphate (obtained from Sigma and stored frozen in the dark) was added per lOml substrate ancf vorte~;ed until the tablet was dissolved. 200u1 of the room temperature solution was quickly added to each well. The reaction was measured at various times.) e.g. t=0) 10, 20) 40, 60, 90 and 120 minutes, using * Trademarks ~.~ ~~71~
a dynatech micro-elisa reader. When p-nitrophenyl phosphate, which is colorless, was hydrolysed by alkaline phosphatase to inorganic phosphate and p-nitrophenol, the latter compound gave the solution a yellow color, which could be spectrophotometrically read at 410nm. The lower limit of detection was less than O.lng.
Example 21 Triparental matings were generally accomplished as described below; other variations known to those skilled in the art are also acceptable. E. coliK802 (pRK290-based shuttle vector) was mated with E. coli(pRK2013) and an A.
tumefaciens strain resistant to streptomycin. The pRK2013 transferred to the shuttle vector carrying strain and mobilized the shuttle vector for transfer to the Agrobacterium. Growth on a medium containing both streptomycin and the drug to which the shuttle vector is resistant, often either kanamycin or chloramphenicol, resulted in the selection of Agrobacterium cells containing shuttle vector sequences. A mating of these cells with E. coli(pPHlJ1) resulted in the transfer of pPHlJ1 to the Agrobacterium cells. pPHlJ1 and pRK290-based shuttle vectors cannot coexist for long in the same cell. Growth on gentamycin, to which pPHlJ1 carries a resistance gene, resulted in selection of cells having lost the pRK290 sequences. The only cells resistant to streptomycin, gentamycin, and either kanamycin or chloramphenicol are those which have Ti plasmids that have undergone double-homologous recombination with the shuttle vector and now carry the desired construction.

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w ~ ~ a~ ~ s~ ~~ ~ ~ c~ a w ~ a w w ~.340'~14 Plasmid BacteriumMade or Ussd Shorn tn Made Out Of Refxences Comments -) ra n) c. T F'~s: -'- or ynonyms ExamPTs:

pKS4 6.2 18 pBR322) pRZ102 pKS4-KB2.4 7,1 21 pBR322, pKS4) P~

pKS4-KB 5,2 7 pBR322, pKS4, = pKS4-3.OKB, =
pKS4-KB3,0 P7.2 pKS-KBc 9 pKS4-KB, pMC6 pKS-5 11.1 26 p8R325 pKS-6 13.1 34 pBR325 pKSil1 (5.1) 12 pRK290) p403 pKS-169 1,2 pBR322-R) p203 pL-B 10.2 23 pKS4, Ilc pLK-prolA 10.4 25 pKS=prolA) pL-B

pMC6 14,5, 9 20 pBR322/phas.cDNAequivalent to pMC36 pNNNi 2,2 8 pKS-noplV) pKS4-KB

pNNN2 4,3 9 pKS-nopVl pNNN4 4,4 pKS4-KB) pNNN2 pPHIJI 21 used to eliminate shuttle vector) same exclusion group as pRK290, carries gene for resistance to gsntamycin) P.R, Htrsh (1973) Thesis) Univ, E.

Anglia) pPBL134 (10) 23 p8R322/IeccDNA

pRK290 common 30 G. Dttta, et al (1980) Proc, Nat,~cea, Sct, USA

77:7347-7357.

pRK2013 21 used to mobilize the shuttle vector) carries tra genes that mobilize a mod site on pRK290 for -conJugaitonai transfer of pRK290 to A robacterium, D) H, Ftgursk , , Helinski) (1979) Proc, Nat, 4cad, Sct, USA 76:1648-1652, -pRZ102 (6,2> ColEt, Tn5 = p7,2 pTtA66 1, 5, 12 octopine-type plasmld, pTtA6 rtth a natural insertion in tms pTt86 -pTi86 (12) pTiC58 (2) 4) nopaltne-type plasmid pTt15955 common 1) 11 octoptne-type plasmld Table 1 ~3~~7'~1~
Plasn~td BactsrtueMads x llsed Shorn tn Made Out Of References Co~ents.

~fra n, c. T~ Exainp~ s: 'FT'gure: -- or ynonya~s p2 13.2 35 p203 p2f 12,2 31 pKSS) p203- ?pKS-oct,can~203 Bglll p2f-rt,/p3e-ift) 39 = pKS-oct,ca~n203 p2f-rt,/p102-lft,12,5 39 p2f, p102 p2f-rt./p103-lft,12.5 39 p2f) p103 pie 12.3 32 pBR322) p202 p3.8 6,1 22) 16 p8R322, 177,4= pJS3.8 p3,8-cDNA 8.1 22 pcDNA3l) p3.8 p7,2 6.1 13 p8R322) 177.4= pSS7,2, S. M, Sun) et al, (1981) Nature 287:32-41, p202 32 pBR322, pTt15955 p203 1) 2, 11, 12) 13, 31, 35) pBR322, 36, id 42 pT115955 p203-Bglll 12.2 31 p203 p401 5 11 def) p8R322) def pT115955 p403 5 2 def pBR322, pT115955 15955-12A 1.7 pTiA66) pKS-OSI-K83.0 Ilc 10.1 23 pBR325, pPYL134 177.4 (6.1) 15 Charon AG-PVPh177,4) S, M, Sun et 24A/phas. al, (1981) Nature gene 289:3 Ta bl a 1 Pa ge 3 -S~-~~~~1714 ,:..

NRRLB-153715 A.tumefaciens/p15955-12A

NRRLB-15394 E.coli C600/pKS4 NRRLB-1539;2 E.coli HB101/p3.8 NRRLB-15391 E..coli HB101/pcDNA31 ATCC 39181 E..coli HB101/pPUL134 ' ~ _ ,.

Claims (32)

1. A DNA vector comprising T-DNA having a plant structural gene inserted therein under control of a T-DNA
promoter.

2. A DNA vector according to claim 1 wherein the plant structural gene comprises an intron.

3. A DNA vector according to claim 1 wherein the plant structural gene is under control of a promoter selected, from the group of T-DNA genes consisting of tmr, tml, tms, nopaline synthase, octopine synthase, or the 1.6 transcript.

4. A DNA vector according to claim 1 wherein the plant structural gene is modified.

5. A DNA vector according to claim 4 wherein the plant structural gene modification comprises removal of an intron.

6. A DNA vector according to claim 4 wherein the plant structural gene comprises cDNA.

7. A DNA vector according to claim 4 wherein the plant structural gene modification comprises a DNA segment insertion.

8. A DNA vector according to claim 4 wherein the plant structural gene modification comprises a DNA segment deletion.

9. A DNA vector according to claim 1 wherein the T-DNA
is modified.

10. A DNA vector according to claim 9 wherein the T-DNA
modification comprises a mutation in tms.

11. A DNA vector according to claim 9 wherein the T-DNA
modification comprises a mutation in tmr.

12. A DNA vector according to claim 9 wherein the T-DNA
modification comprises a deletion in T-DNA.

13. A DNA vector according to claim 4 wherein the T-DNA
promoter includes part of the coding region of the T-DNA
gene normally controlled by said promoter.

14. A DNA vector according to claim 13 wherein the inserted plant structural gene comprises an intron.

15. A DNA vector according to claim 13 wherein the plant structural gene codes for phaseolin.

16. A DNA vector according to claim 15 wherein the plant structural gene coding for phaseolin is inserted under control of a promoter selected from the group of T-DNA
genes consisting of tmr, tml, tms, nopaline synthase, octopine synthase, or the 1.6 transcript.

17. A DNA vector according to claim 1 selected from the group consisting of pKS4, p3.8, pcDNA31, or pPVL134.

18. A bacterial strain containing and replicating a plasmid comprising T-DNA having a plant structural gene inserted therein under control of a T-DNA promoter.

19. The bacterial strain of claim 18 comprising a TIP
plasmid modified to contain within it said T-DNA having a plant structural gene inserted therein under control of a T-DNA promoter.

20. The bacterial strain of claim 19 comprising Agrobacterium tumefaciens or Actrobacterium rhizogenes.

21. The bacterial strain of claim 20 wherein the TIP
plasmid is p15955-12A.

22. The bacterial strain of claim 20 wherein the TIP
plasmid comprises a modification that inactivates the tms gene.

23. The bacterial strain of claim 20 wherein the TIP
plasmid comprises a modification that inactivates the tmr gene.

24. The bacterial strain of claim 22 wherein the TIP
plasmid is pA66-12A.

25. The bacterial strain of claim 18 comprises a TIP
plasmid and a sub-TIP plasmid, the sub-TIP plasmid having a plant structural gene inserted therein under control of a T-DNA promoter.

26. A bacterial strain according to claim 18 wherein the plasmid comprising T-DNA and having a plant structural gene inserted therein under control of a T-DNA promoter is selected from the group: pKS4, p3.8, pcDNA 31 or pPVL 134.

27. A bacterial strain according to claim 18 selected from the group A. tumefaciensp15955-12A, E. coli c600/pKS4, E. coli HB101/p3.8, E. coli HB101/pcDNA31, or E. coli HB101/pPVL134.

28. A DNA molecule comprising in linear sequence;
(a) a first DNA segment comprising plant genomic DNA;
(b) a second DNA segment comprising T-DNA;
(c) a third DNA segment comprising a plant structural gene and a T-DNA promoter in such position and orientation with respect to each other that said plant structural gene is expressible in a plant cell under the control of said T-DNA promoter;
(d) a fourth DNA segment comprising T-DNA; and (e) a fifth DNA segment comprising plant genomic DNA

29. A DNA molecule according to claim 28 that is a plant chromosome.

30. A DNA molecule according to claim 28 wherein said T-DNA promoter is selected from the group consisting of tmr, tml, tms, nopaline synthase, octopine synthase, and the 1.6 transcript.

31. A DNA molecule according claim 28 wherein said plant structural gene contains at least one intron.

32. A DNA molecule according to claim 28 wherein said plant structural gene comprises cDNA.