Process for the site-directed integration of DNA into the genome of plants

The present invention provides a method for site-directed integration of DNA-sequences into the genome of plants via homologous recombination, by transforming said plants using the DNA-transfer system of Agrobacterium, in which the transforming DNA comprises in its most simple form a region homologous to the target locus, as well as a region which is different from the target locus either next to one or between two T-DNA borders. Special constructs are provided, which in its most complete form have the following general structure, ##STR1## in which box 1 and 7 represent T-DNA borders, boxes 2 and 6 comprise functional expression cassettes containing negative selection genes, box 3 provides a region of homology with the target locus promoting recombination, box 4 represents a DNA sequence containing a mutation with respect to the target locus, box 5 represents a functional expression cassette containing a positive selection gene, and box E comprises a DNA sequence which is homologous to a region adjacent of the target locus, or in the vicinity of the target locus, which promotes homologous recombination.

FIELD OF THE INVENTION 
The invention is in the field of recombinant DNA. More in particular, it is 
related to modified plants, processes for the site-directed modification 
of the genome of plants, and DNA constructs used therein. 
BACKGROUND OF THE INVENTION 
During the recent years, techniques have been developed for the genetic 
manipulation of plant cells and the regeneration of these plant cells into 
transgenic plants. On the one hand, direct DNA transformation of plant 
protoplasts may be used for the introduction of the desired DNA into plant 
cells. For this purpose, several methods are available, e.g. Ca/PEG (Krens 
et al., 1982; Negrutiu et al, 1987), electroporation and microinjection 
(Crossway et al., 1986). Using the recently developed microprojectile 
method (Klein et al, 1987) also intact plant tissues may be transformed 
with `naked` DNA. On the other hand, the desired DNA may be introduced 
into the plant cell using the natural DNA transfer system of Agrobacterium 
tumefaciens and Agrobacterium rhizogenes bacteria (for review, see Klee et 
al., 1987). 
Agrobacterium tumefaciens and Agrobacterium rhizogenes, after attachment to 
the plant cell wall, are capable of transferring a piece of DNA to the 
plant cell. Such a piece, the transfer-DNA (T-DNA), is as T-region part of 
a large plasmid (190-240 kbp) in the bacterium, which is called the 
Ti-plasmid in the case of A. tumefaciens and Ri-plasmid in the case of A. 
rhizogenes. The T-DNA becomes integrated into the nuclear genome of the 
plant cell (Tomashow et al., 1980; Chilton et al., 1982). Genes residing 
in the T-DNA are expressed in the plant cell and cause the latter to 
behave as a tumor cell (Ooms et al., 1981; Willmitzer et al., 1982a+b). 
In addition to the genes that are responsible for tumor induction also 
genes are present on the T-DNA which take care of the production of 
so-called opines. Opines, like octopine and nopaline, may serve as energy, 
nitrogen and/or carbon source to Agrobacterium. The enzymes that are 
needed for the catabolism of these opines are encoded by genes that reside 
on the Ti- (Ri-) plasmids (e.g. Bomhoff et al., 1976; Kerr and Roberts, 
1976; Hooykaas et al., 1977). Depending on the opine production, the Ti- 
and Ri-plasmids are classified into groups (for example octopine or 
nopaline plasmids). 
The T-region is confined by two imperfect direct repeats of 25 base pairs, 
also called `borders` (Yadav et al., 1982; Zambryski et al., 1982; Gielen 
et al., 1984; Slightom et al., 1985). The presence of these borders in cis 
is a prerequisite for correct transfer of T-DNA (Wang et al., 1984; 
Peralta and Ream, 1985). 
The presence of the right border is necessary for the efficient T-DNA 
transfer (Ooms et al., 1982; Shaw et al., 1984b; Wang et al., 1984). 
Depending on the test system it was found that deletion of the left border 
in some experiments does (Bakkeren et al., 1989) whereas in other 
experiments does not lead to a lower frequency of T-DNA transfer to the 
plant cell (Hille et al., 1983a; Joos et al., 1983). Next to the right 
border a sequence is present that significantly increases the efficiency 
of T-DNA transfer (Peralta et al., 1986; Van Haaren et al., 1986, 1987; 
Wang et al., 1987). The action of this `enhancer` element is independent 
on position or orientation with respect to the right border (Van Haaren et 
al, 1986). From experiments with synthetic borders it appeared that the 
right and left border sequences are interchangeable and, consequently, the 
`enhancer` determines which border sequence becomes the dominant right 
border (Peralta et al., 1986; Van Haaren et al., 1987). 
In addition to the T-DNA, there are virulence genes that on the one hand 
reside on the chromosome, on the other hand on the Ti-plasmid 
(Vir-region). These genes are involved in attachment of the bacterium to 
the plant cell and in the transfer process of the T-DNA to the plant cell 
(for review see Melchers and Hooykaas, 1987) . 
All the gene transfer systems mentioned above have in common the 
disadvantage that the site of integration of the transforming DNA is 
unpredictable. Thus, as with the other plant transformation techniques 
mentioned above, the DNA that is introduced into the plant cell via 
Agrobacterium appears to become integrated at random locations in the 
genome (Chyi et al., 1986; Wallroth et al., 1986; Spielman and Simpson, 
1986). In certain situations, however, it is desirable or even necessary 
to determine the site of integration beforehand. Thus, the gene to be 
introduced might be targeted to a location where the desired regulation of 
expression is guaranteed. Also the newly introduced DNA could be used to 
mutate or inactivate a specific plant gene. Several methods have been 
described to integrate DNA sequences into the plant genome in a 
site-specific manner. These methods are all based on a mechanism known as 
homologous recombination. 
Homologous recombination is a process that occurs very efficiently within 
bacteria and yeasts. In these organisms it is used for site-directed 
integration of newly introduced DNA (Ruvkun and Ausubel, 1981; Orr-Weaver 
et al., 1981). In yeast it was found that DNA molecules, linearized in the 
area of homology with DNA integrated into the genome, recombine with a 
10-1000 fold higher frequency. More recently, also in mammalian cells 
homologous recombination between genomic and newly introduced DNA was 
found to occur (Smithies et al., 1985; Thomas and Capecchi, 1987; Song et 
al., 1987; Baker et al., 1988; for recent review see Capecchi, 1989). Also 
in these systems it appeared that upon co-transformation of two defective 
mutants linearisation of one of the mutants in the region of homology 
resulted in--on an average--a 10-fold higher recombination frequency 
(Kucherlapati et al., 1984). 
Recombination between two homologous DNA molecules, after their 
simultaneous introduction into a plant cell, has been reported by Wirtz et 
al., 1987. European patent application (EP-A-0 317 509) discloses a method 
for the integration of DNA sequences into the genome of plants through 
homologous recombination. According to the application the introduction of 
the DNA construct into the plant host may occur by known techniques, such 
as the Agrobacterium transfer system. In the Examples, a direct DNA 
transformation method (with "naked" DNA) was actually used to introduce 
the incoming DNA into polyethyleneglycol (PEG) treated tobacco 
protoplasts. 
It was stated that modifications on exactly defined locations in the plant 
genome could be obtained. However, the results of the experiments, using 
different defective APHII genes, conferring kanamycin resistance, were not 
conclusive as to whether restoration of the gene occurred on the desired 
locus ("in situ"). In a later published article on the same experiments by 
one of the inventors, Paszkowski et al. (1988), it was only assumed that 
restoration of the defective APHII gene, due to homologous recombination 
with the incoming defective;APHII gene, could have occurred on locus, "but 
further evidence to confirm this was required". 
There is still a need for an efficient method for in situ modification of 
the plant genome and selection of the desired mutants. 
SUMMARY OF THE INVENTION 
The present invention provides recombinant T-DNA constructs which are 
useful for the integration of defined mutations in desired locations of 
the plant genome. The invention further provides DNA-constructs that 
enable selection of those plants that contain the defined mutations on the 
desired genomic location. These constructs are especially useful if the 
integrated mutation is phenotypically difficult to detect. The invention 
also provides methods to integrate DNA sequences containing defined 
mutations on desired locations of the plant genome, by introducing these 
T-DNA constructs to the plant cell using the DNA-transfer system of 
Agrobacterium tumefaciens, or species related thereto. Also vectors are 
provided, containing said recombinant T-DNA constructs, as well as 
bacteria transformed therewith. In a further aspect of the invention 
genetically modified plants are provided, carrying defined mutations in 
desired locations of their genome, obtained by application of said 
recombinant DNA construct.

The following Examples only serve to illustrate the invention and do not 
mean to limit the scope of its applications. 
EXAMPLE 1 
Transformation of tobacco protoplasts by cocultivation with Agrobacterium 
tumefaciens 
Cocultivation is the plant cell transformation method in which plant 
protoplasts and Agrobacterium are incubated together and where during 
subsequent regeneration from protoplast to callus selection takes place on 
the transfer of T-DNA (Marton et al., 1979; Fraley et al., 1984). 
For the experiments described below the following protocol for 
cocultivation of tobacco protoplasts with A. tumefaciens was used. 
Nicotiana tabacum cv. petit havana SR1 plants were axenically grown in 
Magenta boxes, filled with 50-60 ml Daichin-agar (0.6%) solidified 
MS30-medium (Murashige and Skoog, 1962; contains 30 g sucrose/l). Every 
5-8 weeks apical meristems of the plants were transferred to fresh medium. 
Protoplasts were prepared from leaves of 5-8 week old axenically grown 
tobacco plants by overnight incubation at 26.degree. C. in K3 0.4M sucrose 
medium (Nagy and Maliga, 1976), 1% cellulase R10, 0.1% Macerozyme R10 and 
0.1% MES. The protoplasts were washed one time in K3 sucrose medium, 
diluted to 1.times.10.sup.5 cells/ml in K3 medium containing 0.4M glucose 
(K.sub.3 G) and distributed in batches of 7 ml in 9 cm petridishes. They 
were incubated overnight in the dark prior to cocultivation with the 
bacteria. Agrobacterium strains were grown at 29.degree. C. in LB medium 
containing 20 mg/l rifampicin and 50 mg/l kanamycin. End log phase 
cultures were diluted in K.sub.3 G medium and the bacteria were added to 
the protoplasts at a ratio of approximately 100 bacteria per protoplast. 
After three days of cocultivation the protoplasts were embedded in agarose 
discs by mixing 5 ml protoplasts with 5 ml 0.8% low melting-type agarose 
(Sigma) in SII medium (Muller et al., 1983) containing 0.1M sucrose and 
0.2M mannitol. The bacterial growth was stopped by the addition of 
cefotaxim and vancomycin to final concentrations of 200 mg/l and 100 mg/1, 
respectively. After 10 days 15 ml SII medium containing either 50 mg/l 
kanamycin or 10 mg/l hygromycin was added to the discs. Seven days later 
15 ml SII medium with kanamycin at 100 mg/l and hygromycin at 20 mg/l was 
added. From this moment on the medium was refreshed weekly by replacing 15 
ml old medium with 15 ml fresh SIII medium (100 -150 mg/l kanamycin, 20-30 
mg/l hygromycin). This medium is identical to the SII-medium except for 
the mannitol concentration which is 0.1M instead of 0.2M. The plating 
efficiency was determined by incubation of 1/8 part of an agarose disc on 
liquid medium without selection. The hormone regime in the K3 and SII 
media was 1 mg/l NAA, 0.2 mg/l BAP and 0.1 mg/l 2.4D. In the SIII medium 
2.4D was omitted. Cefotaxim and vancomycin were added to final 
concentrations of 200 mg/l and 100 mg/l, respectively. 
Microcalli were harvested from selective or non selective medium 4 to 5 
weeks after embedding the protoplasts and were transferred to MS30 medium 
(Murashige and Skoog, 1962) containing 3% sucrose, 1.0 mg/l NAA and 0.2 
mg/l BAP and solidified with 0.6% agar (Daichin). Shoots were induced on 
solid MS15 medium containing 1.5% sucrose, 1.0 mg/l BAP and 0.1 mg/l NAA. 
Solid medium also contained 100 mg/l cefotaxim and 50 mg/l vancomycin and 
for selection 100 mg/l kanamycin or 20 mg/l hygromycin was added. Shoots 
were tested for Km.sup.r or Hm.sup.r by allowing them to root on 
MS15-medium to which 20mg/l hygro. or 100mg/l kana. was added. 
EXAMPLE 2 
Construction of defective NPTII genes 
Construction of the vectors containing different defective NPTII genes 
started from the binary vector pMOGEN24 (see FIG. 1). This plasmid was 
derived from the vector pROK1 (Baulcome et al., 1986) and contains between 
the borders of the nopaline Ti- plasmid pTiT37 the genes, functional in 
plants, for kanamycin resistance (Km.sup.r) and hygromycin resistance 
(Hm.sup.r) in opposite orientation. The vector pMOGEN24 is obtained 
through standard recombinant DNA techniques (Maniatis et al., 1982) from 
pROK1 by cloning the coding region of the E. coli hygromycin 
phosphotransferase (Hpt) gene (Gritz et al., 1983) as a BamHI-fragment in 
the BamHI restriction site in pROK1. Consequently, the coding sequence of 
the HPT gene, from 19 basepairs in front of the translation initiation 
codon up to 20 basepairs behind the translationstop codon becomes located 
between the 35S CamV promoter and the transcription terminator of the 
nopaline synthase gene. 
Just in front of the actual translation startcodon (ATG) another ATG codon 
is present. Because this first codon might disturb translation from the 
actual codon the sequence of this codon was changed into ATA via 
oligonucleotide mutagenesis, a standard recombinant DNA technique. 
Accordingly, the BamHI sites on both sides of the HPT fragment was deleted 
by filling in using Klenow polymerase (Maniatis et al., 1982). 
The BglII/HindIII fragment from pMOGEN24 with on it the right border and 
the Km.sup.r gene was transferred to the plasmid pUC12 (Messing, 1983; see 
FIG. 1) after it had been digested with BamHI and HindIII (Maniatis et 
al., 1982). Subsequently, the transcription termination signal of the 
nopaline synthase gene was replaced by the termintion signal of the 
octopine synthase gene. This resulted in the plasmid pSDM2 (FIG. 1) on 
which are located succesively: 1) the right border of pTiT37 (RB), 2) the 
promoter region of the nopaline synthase gene up to the base in front of 
the ATG start codon (5'NOS, Bevan et al., 1983), 3) the coding region of 
the NPTII gene derived from Tn5 from the SauIIIa site .+-.10 basepairs 
(bp) in front of the ATG start codon till the PstI site that is located 
.+-.370 bp behind the TGA stop codon (Beck et al., 1982) and 4) a 700 bp 
PvuII fragment that contains the transcription termination signal of the 
octopine synthase gene (3' OCS, Gielen et al., 1984). 
To simplify reintroduction of the Km.sup.r gene of pSDM2 or defective 
mutants derived therefrom into the binary vector, a XhoI-linker was 
introduced at the EcoRI-site (FIG. 1). This results in plasmid pSDM4. A 
XhoI-site was introduced in the binary vector pMOGEN24 as well, by 
replacing a SphI fragment with a XhoI-linker. SphI cuts in pMOGEN24 just 
before the right border and within the coding region of the Km.sup.r gene. 
Thus, plasmid pSDM5 was obtained (FIG. 3). 
Finally, to be able to isolate the T-DNA constructs integrated in the plant 
genome by recombination via a Lambda vector library (Maniatis et al., 
1982) , the so-called supF gene located on a EcoRI fragment derived from 
plasmid .pi.VX (Seed, 1983) was cloned next to the Km.sup.r gene. Using 
the so-called amber/suppressor system, also used and described by Smithies 
et al., (1985), the Lambda phage library can be enriched for the fragments 
that contain this supF gene. The EcoRI restriction sites of the supF 
fragment were filled in with Klenow polymerase and ligation of 
BamHI-linkers to the fragment was followed by digestion with BamHI. The 
BamHI fragment was ligated into the BamHI site of pSDM4, resulting in 
plasmid pSDM7 (FIG. 1). 
The defective Km.sup.r genes were derived from pSDM7 (see FIG. 2). As an 
illustration the construction of one of the defective genes, namely the 5' 
II construct, is extensively described below. The construction of other 
defective Km.sup.r genes is roughly indicated in FIG. 2. For the 
construction of 5' II, pSDM7 was cut with restriction enzymes TthIII.1 and 
XmaIII, the ends were made blunt by filling in with Klenow polymerase and 
on the site of deletion an EcoRI-linker (10 bp) was inserted. Due to this 
modification a sequence that codes for an active region of the NPTII 
enzyme was deleted (Beck et al., 1982). The sequence at the site of the 
mutation was checked using the dideoxy-sequencing method (Sanger et al., 
1977). Like the other defective Km.sup.r genes 5' II was cloned as a 
XhoI/HindIII fragment into the vector pSDM5 that had been cut beforehand 
with XhoI and HindIII. The resulting plasmid was called pSDM102 (FIG. 3, 4 
and 8). The 3' mutants used in the experiment described in Example 3 are 
depicted in FIG. 5. Starting from the intact Km.sup.r gene located on 
plasmid pSDM4, two types of constructs were made. One type was obtained by 
introducing a 75 bp HindIII/BamHI fragment containing the synthetic 
octopine left border sequence (FIG. 6) behind the intact Km.sup.r gene on 
pSDM4. This resulted in plasmid pSDM8. Through deletion of a EcoRV/BamHI- 
or a EcoRV/RsrII-fragment respectively the plasmids pSDM8* and 3' IIa were 
obtained. The defective gene (3' IIa) lacks part of the coding region of 
the NPTII gene and the transcription termination signal. For the other 
construct type a HindIII/BglII fragment, containing the wildtype octopine 
left border sequence derived from the plasmid pRAL3912 (Hoekema et al., 
1985) was transferred to pSDM4 that had been digested with HindIII and 
BamI. From the obtained plasmid pSDM9 the AccI/RsrII fragment on which 
part of the coding region of the NPTII gene, the transcription termination 
signal (3'OCS) and a part of the HindIII/BglII fragment from pRAL3912 is 
located, is deleted. This results in 3' IIb. An intact Km.sup.r gene with 
the same border fragment behind the 3'OCS was obtained by deleting the 
AccI/BstEII fragment (pSDM9*). The restriction site for BstEII is located 
behind the transcription termination signal at the end of the 3'OCS part. 
The constructs with an intact or defective Km.sup.r gene between the right 
and left border (respectively pSDM8*, pSDM9*, 3' IIa and 3' IIb) were 
transferred as EcoRI/HindIII fragment to plasmid pLM997 (see FIG. 7), that 
was cut beforehand with EcoRI and HindIII. This resulted in the binary 
vectors pSDM200, pSDM210, pSDM201 and pSDM211, respectively Binary 
plasmids were mobilized by a triparental mating (Ditta et al., 1980) to a 
rifampicin resistant (rif.sup.r) Agrobacterium strain, that already 
contained a helper Ti-plasmid without T-DNA (e.g. Hoekema et al., 1983; 
Deblaere et al., 1985). Conjugants were selected on LB agar medium 
(Maniatis et al., 1989) containing 20 mg/l rifampicin and 50 mg/l 
kanamycin. Agrobacterium strains were named after the binary plasmid they 
contain. 
EXAMPLE 3 
Homologous recombination in tobacco protoplasts between two simultaneously 
introduced T-DNA's 
The possibility of homologous recombination between two T-DNAs in a plant 
cell was tested by transforming tobacco protoplasts with two T-DNAs, by 
simultaneously cocultivating the tobacco protoplasts with two different 
Agrobacterium strains. Both strains were derived from the same 
non-oncogenic helper strain, but harbour a different binary vector. The 
T-DNA of each binary vector contained a different defective NPTII gene, 
one with a deletion in the 5'part of the coding region of the gene 
(pSDM102, FIG. 4 and 8) and the other with a deletion in the 3' part of 
the gene (pSDM201, FIG. 5 and 8). In example 2 the construction of the 
different defective NPTII genes is described in extenso. 
Tobacco protoplasts were cocultivated according to the procedure described 
in example 1, with the following strains: 1) 1,5.times.10.sup.6 
protoplasts with SDM102 alone as a negative control, 2) 1,5.times.10.sup.6 
protoplasts with SDM201 alone or SDM211 alone as a negative control, and 
3) 1,5.times.10.sup.6 protoplasts with both SDM102 and SDM201 or both 
SDM102 and SDM211. 
(Co-)transformation frequencies of the various constructs were determined 
in a smaller experiment. Here, 5.times.10.sup.5 protoplasts were 
cocultivated with both SDM102 and SDM200 or both SDM102 and SDM210. For 
strain SDM102 the transformation frequency was determined by using the 
hygromycin resistance gene present on the T-DNA of plasmid pSDM102. 
Strains SDM200 and SDM210 were used to estimate the frequency with which 
the T-DNA of respectively strains SDM201 and SDM211 was transferred to 
tobacco cells. The T-DNA of the binary vector of strain SDM200/SDM210 is 
similar to that of SDM201/SDM211 except that it contains an intact NPTII 
gene construct between right- and left T-DNA border instead of the 3' 
deleted gene. Approximately 5-7% of the protoplasts regenerated to callus. 
Of these surviving calli .+-.20% appeared to be transformed with the 102 
construct, whereas for both the construct 200 (=201) and the construct 210 
(=211) transformation percentages were observed of .+-.15%. Of the calli 
that had already been transformed with one construct (Hm.sup.r or 
Km.sup.r) approximately 30% appears to be transformed with the other 
construct (Hm.sup.r or Km.sup.r). In the cocultivation experiment where 
protoplasts were co-transformed with a 5' -construct (102) and a 3+ 
-construct (201 or 211) restoration was found in 1-4% of the 
co-transformed calli. In the negative controls only one Km.sup.r calli was 
obtained. This callus did not contain a repaired Km.sup.r gene and progeny 
of this callus did no longer show kanamycine resistance. A clear 
difference in transformation frequency between the 201 construct (3' with 
synth. LB) or the 211 construct (3' with wildtype LB) was not observed. 
The obtained Km.sup.r calli were regenerated into plants as described in 
example 1. In leaf extracts from these plants NPTII activity could be 
detected, using non-denaturing gels (Platt and Yang, 1987), at the correct 
position in the gel. The plants were also analysed on the DNA level. 
Accordingly, proof for the presence of a restored Km.sup.r could be 
provided. 
Theoretically, the NPTII gene could have been restored via homologous 
recombination in the bacterial background. This could only be possible if 
transfer of the binary vectors between the two bacterial strains should 
occur. Crossing experiments described in the following example (4) 
excluded this possibility. 
EXAMPLE 4 
The control crossing-experiment 
To test whether transfer of binary vectors occured between Agrobacterium 
strains, a donor- and a recipient-strain were coincubated for 3 days at 
28.degree. C. A total of 10.sup.9 bacteria of each strain was mixed and 
spotted on a nitrocellulose filter lying on either solid LB medium or on a 
layer of tobacco suspension cells that had been plated on solid MS30 
medium containing 0.5 mg/l of the plant hormone 2,4D. In addition similar 
coincubations were performed in the presence of E. coli helper strain 
RK2013 which is used in triparental matings (Ditta et al., 1980). The 
donor strain SDM201 is rif.sup.r and contains the binary vector pSDM201 
that carries a bacterial gene for kanamycin resistance (Km.sup.r). The 
recipient strain LBA285 is a spontaneous spectinomycin resistant 
(spc.sup.r) derivative of strain LBA202 and does not contain any plasmid. 
LBA285 behaves like a wildtype recipient for Ti-plasmids in conjugation 
experiments. (Hooykaas et al., 1980). If transfer of the binary vector 
pSDM201 should occur from SDM201 to LBA285, spc.sup.r Km.sup.r colonies 
would be found on selective plates. The bacteria were plated on LB medium 
containing 250 mg/l spectinomycin and 50 mg/l kanamycin after 
coincubation. Resistant colonies were found at a low frequency 
(0.8.times.10.sup.-8). These were not genuine transconjugants, because 
they were all rif.sup.r. Indeed, incubation of strain SDM201 alone gave 
rise to spc.sup.r rif.sup.r Km.sup.r colonies at a comparable frequency. 
From this we concluded that these colonies represent spontaneous spc.sup.r 
derivatives of strain SDM201. Transfer of the binary vector did occur when 
the donor and recipient strain were coincubated together with E. coli 
helper strain RK2013 (Ditta et al., 1980). This confirmed that genes 
essential for efficient transfer of binary vectors are not present in the 
strains used in our transformation experiments but have to be provided in 
trans to obtain conjugation. When E. coli strain RK 2013 was provided as 
helper, the frequency of transfer after coincubation on MS medium in the 
presence of plant cells (8.times.10.sup.-7 /recipient) was even lower than 
when coincubation was performed on bacterial (LB) medium 
(1.times.10.sup.-3 /recipient). 
Consequently, we can conclude that homologous recombination between the 
T-DNAs had indeed taken place after co-introduction in the plant cell. 
EXAMPLE 5 
Southern blot analysis of plants derived from Km.sup.r -calli 
Plant DNA was isolated from not fully expanded leaves of plants in the 
growth room as described (Mettler, 1987) and purified on a CsCl-gradient. 
The concentration of the obtained DNA suspension was determined by 
measuring the OD.sub.260. Approximately 10 .mu.g of genomic DNA was used 
for digestion with restriction enzymes. Following separation on a 0.7% 
agarose TBE gel (Maniatis et al., 1989) the DNA was transferred to a 
Hybond N membrane (Amersham; Cat. No. RPN.303N ) by capillary blotting and 
the membrane was (pre-)hybridized according to the Hybond N protocol. 
Final washing was performed in 0.3.times. SSC, 0.1% SDS at 65.degree. C. 
DNA probes labelled with .alpha..sup.32 P!-dCTP (specific activity: 
0.5-1.times.10.sup.9 dpm/.mu.g DNA) were obtained using the mixed primer 
method (Boehringer Mannheim kit; Cat. No. 10044 760). The chromosomal DNA 
isolated from plants regerated from kanamycin resistant calli was 
analysedaccording to the method described above. 
Chromosomal DNA isolated from 2 transgenic plants each containing a single 
copy of the 102 construct, from a plant containing the 100 construct and 
from a non-transformed N. tabacum cv. petit havana SR1 were used for 
reconstruction. 
In FIG. 8 is depicted which internal bands are to be expected after 
digestion with EcoRI and BclI. The 5'deletion construct (102) gives an 
internal band of 1.6 kilobasepairs (Kb), while the intact (correctly 
repaired) Km.sup.r gene should give a band of 2.1 Kb. Digestion with 
HindIII only generates so-called junction fragments. When the probe only 
comprised the 3'part of the Km.sup.r gene (e.g. the 0.7 Kb PvuII fragment 
with the 3'OCS, Gielen et al., 1983) integrated copies of the 3'deletion 
mutant were not observed on the blot, which simplified the interpretation. 
The expected 2.1 Kb fragment, corresponding with a repaired NPTII gene, was 
present in almost all Km.sup.r plants tested (FIG. 8). In case the 2.1 Kb 
fragment could not be detected the presence of a repaired gene could be 
shown by PCR-analysis using primers 2 and 3 (FIG. 8). 
EXAMPLE 6 
Construction of transgenic tobacco plants containing a defective NPTII gene 
as target-locus for homologous recombination 
Transgenic tobacco plants were obtained by co-cultivating leaf discs of 
axenically grown tobacco plants (Horsch et al., 1985) with bacterial 
strain SDM104. After cocultivation, the leaf discs were placed on solid 
MS30-medium with callus inducing hormones (1.0 mg/l NAA and 0.2 mg/l BAP) 
and the antibiotics cefotaxim (200 mg/l) and vancomycin (100 mg/l). After 
one week the leaf discs were transferred to callus-inducing medium with 20 
mg/l hygromycin. Resistant calli were cut and transferred to 
shoot-inducing medium (MS15, 1 mg/l BAP, 0.1 mg/l NAA, 100 mg/l cefotaxim 
and 50 mg/l vancomycin). Shoots were cut and tested for Hm.sup.r by 
allowing them to root on hormoneless MS30-medium with 20 mg/l hygromycin. 
The transgenic plants obtained with the method described above were 
analysed at the DNA level (see example 5). Subsequently, plant lines were 
selected that showed a simple T-DNA integration pattern. Plant line 
104(.1.6) transformed with the 104-construct was used as acceptor plant 
for the targeting experiments. This line appeared to have 2 T-DNA copies 
integrated in inverted orientation at the same position in the plant 
genome (FIG. 9). 
EXAMPLE 7 
Restoration of a defective NPTII gene in a transgenic tobacco plant via 
homologous recombination using Agrobacterium-mediated DNA-transfer 
Protoplasts of transgenic plant 104.1.6 were co-cultivated with 
Agrobacterium strain SDM101. Cocultivations were carried out with 
approximately 2.times.10.sup.7 protoplasts. To determine the 
transformation frequency 1.times.10.sup.6 protoplasts were cocultivated 
with Agrobacterium strain SDM100. The cocultivation experiments were 
carried out according to the procedure described in example 1. In two 
independent experiments, protoplasts of plant line 104 were cocultivated 
with an Agrobacterium strain harbouring the binary vector pSDM101. The 
plasmid pSDM101 contains a NPTII gene with a 5'deletion, next to the 
hygromycin resistance marker (FIG. 9). The transformation experiments 
resulted in 285 and 281 kanamycin resistant calli, respectively. In most 
of these calli gene targeting had not occurred. Results which are not 
shown here suggested that the 5'deleted NPTII gene at the repair T-DNA had 
been fused to an endogenous plant gene. 
In recent articles on homologous recombination in animal cells the 
polymerase chain reaction (PCR) technique is used for quick detection of a 
homologous recombination event (Kim & Smithies, 1988; Zimmer and Gruss, 
1989). Also in our experiments we used the PCR method to screen for 
kanamycin resistant calli in which an intact NPTII gene had been formed 
via homologous recombination (FIG. 9). A PCR with two primers that anneal 
within the regions deleted in either the target NPTII gene or the repair 
construct should result in amplification (of a 979 bp size fragment) only 
if an intact NPTII gene is present. In this way a total number of 213 
calli was screened. Three calli appeared to be PCR-positive and plants 
were regenerated from these calli resulting in plant lines 1, 2 and 3, 
respectively. 
EXAMPLE 8 
Molecular analysis to detect targeting events 
The chromosomal DNA of the plant lines was analysed using the Southern blot 
method described in example 5. DNA was cut with the restriction enzymes 
EcoRI/BclI and HindIII, the fragments were separated on gel and 
subsequently transferred to Hybond-N membrane. Hybridisation was performed 
with an internal NPTII probe (the 610 bp XmaIII/RsrII fragment, see FIG. 
8). 
In plant line 1, one of the 3' deletion mutant copies of the NPTII gene 
present on the target locus had been restored by the incoming T-DNA via 
homologous recombination. Wildtype NPTII activity could be detected in 
leaves of this plant line (Platt and Yang, 1987), thereby confirming the 
presence of an intact NPTII gene. 
EXAMPLE 9 
Construction of the SSU-fusion genes 
In order to isolate an active member of the SSU multigene family a 
subgenomic library of N. tabacum SR1 was constructed in lambda phage PDJII 
(Maniatis et al., 1982). Using a probe specific for a SSU cDNA-clone of N. 
tabacum cv. petit havana SR1, a clone containing a SSU-gene with 3 introns 
(Mazur et al., 1985) was isolated from this library. The active gene is 
located on a 2.4 Kb basepair HindIII fragment. Both the restriction map 
and the DNA-sequence of the clones isolated by us corresponded with the 
published data (Mazur et al., 1985). The HindIII fragment was cloned in 
pIC19H (Marsh et al., 1984) resulting in plasmid pSIC1. This gene was used 
for the construction of chimaeric SSU-NPTII genes (FIG. 10). To do this a 
NPTII insertion module was constructed (clone pSDM53) by: 
a) replacing the EcoRI/XmaIII fragment of pSDM4 (see FIG. 1) containing the 
nopaline synthase promoter and the start of the coding region by a 51 
basepair synthetic EcoRI/XmaIII fragment (see FIG. 11B) on which a unique 
BamHI and a unique BglII restriction site are located. 
b) removing both PstI restriction sites from the NPTII-sequence (Beck et 
al., 1982). The PstI site in the coding region was removed by changing a G 
into an A on position 1733 in the Tn5 sequence (Beck et al., 1982), using 
M13/oligonucleotide mutagenesis (mutagenesis kit from Biorad). 
Consequently, no changes were introduced in the amino acid sequence of the 
NPTII protein. The other PstI site is located outside the coding region 
and was removed by cutting with PstI, blunt-ending with Klenow in the 
presence of dCTP, subsequently cutting with SmaI and closing by ligation. 
Due to this the fragment ranging from base 2519 till base 2656 in the Tn5 
sequence (Beck et al., 1982) of the 3' non-coding region was deleted. 
For the construction of one fusion gene the 1,7 Kb BamHI fragment of the 
promoterless NPTII and the 3' OCS terminator was inserted into a BamHI 
site of the rbcS-gene on pSIC1. The resulting plasmid pNS1 contains a 
translational fusion between the rbcS and the NPTII in the fourth exon of 
the rbsS gene. 
For the construction of the other SSU-NPTII fusion in the second exon, in 
pSIC1 the BamHI site was removed from the fourth exon by filling in with 
Klenow-polymerase This resulted in plasmid pSIC2. Subsequently, in the 
second exon of pSIC2 a new BamHI site was introduced using 
M13/oligonucleotide mutagenesis (mutagenesis kit of Biorad). Due to this a 
G was changed into a T and an A into a C, respectively on positions 1383 
and 1385 in the sequence of Mazur et al., (1984). In the resulting plasmid 
pSIC3, the 1,7 Kb BglII/BamHI fragment from pSDM53 was cloned into the new 
BamHI restriction-site. The clone pNS2 thus obtained contains a 
translational fusion between the rbcS and the NPTII in the second exon of 
the rbcS-gene. 
EXAMPLE 10 
Construction of the binary vector pSDM14 
The plasmid pSDM10 (see FIG. 7) served as a basis for the construction of 
the binary vector pSDM14. Synthetic borders and a fragment containing the 
overdrive sequences were transferred to pSDM10 by the following method. 
The overdrive sequence of pTiAch5 is located on a BclI/SacI fragment 
(14087-14710, Barker et al., 1983). This fragment was cloned in pIC20R 
(Marsh et al., 1984) cut with SacI and BamHI. From pIC20R, the `overdrive` 
was cloned as a SacI/EcoRI fragment to pUC19 (xSacIxEcoRI). The resulting 
plasmid was digested with SacI and KpnI and a synthetic KpnI/SacI fragment 
containing the right T-DNA border was ligated into it. By cutting the 
resulting plasmid with HindIII and KpnI a HindIII/KpnI fragment containing 
the left T-DNA border could be cloned. From this plasmid pBINSB2 the 
EcoRI/HindIII fragment was excised. The ends of the fragment were filled 
in with Klenow-polymerase, BglII-linkers were ligated to it, and following 
digestion with BglII the fragment was ligated in pSDM10. In this way 
pSDM14 was obtained. 
EXAMPLE 11 
Cloning of the rbcS-NPTII fusion genes in pSDM14 
From pNS1 and pNS2 the HindIII fragments with the intact fusion genes and 
the PstI/HindIII fragments with the promoterless rbcS-NPTII fusions were 
cloned into pIC20R (Marsh et al., 1984), resulting in plasmids pNTSS1, 2, 
3, and 4 respectively (see FIG. 10). From there, the fusion genes were 
ligated as SalI/XhoI fragments in the binary vector pSDM14 (FIG. 11) that 
had been cut with SalI/XhoI. 
The resulting plasmids pNTSS11 (A/B), pNTSS12 (A/B), pNTSS21 (A/B), and 
pNTSS22 (A/B) contain between a synthetic right and left border of the 
octopine Ti-plasmid, the intact 4th. exon fusion gene, the promoterless 
4th. exon fusion gene, the intact 2nd. exon fusion gene, and the 
promoterless 2nd exon fusion gene, respectively. A and B refer to the 
orientation of the XhoI/SalI fragment in pIC20R. In FIG. 10 only the B 
orientation is depicted. The binary plasmids were crossed to a 
Agrobacterium helper strain that contains the Vir-region on a helper 
Ti-plasmid (example 2), resulting accordingly in strains NTSS11, NTSS12, 
NTSS21, and NTSS22. 
EXAMPLE 12 
Site-directed mutagenesis of one of the rbcS-genes via cocultivation of the 
protoplasts of Nicotiana tabacum cv. petit havana SR1 with Agrobacterium 
In separate transformation experiments 10.sup.7 tobacco protoplasts were 
co-cultivated with the Agrobacterium strain NTSS12 and with the strain 
NTSS22. These strains respectively contain the promoterless 4th exon 
rbcS-NPTII fusion gene and the promoterless 2nd. exon rbcS-NPTII fusion 
gene between the borders of the T-DNA residing on the binary vector. As 
positive controls the strains NTSS11 and NTSS21 were co-cultivated with 
10.sup.6 tobacco protoplasts. The cocultivation method used is extensively 
described in example 1. Five percent of the protoplasts regenerated to 
callus and from the positive control it appeared that 15% of the calli had 
been transformed. In the cocultivation experiments with NTSS12 and NTSS22 
Km.sup.r calli were obtained. PCR-analysis was performed on chromosomal 
DNA of pooled tissue of 10 calli (for the method see Lassner et al., 
1989). In some calli the transformed T-DNA, which carries the promoterless 
fusion construct had recombined within the target locus, giving rise to a 
functional fusion between the rbcS-promoter and the structural part of the 
NPTII gene. From these calli plants were regenerated and Southern analysis 
was performed on genomic DNA isolated from these plants. In some of these 
plants the rbcS-NPTII-part of the T-DNA was found to be correctly 
integrated at the target locus via homologous recombination. 
EXAMPLE 
An alternative T-DNA construct to achieve gene targeting to the SSU locus. 
In this part an alternative T-DNA construct to target the SSU locus is 
described (FIG. 13). In contrast with the SSU-NPTII fusion contructs this 
new construct does not comprise a translational fusion between the SSU 
gene and the coding region of the NPTII gene but has the NPTII coding 
region under the nopaline synthase promoter inserted in the 4th exon of 
the SSU gene. A small portion of the SSU promoter (.+-.365 bp) is deleted 
to permit screening for recombinants via PCR. In addition the T-DNA aux-2 
gene is introduced into the construct as a negative selection marker to 
enrich for recombinant calli. The aux-2 gene product converts 
.alpha.-naphtalene acetamide (NAM) to the auxin (NAA). At high 
concentrations (10-20 mg/l) NAM is capable to promote growth of tobacco 
cells in auxin-free medium. However, cells that contain the aux-2 gene 
will efficiently convert NAM to the more potent auxin NAA which is toxic 
for plant cells at high concentrations (Depicker et al., 1988). Thus, by 
using auxin-free medium containing NAM at a high concentration it is 
possible to select for cells, that do not express the aux-2 gene. 
A 2.5 Kb HindIII partial of the T-DNA of pTiAch5 (3390 to 5933, Barker, et 
al., 1983; Gielen et al., 1984) which contains the aux-2 gene was cloned 
into the HindIII site of pIC20R (Marsch et al., 1984). Sequences upstream 
of the aux-2 gene comprising the promoter and part of the coding regon of 
the aux-1 gene were deleted. This was done by digestion with PstI (in the 
polylinker of pIC), producing blunt ends by using the exonuclease activity 
of `Klenow`-polymerase, digestion with HincII (5721, Barker et al., 1983; 
Gielen et al., 1984) and re-ligation of the plasmid. In this way the 
promoter sequences of the aux-2 gene were left intact. Subsequently, the 
aux-2 gene was cloned as a SalI/XhoI fragment into the unique SalI site of 
pNTSS11B. In the resulting plasmid pNTSS112 the transcription of the aux-2 
gene is directed towards the left T-DNA border. 
Plasmid pNTSS1 was digested with XhoI followed by a partial digestion with 
BglII and a 3,9 Kb XhoI/BglII fragment was obtained that contained most of 
the SSU/NPTII fusion gene exept for 365 bp which were deleted from the 
5'end of the SSU promoter region. This fragment was cloned into the pUC 
derivative pIC19H (Marsch et al., 1984) cut with XhoI and BamHI. The BamHI 
insertion module containing the NPTII coding region and the OCS terminator 
was replaced by a BglII/BamHI fragment which consisted of the NPTII coding 
region behind the NOS promoterregion without the right T-DNA border 
sequence. The construction of this new insertion module is described 
below. In the resulting plasmid pNTSS5 the transcription of the NPTII gene 
from the NOS promoter is directed towards the 3' end of the SSU gene which 
can serve as terminator of transcription. In this way the OCS terminator 
can be omitted which results in a reduction of the size of the insertion 
module. The SalI/BamHI fragment of pNTSS5 was isolated and ligated into 
the vector part of pNTSS112 cut with SalI and BamHI. Thus, the binary 
vector pNTSS512 was obtained which contained the alternative targeting 
construct (see FIG. 13). 
For the construction of the new NPTII insertion module the BclI/SmaI 
fragment of pSDM4 containing the NOS promoter and the restored NPTII 
coding region was cloned into pIC20H (Marsch et al., 1984) cut with SmaI 
and BglII. From the resulting plasmid the fragment was excised with XhoI 
and BamHI and was recloned in pIC19R (Marsch et al., 1984) cut with XhoI 
and BamHI. Finally, the new NPTII insertion module was introduced as a 
BglII/BamHI fragment into pNTSS5. 
The binary vector pNTSS512 (FIG. 13) was transferred via triparental mating 
to an Agrobacterium strain that contains the Vir-region on a non-oncogenic 
helper Ti-plasmid (example 2). 
EXAMPLE 14 
Transformation with T-DNA construct of plasmid pNTSS512. 
Tobacco protoplasts were co-cultivated with Agrobacterium strain NTSS512. 
To determine the regeneration and transformation frequencies protoplasts 
were regenerated with or without kanamycin as described in example 1. 
Non-transformed tobacco cells were cultivated on NAM containing medium to 
determine the regeneration frequency on this medium. In a larger 
experiment protoplasts were co-cultivated with strain NTSS512 and grown on 
auxin-free medium containing kanamycin and 10-20 mg/l NAM. Integration of 
the T-DNA construct via illegitimate recombination in the genome of the 
plant cells resulted in kanamycin resistant and aux-2.sup.+ cells. Some of 
these cells were able to grow on NAM-medium because the aux-2 gene was not 
expressed due to incomplete integration of the T-DNA or inactivation of 
the gene by methylation. In case the NPTII marker was inserted correctly 
at the target locus via homologous recombination the aux-2 gene was lost. 
The resulting cells were resistant to kanamycin and the absence of the 
aux-2 gene enabled them to grow on auxin-free medium containing a high 
concentration of NAM. 
From the smaller control experiments it was estimated that 5% of the 
initial protoplasts survived (equal for NAA- and NAM- containing medium) 
and 15% of the regenerated calli were resistant to kanamycin. In the large 
targeting experiment the regeneration of protoplasts on NAM-containing 
medium gave a 10 to 100 fold enrichment for cells that did not express the 
aux-2 gene. The PCR technique was used to identify the calli obtained 
through the desired recombination event. Genomic DNA was extracted from 
pooled tissue of 10 calli (Lassner et al., 1989) and tested in a PCR. The 
primers used in the reaction respectively annealed in the NPTII coding 
region and in the upstream region of the SSU promoter that was deleted in 
T-DNA construct pNTSS512. PCR will only result in amplification of a 
fragment of expected size if recombination has occurred between incoming 
T-DNA and target-locus. In fact, in several calli a recombination event 
was detected. Plants were regenerated from these calli as described and 
Southern analysis was performed on these plants and on progeny of these 
plants. In a few plant lines the NPTII module was found to be inserted 
correctly via homologous recombination at the target locus. It was 
estimated that the gene targeting frequency ranged from 10.sup.-4 to 
10.sup.-5. 
Deposition 
For the purpose of enablement the following E. coli strains have been 
deposited at the "Centraal Bureau voor Schimmelcultures" CBS at Baarn, The 
Netherlands: (strain Dh5.alpha.; genotype: F.sup.-, endA1, hsdR17 (r.sup.- 
k m.sup.+ k) supE44, thi-1, lambda.sup.-, recA1, gyrA96, relA1, / 
(argFlaczya)U169, phi80dlacz/ M15). 
______________________________________ 
E. coli strain 
useful for 
DH5.alpha. with plasmid 
(Date of (Deposition 
construct Deposition) 
Number) 
______________________________________ 
pSDM100 July 21 1989 
CBS 348.89 
pSDM4 
pSDM10 
pSDM200/201 
pSDM7 
pSDM101 
pSDMI02 
pSDM104 
pSDM9 July 21st 1989 
CBS 346.89 
pSDM210/211 
pSDM14 July 21st 1989 
CBS 347.89 
pNTSS11 
pNTSS12 
pNTSS21 
pNTSS22 
pNTSS512 
PSIC1 July 21st 1989 
CBS 349.89 
pNTSS11 
pNTSS12 
pNTSS21 
pNTSS22 
pNTSS512 
______________________________________ 
The Agrobacterium strain LBA4404, which is a good acceptor strain for all 
binary plant transformation vectors, has already been deposited earlier 
(Feb. 24th. 1983) and is available via the "Centraal Bureau voor 
Schimmelcultures" (CBS) at Baarn, The Netherlands, under number CBS 
191.83. 
______________________________________ 
Legend 
List of symbols and abbreviations used in the figures. 
______________________________________ 
##STR2## 
##STR3## 
##STR4## 
##STR5## 
______________________________________ 
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