Mol Gen Genet (1990) 223:369 378 © Springer-Verlag 1990

Site-directed recombination in the genome of transgenic tobacco Joan Odell 1, Perry Caimi ~, Brian Sauer 2, and Sandra Russell 2 1 Agricultural Biotechnology,Agricultural Products Department and 2 Molecular Biology, Central Research and Development Department, ExperimentalStation, E.I. DuPont de Nemours and Co, Wilmington,Delaware 19880-0402, USA Received April 16, 1990

Summary. The plant genome responds to the bacteriophage Pl-derived loxP-Cre site-specific recombination system. Recombination took place at loxP sites stably integrated in the tobacco genome, indicating that the Cre recombinase protein, expressed by a chimeric gene also stably resident in the genome, was able to enter the nucleus and to locate a specific 34 bp DNA sequence. An excisional recombination event was monitored by the acquisition of kanamycin resistance, which resulted from the loss of a polyadenylation signal sequence that interrupted a chimeric neomycin phosphotransferase II gene. Molecular analysis confirmed that the excision had occurred. Recombination occurred when plants with the integrated loxP construction were stably re-transformed with a chimeric cre gene and when plants with the introduced loxP construction were cross-bred with those carrying the chimeric cre gene. As assayed phenotypically, site-specific recombination could be detected in 50%100% of the plants containing both elements of the system. Kanamycin resistance was detected at 2-3 weeks after re-transformation and in the first leaf of hybrid seedlings. This demonstration of the effectiveness of the loxP-Cre system in plants provides the basis for development of this system for such purposes as directing sitespecific integration and regulation of gene expression. Key words: Site-specific recombination - l o x P - C r e Plant transformation - Sulfonylurea resistance marker

Introduction The plant cell is capable of supporting recombination, in addition to natural meiotic homologous recombination, as documented by studies involving the introduction of various derivatives of the cauliflower mosaic virus genome into plant cells (Howell et al. 1981 ; Lebeurier et al. 1982). The recombination which was observed Offprint requests to: S.H. Russell

involved DNA that was extrachromosomal, as does recombination detected in DNA introduced during transformation procedures. DNA introduced into plant cells by Agrobacterium tumefaciens (Spielmann and Simpson 1986; Odell et al. 1987), direct DNA uptake (Krens et al. 1985; Riggs and Bates 1986), or particle bombardment (Christou et al. 1988) may undergo rearrangements during the transformation process. Homologous recombination between extrachromosomal DNA and plant genomic sequences has been shown to occur at a low frequency (Paszkowski et al. 1988). To determine the accessibility of plant genomic DNA to directed intramolecular recombination events we introduced the elements of a site-specific recombination system from bacteriophage Pl into the plant genome as stable components. The bacteriophage P1 uses the loxP-Cre site-specific recombination system to maintain its unit-sized genome (Austin et al. 1981), and the components of this system have been well characterized (Sternberg and Hamilton 1981). A 34 bp loxP site (locus of crossing over(x), P1) consisting of 13 bp inverted repeats separated by an 8 bp asymmetric spacer region, is recognized by the 38 kDa Cre (causes recombination) recombinase protein. These two elements have been shown to interact to effect recombination in vitro, resulting in excision, inversion, or insertion of DNA depending on the location and orientation of the loxP sites (Abremski etal. 1983; Hoess et al. 1986). If two loxP sites surrounding a fragment of DNA are inverted with respect to each other, the intervening DNA will be inverted by Cre activity. If the two loxP sites are in the same orientation, the intervening DNA will be deleted. If the loxP sites are on separate molecules, one being a small circle, the DNA on that circle will be integrated into the other molecule and will then reside between two directly repeated loxP sites. This integrated DNA will again be resolved into a separate molecule if Cre continues to function. Credependent excision of DNA located between loxP sites within the eukaryotic genome has been reported in Saccharomyces cerevisiae (Sauer 1987) and recently in a mouse cell culture line (Sauer and Henderson 1989).

370 The functions of the loxP-Cre recombination system could potentially be applied to direct several types of events, including the following. A marker or trait gene could be deleted from an organism in a specific tissue or at a particular time in development, depending on the control of cre expression. Gene expression could be regulated by inversion of the promoter with respect to the coding region, or vice versa, also dependent on the timing and localization of cre expression. Integration of D N A could be directed to a site where loxP had been inserted, thereby reducing the variability that is observed between independent transformants in expression of introduced genes. To determine whether it might be possible to make use of the loxP-Cre system for these applications in plants, we first tested the ability of the elements to function as stably integrated elements in the plant genome. We were able to demonstrate that the excision function is highly effective when the loxP and cre elements are brought together by successive double transformation of tobacco cells in tissue culture or by cross-pollination of transgenic loxP- and cre-containing tobacco plants. This is the first report of loxP-Cre function in a multicellular eukaryotic organism.

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were followed (Maniatis et al. 1982) unless otherwise specified. The cre/Hpt-A (hygromycin phosphotransferase) vector (Fig. 1 A) was made by first replacing the chloramphenicol acetyltransferase (CAT) coding region between the 35S promoter and a nopaline synthase (NOS) 3' region in pK35CAT (Lin et al. 1987) with the cre coding region, generating pK35cre. A 1.2 kb XhoISalI fragment containing the cre coding region from pBS7 (Sauer 1987) was ligated into HindIII and BamHI digested pK35CAT, after adding a HindIII linker to its 5' end and partially filling-in the SalI and BamHI ends. Next, a ClaI-SalI fragment containing a chimeric NOS/ P-Hpt-NOS 3' gene and the neomycin phosphotransferase I (NptI) gene from pAGS122 (essentially the same as pAGS120: van den Elzen etal. 1985), was ligated into the SalI site of the pK35cre plasmid following the addition of a SalI linker to the ClaI end. This pBR322based vector was mobilized into A. tumefaciens strain GV3850 (Zambryski et al. 1983) by tri-parental matings (Ditta et al. 1980) and colonies were selected on LB plates containing 100 gg/ml rifampicin and 25 lag/ml kanamycin. Co-integrates were verified by Southern blot analysis. The cre/Hpt-B vector (Fig. 1B) was made by first ligating a HindIII fragment containing the cre coding region, made by adding HindIII linkers to the 1.2 kb XhoI-SalI cre fragment from pBS7, into the XbaI site between the 35S promoter and CaMV 3' region in pDH51 (Pietrzak et al. •986). The HindlII and XbaI ends were partially filled-in for this ligation. Clones with the correct orientation of the cre coding region were identified using the BamHI site located 430 bp from the 5' end of the fragment. The entire chimeric gene was

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dIII; P, PstI; S, SalI; X, XbaI. A cre/Hpt-A plasmid. The boxes represent the chimeric 35S/P-cre-NOS 3' and NOS/P-Hpt-NOS 3' chimeric genes. The arrows represent the transcripts expressed by these chimeric genes. The NptI gene is derived from Tng03. These genes are cloned into the pBR322 vector. B cre/Hpt-B plasmid. The boxes represent the chimeric 35S/P-cre-CaMV 3' and I'/P-HptNOS 3' genes. The T-DNA left and right borders are marked as filled boxes. The slashes indicate unrepresented sequences of the binary vector pJJ2644, which includes a tetracyclineresistance gene (tet). C loxP/NptII/Hra plasmid. The open boxes represent the chimeric NOS/P-NptII-NOS 3' gene that is interrupted between the promoter and coding region by a Rubisco small subunit gene polyadenylation signal sequence, shown as the stippled box, which is surrounded by two loxP sites, represented by arrows. The asterisk marks the polyadenylation site. The sulfonylurea resistant ALS gene called Hra and the streptomycin/spectinomycin resistance marker were also cloned into the pBR322 vector. The orientation of this PstI insert was not determined then isolated as an EcoRI fragment, HindIII linkers were added, and it was ligated into the HindIII site of pJJ2644, a binary vector for Agrobacterium transformation. The vector p J J2644 (kindly provided by J. Jones) carries a chimeric I'/P-Hpt-NOS 3' gene and the HindIII site between T-DNA borders, as well as a tetracycline resistance gene and a broad host range origin of replication. The -/Hpt-B vector is p J J2644 without the chimeric

371

cre gene. These vectors were mobilized into A. tumefaciens strain LBA4404 (Hoekema et al. 1983) by tri-parental matings and colonies were selected on minimal media containing 100 gg/ml rifampicin and 10 gg/ml tetracycline. Agrobacterium clones containing the cre/HptB and -/Hpt-B binary vectors were verified by restriction digests of minipreparation DNA. The loxP/NptII/Hra vector (Fig. 1 C) was made by first ligating a 380 bp BamHI-XbaI fragment containing the polyadenylation signal sequence of a tobacco ribulose bis-phosphate carboxylase/oxygenase (Rubisco) small subunit gene (Mazur and Chui 1985) between the two loxP sites in BamHI-EcoRI digested pBS69 (Sauer and Henderson 1989). The XbaI and EcoRI ends were joined after being filled-in. Next, the XhoI site of the XhoI-HindIII fragment containing the loxP-polyA-loxP region was converted to a HindlII site by linker ligation, and the resulting HindIII fragment was cloned into the HindIII site located between the NOS promoter and NptII coding region of p K N K (Lin et al. 1987). A 6 kb PstI fragment containing the Hra gene, derived from pALS032BV (Lee et al. 1988), and a streptomycin/spectinomycin resistance gene from the R100.1 plasmid (Prentki and Krisch 1984), was filled-in and ligated into the filled SalI site of the loxP/NptII vector. This pBR322based vector was then mobilized into Agrobacterium strain GV3850, colonies were selected on 100 gg/ml rifampicin and 100 gg/ml spectinomycin, and co-integrates were verified by Southern blot analysis. RNA/DNA isolation and analysis. Both R N A and D N A were extracted from the same leaf samples by combining methods for extraction of each as follows. One to five grams of leaf tissue was frozen in liquid nitrogen and ground. Frozen tissue was added to 15 ml of extraction buffer (100mM TRIS-HC1 pH 8.0, 50mM EDTA, 100 mM NaC1, 1% SDS, 200 gg/ml proteinase K) and heated at 65 ° C for 10 min. Five milliters of 5 M potassium acetate was added, and the samples were placed on ice for 20 rain. The samples were centrifuged 25 000 x g for 20 rain and the supernatant solution was poured through cheesecloth into a tube containing 1 ml of 5 M sodium acetate and 10 ml of isopropanol. The tubes were left overnight at - 20 ° C. The R N A / D N A was pelleted by centrifugation at 20 000 g for 15 rain. The pellets were resuspended in 10 ml of water and an equal volume of 4 M lithium chloride was added. The solutions were placed on ice for 1-2 h, then centrifuged for 20 min at 20000 x g. The supernatant solution was collected and an equal volume of isopropanol was added. After an overnight incubation at - 2 0 ° C, the DNA was pelleted and resuspended in TE (10mM TRIS-HC1 pH 8.0, I mM EDTA). The samples were extracted with an equal volume of TRIS-HC1 pH 8.0 buffered phenol and precipitated with ethanol. DNA was digested and the fragments were separated by gel electrophoresis, transferred to Zeta-Probe filters (Bio-Rad Laboratories, Richmond, Calif., USA), and hybridized with nicktranslated probes as described by Maniatis et al. (1982). The lithium chloride pellet was resuspended in one half the original volume of water, an equal volume

of lithium chloride was added, and the mixture was placed on ice for 1 h. The R N A was pelleted by centrifugation, resuspended in water, extracted with buffered phenol, and precipitated with ethanol. R N A was separated by gel electrophoresis in formaldehyde as described by Rave et al. (1979), transferred to Zeta-Probe filters, and hybridized to nick-translated probes (Maniatis et al. 1982).

Plant transformation. Young (10-15 cm) leaves from 5 8 week old Nicotiana tabacum cv. Xanthi plants grown in a growth chamber at 21 ° C day, 18° C night, 14/10 h photoperiod, and with 65%-85 % relative humidity were used as source material for leaf disk transformations. The procedure used was essentially as described by Horsch et al. (1985) with the following modifications. Nurse cultures were omitted and inoculated leaf disks were placed directly on .1NIB medium (MS salts, 0.5 mg/1 thiamine, 0.5 mg/1 pyridoxine, 0.5 rag/1 nicotinic acid, 100 rag/1 i-inositol, 3% sucrose, 0.! rag/1 ~-naphthaleneacetic acid (NAA), i rag/1 6-benzylaminopurine (BAP), 0.8% agar, pH 5.8). After incubation for 3 days at 26 ° C, leaf disks were transferred to .IN1B medium containing 500 rag/1 cefotaxime and either 20 50 ppb chlorsulfuron (loxP/NptII/Hpt transformation) or 20 rag/1 hygromycin (cre/Hpt transformation). Shoots were rooted on M X - medium (.IN1B medium without plant growth regulators) containing 20 ppb chlorsulfuron or 10 rag/1 hygromycin in Magenta vessels (Magenta, Chicago, Ill., USA). Plants were transferred to soil and allowed to grow to maturity in a growth chamber under the conditions described above. Inflorescences were bagged to ensure self-pollination. This same leaf disk transformation method was used for the re-transformation experiments with the following modifications. Leaf disks were obtained from loxP plants (primary transformants) grown on M X - medium in Magenta vessels, inoculated with A. tumefaciens harboring the -/Hpt-B and cre/Hpt-B vectors (Fig. 1), incubated for 3 days, and then selected on 30 mg/1 hygromycin and on 100 rag/1 kanamycin. Leaf disk callus growth assay. Leaf disks cut with a hole punch were placed on B medium (MX- medium with 1 rag/1 NAA and 0.2 mg/1 BAP) supplemented with 50100 rag/1 kanamycin, 3~50mg/1 hygromycin, or 1030 ppb chlorsulfuron in 25 × 100 mm petri dishes. After 3 weeks incubation (16 h light/8 h dark, 26 ° C), leaf disks and associated calli were visually inspected and weighed to determine fresh weight of leaf disk and associated callus growth, a measure of resistance to the antibiotic. Cross-pollinations. Hand pollinations were performed using a slight modification of the procedure of Wernsman and Matzinger (1980). After removing the anthers, the stigma was pollinated with pollen from flowers that were allowed to anthese either on the plant or overnight in a beaker of water. Then, a 4 cm length of cocktail stirrer, one end of which was plugged with modelling clay, was slipped over the stigma and style and held

372 in place by the corolla to prevent other pollen from reaching the stigma. Each flower was tagged. Capsules were allowed to grow to maturity and then harvested.

Seed germination assay. Seed was sterilized for 30 min in a solution of 50% bleach and 0.1% SDS, rinsed thoroughly (3-5 times) in sterile water, allowed to dry, and then sown on M X - medium with either 50-300 ppb chlorsulfuron, 20-50 rag/1 hygromycin, or 50-100 rag/1 kanamycin. After 3-4 weeks, seedlings were scored for resistance (green leaves and well-developed roots) and sensitivity (chlorotic, no true leaves, few to no roots), to the antibiotic.

Results

Design of Cre-lox recombination assay To demonstrate excisional site-specific recombination in plants using a phenotypic marker, three selectable markers were needed: one for selection and monitoring of transgenic plants containing the two loxP sites, a second for selection and monitoring of transgenic plants containing the cre recombinase gene, and a third to select or assay for the recombination event. Selection procedures were already available for two markers; resistance to kanamycin (Herrera-Estrella et al. 1983; Fraley et al. 1983; Horsch et al. 1985) and to hygromycin (van den Elzen et al. 1985; Rothstein et al. 1987). We developed a third selection system using a sulfonylurea herbicide resistant form of the acetolactate synthase (ALS) gene, designated Hra (Lee et al. 1988). The selective agent used was chlorsulfuron, the active ingredient in Glean® herbicide. Various concentrations between 5 and 100 ppb chlorsulfuron were tested in Agrobacterium-mediated leaf disk transformations using vectors with and without the Hra gene. Selection with chlorsulfuron in tobacco was not as unambiguous as with kanamycin. On some leaf disks inoculated with Agrobacterium lacking the Hra gene, small shoots developed at all concentrations tested, and a few large shoots formed on 5 and 10 ppb chlorsulfuron. However, with 20 and 50 ppb chlorsulfuron, we were able to differentiate large, transformed shoots which formed on leaf disks inoculated with Agrobacterium harboring the Hra gene from the small shoots which formed as on the controls. An assay for root formation in medium containing 20 ppb chlorsulfuron was used as a secondary screen of selected shoots to confirm their resistance to chlorsulfuron. Shoots from 23 out of 32 independent putative transformation events (72%) formed roots, which indicates that the majority of the shoots selected expressed the Hra gene. Plant transformation vectors were constructed making use of the three selectable markers as shown in Fig. 1. Two different plasmids containing chimeric ere genes were used during the course of these experiments, each incorporating a chimeric hygromycin phosphotransferase (Hpt) gene as the plant cell selection marker. In the first cre vector that was constructed, cre/Hpt-A, the ere coding region was placed between the highly active 35S

promoter of cauliflower mosaic virus (CaMV) (Odell et al. 1985) and a nopaline synthase gene polyadenylation region (NOS 3') (Fig. 1 A). The Hpt coding region was controlled by a NOS promoter and a NOS 3' sequence (van den Elzen et al. 1985). This vector was used to obtain some hygromycin resistant transformants that could be used for genetic crosses, but the efficiency of transformation was too low to carry out an effective re-transformation experiment. Therefore a second cre vector, cre/Hpt-B, was constructed which conferred superior hygromycin resistance, due to the incorporation of a chimeric Hpt gene which lacked an ATG located upstream of and out of fi'ame with the translation initiator ATG (P. van den Elzen, unpublished result). This Hpt coding region was controlled by a T-DNA I' promoter (Harpster et al. 1988) and a NOS 3' sequence (Fig. 1 B). In this vector, cre expression was controlled by a CaMV 35S promoter and a CaMV 3' sequence. The vector containing the loxP sites, loxP/NptII/ Hra, makes use of the Hra gene as the plant cell selection marker (Fig. 1 C). In this vector we inserted a 1.4 kb fragment that is bounded by two loxP sites, oriented in the same direction, between the NOS promoter and the neomycin phosphotransferase II (NptII) coding region. This fragment includes the polyadenylation signal sequence from the Rubisco small subunit gene (Mazur and Chui 1985). We anticipated that prior to site-specific recombination, no viable transcript containing the NptII sequence would be produced due to the presence of the polyadenylation signal sequence between the NOS promoter and NptII coding region. Following excisional recombination at the loxP sites this sequence would be removed and only a single loxP site, which contains no ATG translation initiation codons, would remain. The NOS promoter could then direct NptII transcription resulting in kanamycin resistance.

Production and analysis of primary transformants Transgenic tobacco plants containing the cre/Hpt-A and loxP/NptII/Hra constructions were generated using Agrobacterium tumefaciens-mediated leaf disk transformation. Callus growth assays on leaf disks from each of the transformants were carried out to verify resistance to the introduced marker gene and sensitivity to kanamycin. Twenty independent transformants containing the loxP/NptII/Hra construction (hereafter called loxP plants) were resistant to chlorsulfuron, and all but one of them retained sensitivity to kanamycin (data not shown). The exceptional transformant produced, by weight, only 12% of the callus produced by the average kanamycin resistant transformant. Over 1500 T2 seeds from primary transformants (T1) were tested on kanamycin in a seed germination assay and none was resistant, except for progeny of the same loxP plant that showed resistance in the callus assay. This transformant was not used in further experiments. The fact that kanamycin resistance was not expressed in any other of the loxP transformants demonstrates that, once the foreign DNA was integrated into the plant genome, the loxP

373

• 2.0 kb - 1.5 kb

1

2

3

4

5

6

7

Fig. 2. Northern blot analysisof cre plants. Lanescontain approximately 10 Ixg of total RNA from plants transformedwith the cre/ Hpt-A construction.The 2.0 kb cre mRNA and 1.5 kb Hpt mRNA bands that hybridizeto the NOS 3' probe, as illustrated in Fig. 1A, are marked site construction was stable and that the polyadenylation signal sequence was effective in blocking expression of the NptII gene. This also verifies that the NptI marker, used to select Agrobacterium carrying the transforming plasmid sequences, does not confer kanamycin resistance on plant cells. Seven independent tobacco plants transformed with the cre/Hpt-A construction (henceforth called cre plants) were tested in the callus growth assay and all were resistant to hygromycin and sensitive to kanamycin, as expected. These plants were assayed for expression of the cre gene by Northern blots. Since in the transforming plasmid both cre and Hpt chimeric genes have a NOS 3' sequence (See Fig. 1 A), a 700 bp BamHI-ClaI fragment containing the complete NOS 3' end, which includes 600 bp of untranslated transcribed sequences, was used as a probe to detect transcripts from both genes. The 2.0 kb band seen in Fig. 2 is of the size expected for the cre mRNA, and the 1.5 kb band corresponds to the Hpt mRNA. The identity of the 2.0 kb band as the cre mRNA was verified using a cre-specific probe (data not shown). The levels of cre and Hpt transcripts varied between independent transformants and also with respect to each other in each plant. However, all but one of the hygromycin resistant plants assayed produced a detectable level of cre transcript. In one plant (lane 5) no Hpt transcript was detected. Since this plant was hygromycin resistant, it probably expresses a level of the Hpt transcript that is below the detection level of this Northern blot. This could also be the case for the cre transcript in the plant in lane 7, but since we required higher levels of cre expression, this plant was not used in further experiments.

Re-transformation ofloxP plants

To test for Cre-mediated site-specific recombination we transformed transgenic loxP plants with the cre/Hpt-B

vector. Leaf disks from two independent loxP transformants called L1 and L2, and a wild-type plant (WT) were inoculated with A. tumefaciens containing the Hpt plasmid with or without the chimeric ere gene (cre/Hpt-B and -/Hpt-B, respectively), and placed on selective medium containing either kanamycin or hygromycin. When inoculated disks were placed on kanamycin, no calli or shoots developed on any WT leaf disks, or on loxP disks transformed with -/Hpt-B. Only explants from loxP plants transformed with the cre/Hpt-B plasmid formed calli and shoots on kanamycin. Thus, both the loxP construction and the introduced cre gene were necessary for growth on kanamycin, suggesting that the polyadenylation signal sequence blocking NptII expression was deleted by an interaction between Cre and the two loxP sites, thereby restoring a functional NptII gene. In fact, there was as much growth on the cre-transformed L1 and L2 leaf disks as we normally observe when using an intact kanamycin resistance marker gene. When inoculated leaf disks were placed on hygromycin, shoots and calli formed on both Wt and loxP leaf disks inoculated with Agrobacterium harboring either plasmid. Shoots taken from these disks were screened to determine the proportion that were kanamycin resistant. Twenty-four representative plants were generated from the -/Hpt-B and cre/Hpt-B re-transformed leaf disks that had been grown on medium containing hygromycin. Leaf disks from these plants were tested for callus growth on hygromycin and on kanamycin. All 24 plants tested formed callus on hygromycin, as expected. Plants from the transformation of wild-type tobacco, with either -/Hpt-B (five plants) or with cre/Hpt-B (four plants), were all kanamycin sensitive. Three L1 and L2 re-transformants from the -/Hpt-B inoculation were also kanamycin sensitive. Kanamycin resistance was detected only in plants derived from the IoxP leaf disks that received cre. Two out of four, and eight out of eight plants from re-transformation of L1 and L2, respectively, with cre/Hpt-B were kanamycin resistant. Callus growth data from representative shoots is shown in Fig. 3 A. Since no kanamycin selection was applied during the generation of these plants, this result indicates that a functional NptII gene has been restored in a high percentage of those transformed cells derived from the L1 and L2 leaf disks that received cre. Leaf tissue from these same plants was collected for Southern blot analyses. Fragments that hybridize to an NptII probe on gel blots of B a m H I digested genomic DNA can be used to distinguish between the intact loxP construction and a post-recombination product. The loss of a B a m H I site between the two loxP sites results in the shift of the detected band from a 2.4 kb fragment in the original construction, to a 5.7 kb fragment following recombination, as illustrated in the diagram in Fig. 3B. Parent plants L1 and L2 show the original 2.4 kb B a m H I fragment (Fig. 3C) indicating that the B a m H I restriction site between the two loxP sites is positioned as expected following insertion into the plant genome. No recombination was detected at the loxP sites in these plants. Plants resulting from re-transformation of La and L2 with the -/Hpt-B vector also show the

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2.4 kb band, demonstrating that the l o x P sites were stable through the re-transformation procedure. However, in kanamycin resistant plants resulting from the re-transformation of L1 and L2 with c r e / H p t - B , the fragment size shifts from 2.4 to 5.7 kb. A kanamycin sensitive plant resulting f r o m c r e / H p t - B re-transformation of a l o x P parent (LI) retained the 2.4 kb hybridizing band (see lane 2, Fig. 3 C). This demonstrates a consistency between the size of the band detected on the Southern blot and the response to kanamycin.

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5.7kb2.4kb1 2 3 4 5 6 7 8 9 1 0 Fig. 3A-C. Evidence for site-specific recombination in loxP plants re-transformed with Agrobacterium tumefaciens harboring the cre/ Hpt-B vector. A Callus induction assay. Each bar represents the total weight of five leaf disks grown on callus induction medium containing kanamycin. Weight includes that of the original leaf disks, which account for weights up to 0.4 g. Disks were taken from hygromycin resistant plants resulting from re-transformation of loxP plants (L1 and L2), or untransformed tobacco plants (WT), with either the cre gene: cre/Hpt-B vector (i.e. LI*C/H or L2*C/ H), or without the cre gene: -/Hpt-B vector (i.e. L I * - / H or L2* --/H). B Map of the lox region of the loxP/NptII/Hra vector. Distances between the BamHI sites and between the loxP sites in the original construction are shown above. Below is a map of the expected configuration following recombination between loxP sites, with the loss of a BamHI site and resulting change in distance between remaining BamHI sites shown. The 2.4 kb and 5.7 kb fragments marked with bold lines are those detected by the probe shown as a checked box. C Southern blot analysis of re-transformed plants. Lanes contain approximately 10 pg of BamHI digested DNA from the plants described in A, in the same order, except that lanes 1 and 6 contain additional samples from the original L1 and L2 plants, respectively. Positions of the 2.4 kb and 5.7 kb bands detected with the NptII probe, as described in B, are marked

The second method utilized to test excisional recombination was to unite the Cre recombinase with the l o x P / N p t I I construction genetically by sexual hybridization of l o x P and cre plants. Initially, primary transformants were used. First, the number of independent genetic loci for the integrated markers was determined by testing seed f r o m primary transformants in a germination assay on either hygromycin or chlorsulfuron. The H p t marker gene segregated as one locus in seven out of eight of the independent cre transformants. In contrast, the H r a marker segregated as three or more loci in over 50% of the l o x P transformants and as one locus in less than one third of the plants tested, possibly due to a more stringent selection which required high expression of the H r a gene. The four l o x P plants used in the crosses between heterozygous parents had segregation ratios that suggest the presence of three or more independent loci. All of the cre plants used had segregation ratios indicating insertion of the cre gene at only one genetic locus. Since both the l o x P and cre parents were heterozygous, the seed produced from hand pollinations of l o x P pollen onto cre plants could carry none, both, or either one of the foreign D N A insertions. To identify only those progeny from cross-pollinations that carried both markers, 100-150 seed from each cross were first screened on chlorsulfuron in a germination assay. Then, shoot cuttings of seedlings resistant to chlorsulfuron were tested for root formation in medium containing hygromycin. Seeds from self-crossed cre and l o x P plants were used as controls at each step. Cre x cre seed produced only bleached seedlings on chlorsulfuron, indicating herbicide sensitivity. None of the l o x P x l o x P seedlings rooted on hygromycin. Selected seedlings that were resistant to both compounds, together with controls, were tested for k a n a m y cin resistance using a callus growth assay. A total of 83 out of 90 seedlings (92%) from eight crosses involving six different cre and four different l o x P parents, were found to be resistant to kanamycin. Table ] shows the number of seedlings that were kanamycin resistant for each cross. In six of these crosses all of the plants tested were kanamycin resistant (53/53). Progeny from two crosses yielded about 80% kanamycin resistant progeny (20/24 and 10/13). All of the 59 seedlings tested from self-crosses of the l o x P and cre plants were sensitive to kanamycin. Southern blot analysis of representative seedlings is

375 Table 1. Numbers of kanamycin resistant progeny from crosses between heterozygous cre and loxP parents Female

Male

Self 0/9 c 0/9 0/7 0/9 0/5 0/5

CI b C2 c3 C4 C5 C6

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0/5

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4/4 20/24

16/16

the region between the l o x P sites was excised, blots were hybridized to a probe of the Rubisco small subunit polyadenylation sequence. Progeny of selfed l o x P plants did show the expected 2.4 kb band, while neither this band nor the 5.7 kb band is detected in kanamycin resistant cre x l o x P progeny (data not shown). Therefore, both the phenotypic and molecular evidence indicate that Cre-mediated site-specific recombination has occurred in these tobacco seedlings, and the phenotypic data suggests that recombination has occurred in 8 0 % 100% of the progeny.

13/13 10/13

a L3 L6 are independent heterozygous loxP transformants used as the pollen parent b C1-C6 are different heterozygous cre transformants used as the female parent c Number of kanamycin resistant progeny/number of hygromycin resistant, chlorsulfuron resistant progeny tested. Resistance was defined as >0.5 g callus growth in at least two of the three leaves tested. Disks were taken from leaves from the distal, middle and proximal portions of the plant to assay for resistance throughout the plant

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Fig. 4. Southern blot analysis of cre x loxP progeny. Lanes contain approximately 10 gg of BamHI digested DNA from progeny of selfed loxP parents, lanes 2 and 4, and from progeny of different crosses as marked above each lane. L3 and L4 are independent loxP/NptII/Hra transformants. C1, C2, C3, and C5 are independent cre/Hpt-A transformants. Positions of the 2.4 kb and 5.7 kb bands detected with the NptII probe, as described in Fig. 3 B, are marked

shown in Fig. 4. Hybridization of the NptII probe to gel blots of B a m H I digested genomic D N A shows the presence o f the 2.4 kb band in chlorsulfuron resistant progeny from self-crossed l o x P plants (L3 x L 3 and L4 x L4), indicating that the integrated l o x P construction is stable through meiosis. Progeny from cre x l o x P crosses that are resistant to kanamycin all exhibit the predicted 5.7 kb B a m H I band following site-specific recombination at the l o x P sites (Fig. 2 B). To confirm that

S e x u a l hybridization o f homozygous loxP and cre plants

Although the analysis of progeny from heterozygous l o x P and cre parents showed that loxP-specific recombination did occur following sexual hybridization, we were interested in assaying very young seedlings to assess the developmental timing of the recombination event. Crosspollination using homozygous parents would ensure the presence of both l o x P and cre D N A insertions in all progeny, thereby making initial selection for the Hra and Hpt markers unnecessary. Individual cre and l o x P primary transformants (T1) were used which had the genes integrated at a single locus, as measured by a 3:1 segregation of the marker gene in a seed germination assay. Resistant T2 seedlings were grown to maturity and selfed. T2 plants that produced 100% resistant progeny for the introduced marker gene were identified as homozygotes. Pollen from one homozygous cre plant, as well as from one W T plant, was used to fertilize three homozygous l o x P plants that were derived from independent primary transformants. To confirm that the seed carried both markers, seeds from l o x P x cre crosses were germinated on chlorsulfuron and on hygromycin. All of the germinated seedlings bore true leaves and roots, indicating 100% resistance to both selections. As expected, the progeny of a l o x P x W T cross were all chlorsulfuron resistant but hygromycin sensitive. To determine whether site-specific recombination resulting in kanamycin resistance occurred during embryo development or during germination, a seed germination assay was carried out. On kanamycin medium, hybrid l o x P x cre seed gave rise to small seedlings which did not produce roots or true leaves indicating their sensitivity to the selection. Therefore, site-specific recombination was not detected at this early stage. To test leaf tissue from young seedlings, hybrid l o x P x cre seeds were germinated and grown on medium without selection. Tissue from the first through third leaves was tested for growth on kanamycin in a callus growth assay at 25 days after imbibition and tissue from the fourth through sixth leaves was tested at 40 days. The total weight of the leaf disks after 3 weeks of callus growth is shown in Fig. 5. All seven l o x P x W T control progeny tested were sensitive to kanamycin. Most of the 28 progeny from the homozygous l o x P x cre crosses had kanamycin resistant tissue. Callus growth from older leaves tended to be greater than that from younger leaves; perhaps the older leaves were more robust or

376

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Discussion

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Fig. 5. Kanamycin resistance in l o x P x cre hybrids from homozygous parents. C7, L7, and L8 are single copy homozygous plants that were derived from C3, L1, and L2 heterozygous plants, respectively. L9 is a homozygous l o x P plant derived from a primary transformant not used in previous experiments. Each bar represents the weight of four leaf disks from an individual offspring, one

from each of the first two leaves and two disks from the third leaf, after incubation on callus induction medium. About 0.2 g is contributed by the original leaf disks. The asterisks mark two progeny that exhibit kanamycinsensitivity

recombination had occurred in a greater percentage of the cells later in development. In two crosses, 100% of the progeny exhibited kanamycin resistance in at least five of six leaves tested (8/8 and 4/4, respectively). Fourteen out of 16 progeny in another cross showed resistance to kanamycin in at least four out of six leaves tested. The two plants which appeared to be kanamycin sensitive produced more callus than did the controls, but did not exhibit the amount of callus growth associated with resistance (Fig. 5). In these two plants, it is possible that recombination occurred during the callus induction assay explaining the relatively small increase in level of growth over the control. Recombination during the assay would not be expected to produce the greater levels of growth seen for most of the tissue assayed. To get an idea of the frequency of kanamycin sensitivity among progeny from a cross between homozygous cre and IoxP parents, the first, second, and third leaves from 94 seedlings from one cross were tested for kanamycin resistance. Seventy-one out of the 94 seedlings were kanamycin resistant in all three leaves tested, indicating that recombination had occurred early in development in 75% of the progeny. However, recombination seems also to have occurred later in development; 90% of the seelings (85/94) exhibited resistance in the third leaf. Less than 8% of the progeny were sensitive in all three leaves. To assess the incidence of spontaneous kanamycin resistance, 14 self-crossed loxP and 13 selfcrossed cre seedlings along with 16 loxP x WT progeny were tested in a callus growth assay; none was resistant (data not shown). This confirms again that the loxP construction is stable through meiosis, even following cross-pollination.

We have shown that the plant cell is receptive to bacteriophage P1 site-specific recombination elements, suggesting that Cre protein can enter the nucleus and interact with loxP sites that are integrated in the plant genome, thereby causing an excisional recombination event. This event was assayed by monitoring the loss of a polyadenylation signal sequence flanked by loxP sites and placed between the NptII coding region and the promoter regulating its expression. The polyadenylation signal was very effective in blocking expression of kanamycin resistance in tobacco transformants containing this interrupted chimeric NptII gene. Only i of the 20 independent loxP primary transformants exhibited any growth in the callus assay and some kanamycin resistant progeny in the seed germination assay. This transformant was not used in any further experiments. No other primary loxP transformants, control loxP retransformants, or progeny of loxP plants showed any kanamycin resistance. Therefore, once integrated, the loxP construction was a stable part of the plant genome during manipulations in tissue culture and throughout the complete plant life cycle. No endogenous recombination system was observed to act at these sites. This is unlike the case in mammalian cells where G418 resistance could sometimes be generated under selection pressure by amplification of the NptII gene or, in one example, by low frequency homologous recombination between the 50 bp DNA segment containing the loxP sites (Sauer and Henderson 1989). Following introduction of a chimeric cre gene into plant cells containing loxP/NptII/Hra, either by retransformation or by cross-breeding, kanamycin resistance could be detected in 50%-100% of the progeny, although the number of plants analyzed was low in some cases. Molecular data confirmed that kanamycin resistance was a true indicator of recombination between loxP sites. Differences in the frequency of kanamycin resistance in both re-transformations and sexual hybridizations were related to specific loxP and cre parents. Varying levels of expression in independent cre transformants would be expected to affect the amount of Cre protein available to interact with loxP sites, thereby affecting the recombination frequency. Since each loxP plant used in these experiments is an independent transformant, and since integration of foreign DNA generally occurs at random (Zambryski et al. 1982; Chyi et al. 1986), each is likely to have the l o x P / N p t l I construction integrated at different sites of the plant genome. Thus the loxP sites in one plant may be integrated at positions that are less accessible to the Cre protein and/or to recombination. Altogether, nine independent loxP transformants were used in our experiments and all underwent recombination, indicating that the plant genome is generally receptive to the loxP-Cre system, at least at sites where T-DNA has integrated. Effects of the position of the loxP site integration were not addressed in the mammalian study cited above, since only one IoxP parent cell line was assayed. Recombined progeny cells were observed at a frequency of 17%

377

after 4 days of induced cre expression. In subsequent experiments, using a more efficient electroporation procedure, a frequency of 30% recombined progeny is routinely observed (unpublished results). The methods of leaf disk re-transformation and sexual hybridization for combining ere with the loxP sites in plant cells do not allow a direct comparison of recombination frequencies between plant and animal cells. In our experiments, the most rapid type of assay was to place loxP leaf disks inoculated with Agrobacterium containing the cre vector directly onto kanamycin selection medium. The first growth of resistant callus and shoots could be observed 2-3 weeks after inoculation, which is the same time period required to observe callus growth under kanamycin selection on tissue from plants receiving a non-interrupted chimeric NptII gene. The fact that no delay in callus growth was observed suggests that the recombination event occurred soon after inoculation. The second assay that we carried out to test the timing of Cre-directed recombination was to germinate seeds, derived from cross-breeding homozygous loxP and cre plants, on kanamycin medium. No seedlings showed resistance in this experiment. This could be due to a lack of 35S/P-ere expression in the embryonic root primordium and young root, the tissue that comes into contact with the selective agent. Although Benfey et al. (1989) reported that the 35S promoter is active throughout the embryo, we have evidence indicating that not all 35S promoters are active in this tissue (unpublished data). The earliest assay of leaf tissue, taken 3 weeks after germination, did result in plants showing kanamycin resistance. In a few plants, the leaves which developed first were kanamycin sensitive or produced very little callus, while developmentally older leaves were resistant. These leaf disks could have been chimeric, containing cells which had undergone recombination as well as cells which had not. However the analysis of D N A from mature leaves showed no unrecombined loxP/ NptII region remaining, indicating that recombination occurred early in development or in the vast majority of the leaf cells. Dicotyledonous plant cells have been shown to be capable of responding to other two-component systems: the Tnl0-encoded tetracycline repressor-operator system (Gatz and Quail 1988), and the maize Activator transposable element system (Baker et al. 1986). The tet repressor protein and operator sequence were shown to function when assayed as non-integraded elements in a transient protoplast assay. The Ac system functioned as an integrated part of the plant genome. In one study (Taylor et al. 1989), differences in Ac transposition frequency were observed in the progeny of independent transformants, perhaps related to integration position as we also suggest for loxP recombination. The two components of the naturally occurring Ac system are linked, with the transposase gene being a part of the transposable element. In a report that appeared after this project was completed (Lassner et al. 1989), the two components of the Ac system were transformed separately into plant genomes, then brought together by cross-breeding heterozygous transgenic plants as we also describe. Progeny

containing both Ac components showed a new shifted DNA band on Southern blots resulting from excision of the transposable element. Unlike our results demonstrating complete disappearance of the DNA band representing the original intact loxP/NptII/Hra construction, a substantial amount of the original DNA band remained in the hybrid Ac progeny. This result indicates that the loxP-Cre recombination system may be more efficient than the Ac transposable element system in tobacco cells. Since this system is highly effective in plants, it has great potential in a number of applications. We are currently testing the ability of Cre to activate gene expression by inverting a coding region from an inactive to an active configuration with respect to its regulating promoter. Turning on expression of a marker gene, such as/~-glucuronidase, by controlling cre in a developmental or tissue-specific fashion would provide the ability to follow cell lineages in the plant. Also we are testing the effectiveness of deletion of the transformation marker gene, which would result in transformed plants that carry only an introduced, non-selectable trait. This type of transformant may be desirable for field releases. Finally we, in common with many researchers in the field, have experienced tremendous variability in gene expression between independent transformants receiving exactly the same construction (Jones et al. 1985; Odell et al. 1987; Landsmann et al. 1988; Broglie et al. 1989; Keller et al. 1989). This variability, which is thought to be due to effects of the surrounding D N A at the integration site, could possibly be reduced if introduced D N A could be directed to an integration site via the loxP-Cre system.

Acknowledgements. We wish to thank R. Quatrano and N. Yadav for their valuable discussions, B. Mazur for the Rubisco small subunit clone and for critically reading the manuscript, I. Potrykus for pDH51, and J. Jones for pJJ2644. We also thank D. Delduco and J. Hoopes for their excellent technical assistance.

References Abremski K, Hoess R, Sternberg N (1983) Studies on the properties of P1 site-specific recombination: Evidence for topologically unlinked products following recombination. Cell 32 : t 301-1311 Austin S, Ziese M, Sternberg N (1981) A novel role for site-specific recombination in maintenance of bacterial replicons. Cell 25 : 729-736 Baker B, Schell J, Lorz H, Fedoroff N (1986) Transposition of the maize controlling element "Activator" in tobacco. Proc Natl Acad Sci USA 83:4844-4848 Benfey PN, Ren L, Chua N-H (1989) The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. EMBO J 8: 2195-2202 Broglie KE, Biddle P, Cressman R, Broglie R (1989) Functional analysis of DNA sequences responsible for ethylene regulation of a bean chitinase gene in transgenic tobacco. Plant Cell 1 : 599-607 Christou P, McCabe DE, Swain WE (1988) Stable transformation of soybean callus by DNA-coated gold particles. Plant Physiol 87:671 674 Chyi Y-S, Jorgensen RA, Goldstein D, Tanksley SD, Loaiza-Fi-

378 gueroa F (1986) Locations and stability of Agrobacterium-mediated T-DNA insertions in the Lyeopersieon genome. Mol Gen Genet 204: 64-69 Ditta G, Stanfield S, Corbin D, Helinski D (1980) Broad host range DNA cloning system for Gram-negative bacteria: Construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347 7351 Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick JS, Adams SP, Bittner ML, Brand LA, Fink CL, Fry JS, Galluppi GR, Goldberg SB, Hoffmann NL, Woo SC (1983) Expression of bacterial genes in plant cells. Proc Natl Acad Sci USA 80:48034807 Gatz C, Quail PH (1988) Tnl0-encoded tet repressor can regulate an operator-containing plant promoter. Proc Natl Acad Sci USA 85:1394-1397 Harpster MH, Townsend JA, Jones JDG, Bedbrook J, Dunsmuir P (1988) Relative strengths of the 35S cauliflower mosaic virus, 1', 2', and nopaline synthase promoters in transformed tobacco sugarbeet and oilseed rape callus tissue. Mol Gen Genet 212:182-190 Herrera-Estrella L, De Block M, Messens E, Hernalsteens J-P, Van Montagu M, Schell J (1983) Chimeric genes as dominant selectable markers in plant cells. EMBO J 2:987-995 Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303 : 179-180 Hoess RH, Wierzbicki A, Abremski K (1986) The role of the loxP spacer region in P1 site-specific recombination. Nucleic Acids Res 14:2287-2300 Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229-1231 Howell SH, Walker LL, Walden RM (1981) Rescue of in vitro generated mutants of cloned cauliflower mosaic virus genomes in infected plants. Nature 293:483-486 Jones JDG, Dunsmuir P, Bedbrook J (1985) High level expression of introduced chimaeric genes in regenerated transformed plants. EMBO J 4: 2411-2418 Keller B, Schmid J, Lamb C (1989) Vascular expression of a bean cell wall glycine-rich protein-/~-glucuronidase gene fusion in transgenic tobacco. EMBO J 8:1309 1314 Krens FA, Mans RMW, van Slogteren TMS, Hoge JHC, Wullems GJ, Schilperoort RA (1985) Structure and expression of DNA transferred to tobacco via transformation of protoplasts with Ti-plasmid DNA: co-transfer of T-DNA and non T-DNA sequences. Plant Mol Biol 5:223-234 Landsmann J, Llewellyn D, Dennis ES, Peacock WJ (1988) Organ regulated expression of the Parasponia andersonii haemoglobin gene in transgenic tobacco plants. Mol Gen Genet 214:68-73 Lassner MW, Palys JM, Yoder JI (1989) Genetic transactivation of Dissociation elements in transgenic tomato plants. Mol Gen Genet 218:25-32 Lebeurier G, Hirth L, Hohn B, Hohn T (1982) In vivo recombination of cauliflower mosaic virus DNA. Proc Nat1 Acad Sci USA 79:2932-2936 Lee KY, Townsend J, Tepperman J, Black M, Chui CF, Mazur B, Dunsmuir P, Bedbrook J (1988) The molecular basis of sulfonylurea herbicide resistance in tobacco. EMBO J 7:12411248 Lin W, Odell JT, Schreiner RM (1987) Soybean protoplast culture and direct gene uptake and expression by cultured soybean protoplasts. Plant Physiol 84:856-861

Note added in proof

Dale and Ow have shown that the loxP-Crc system can mediate inversion, excision, and integration events when transiently expressed in tobacco protoplasts (Gene, in press).

Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Mazur BJ, Chui C-F (1985) Sequence of a genomic DNA clone for the small subunit of ribulose bis-phosphate carboxylaseoxygenase from tobacco. Nucleic Acids Res 13:2373-2386 Odell JT, Nagy F, Chua N-H (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810-812 Odell JT, Nagy F, Chua N-H (1987) Variability in 35S promoter expression between independent transformants. In: Key JL, McIntosh L (eds) Plant Gene Systems and Their Biology. Alan R. Liss, New York, NY, pp 321-330 Paszkowski J, Baur M, Bogucki A, Potrykus I (1988) Gene targeting in plants. EMBO J 7:40214026 Pietrzak M, ShilIito RD, Hohn T, Potrykus I (1986) Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucleic Acids Res 14:585%5868 Prentki P, Krisch HM (1984) In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303-313 Rave N, Crkvenjakov R, Boedtker H (1979) Identification of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels. Nucleic Acids Res 6:3559 3569 Riggs CD, Bates GW (1986) Stable transformation of tobacco by electroporation: Evidence for plasmid concatenation. Proc Nat1 Acad Sci USA 83 : 5602-5606 Rothstein SJ, Lahners KN, Lotstein RJ, Carozzi NB, Jayne SM, Rice DA (1987) Promoter cassettes, antibiotic-resistance genes, and vectors for plant transformation. Gene 53:153-161 Sauer B (1987) Functionai expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol Cell Biol 7:2087-2096 Sauer B, Henderson N (1989) Cre-stimulated recombination at loxP-containing DNA sequences placed into the mammalian genome. Nucleic Acids Res 17:147-161 Spielmann A, Simpson RB (1986) T-DNA structure in transgenic tobacco plants with multiple independent integration sites. Mol Gen Genet 205: 34-41 Sternberg N, Hamilton D (1981) Bacteriophage P1 site-specific recombination I. Recombination between loxP sites. J Mol Biol 150: 467-486 Taylor BH, Finnegan EJ, Dennis ES, Peacock WJ (1989) The maize transposable element Ac excises in progeny of transformed tobacco. Plant Mol Biol 13 : 109-118 van den Elzen PJM, Townsend J, Lee KY, Bedbrook JR (1985) A chimeric hygromycin resistance gene as a selectable marker in plant cells. Ptant Mol Blot 5 : 299-302 Wernsman EA, Matzinger DF (1980) Tobacco. In: Fehr WR, Hadley HH (eds) Hybridization of Crop Plants. American Society of Agronomy, Madison, Wis., pp 657-668 Zambryski P, Depicker A, Kruger K, Goodman HM (1982) Tumor induction by Agrobacterium tumefaeiens: Analysis of the boundaries of T-DNA. J Mol Appl Genet 1 : 361-370 Zambryski P, Joos H, Gentello C, Leemans J, Van Montagu M, Schell J (1983) Ti-plasmid vector for the introduction of DNA into plant cells without altering their normal regeneration capacity. EMBO J 2:2143-2150

Communicated by E. Meyerowitz