Abstract:
The invention relates to glyphosate tolerant sugar beet plants, plant material and seeds. It is the object of the invention to provide a transgenic sugar beet plant event that shows a high level of tolerance against glyphosate, but is not impaired in other important agronomic properties such as growth, yield, quality, pathogen resistance etc.

Description:
BACKGROUND OF THE INVENTION 
     Field of the Invention 
     The invention relates to glyphosate tolerant sugar beet plants, plant material and seeds. 
     Sugar beet ( Beta vulgaris ) is grown as a commercial crop in many countries, with a combined harvest of over 240 million metric tons. 
     N-phosphonomethyl-glycine, commonly referred to as glyphosate, is a broad spectrum herbicide, which is widely used due to its high efficiency, biodegradability, and low toxicity to animals and humans. Glyphosate inhibits the shikimic acid pathway which leads to the biosynthesis of aromatic compounds including amino acids and vitamins. Specifically, glyphosate inhibits the conversion of phosphoenolpyruvic acid and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the enzyme 5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSP synthase or EPSPS). When conventional plants are treated with glyphosate, the plants cannot produce the aromatic amino acids (e.g. phenylalanine and tyrosine) needed to grow and survive. EPSPS is present in all plants, bacteria, and fungi. It is not present in animals, which do not synthesize their own aromatic amino acids. Because the aromatic amino acid biosynthetic pathway is not present in mammals, birds or aquatic life forms, glyphosate has little if any toxicity for these organisms. The EPSPS enzyme is naturally present in foods derived from plant and microbial sources. 
     Glyphosate is the active ingredient in herbicides such as Roundup®, produced by Monsanto Company, USA. Typically, it is formulated as a water-soluble salt such as an ammonium, alkylamine, alkali metal or trimethylsulfonium salt. One of the most common formulations is the isopropylamine salt of glyphosate, which is the form employed in Roundup® herbicide. 
     It has been shown that glyphosate tolerant plants can be produced by inserting into the genome of the plant the capacity to produce an EPSP synthase which is glyphosate tolerant, e.g. the CP4-EPSPS from Agrobacterium sp. strain CP4. 
     DESCRIPTION OF THE RELATED ART 
     A glyphosate tolerant sugar beet plant may be produced by Agrobacterium mediated transformation, introducing a gene coding for a glyphosate tolerant EPSPS such as CP4-EPSPS into the plant&#39;s genome. Such a sugar beet plant, expressing CP4-EPSPS, has been described in WO 99/23232. However, sugar beet plants grown from cells transformed with the gene for CP4-EPSPS in this manner, differ widely in their characteristics, due to the fact that the gene is inserted at a random position in the plant genome. The insertion of a particular transgene into a specific location on a chromosome is often referred to as an “event”. The term “event” is also used to differentiate genetically engineered crop varieties. Desirable events are very rare. By far the most events are discarded because the transgene inserted into a plant gene important for growth, causing the gene to be disrupted and not expressed, or the transgene inserted into a portion of the chromosome that does not allow for expression of the transgene or expression that is too low. For this reason, it is necessary to screen a large number of events in order to identify an event characterized by sufficient expression of the introduced gene. This procedure is very time and cost consuming, and it is in no way guaranteed that a plant with satisfactory properties may be found. 
     SUMMARY OF THE INVENTION 
     Therefore, it is the object of the present invention to provide a sugar beet plant that shows a high level of tolerance against glyphosate, but has no disadvantages with respect to other important agronomic properties such as growth, yield, quality, pathogen resistance etc. 
     The glyphosate tolerant sugar beet plant according to the invention is characterized in that a) the sugar beet plant is obtained from seed deposited with the NCIMB, Aberdeen (Scotland, U.K.) and having the accession number NCIMB 41158 or NCIMB 41159, and/or b) a DNA fragment of between 630-700 bp, preferably 664 bp, can be amplified from the genomic DNA of said sugar beet plant, parts or seeds thereof, using polymerase chain reaction with a first primer having the nucleotide sequence of SEQ ID NO: 1 and a second primer having the nucleotide sequence of SEQ ID NO: 2, and/or c) a DNA fragment of 3500-3900, preferably 3706 bp, can be amplified from the genomic DNA of said sugar beet plant, parts or seeds thereof, using polymerase chain reaction with a first primer having the nucleotide sequence of SEQ ID NO: 3 and a second primer having the nucleotide sequence of SEQ ID NO: 4, and/or d) a DNA fragment of 270-300 bp, preferably 288 bp, can be amplified from the genomic DNA of said sugar beet plant, parts or seeds thereof, using polymerase chain reaction with a first primer having the nucleotide sequence of SEQ ID NO: 7 and a second primer having the nucleotide sequence of SEQ ID NO: 8, and/or e) a DNA fragment of 710-790 bp, preferably 751 bp, can be amplified from the genomic DNA of said sugar beet plant, parts or seeds thereof, using polymerase chain reaction with a first primer having the nucleotide sequence of SEQ ID NO: 9 and a second primer having the nucleotide sequence of SEQ ID NO: 10, and/or f) a DNA fragment of 990-1100 bp, preferably 1042 bp, can be amplified from the genomic DNA of said sugar beet plant, parts or seeds thereof, using polymerase chain reaction with a first primer having the nucleotide sequence of SEQ ID NO: 14 and a second primer having the nucleotide sequence of SEQ ID NO: 16. 
     Polymerase chain reaction (PCR) is a well-known standard method used to amplify nucleic acid molecules (see for example U.S. Pat. No. 4,683,202). 
     The sugar beet plant according to the invention (in the following referred to as “event H7-1”) shows a high tolerance to glyphosate herbicide. In addition, growth characteristics and other important agronomic properties of event H7-1 are not affected by the transformation process. Event H7-1 expresses a high level of the Agrobacterium-CP4-EPSPS-gene, which is stably incorporated within the plant genome, and which confers glyphosate tolerance to the plant. The plant has been produced by Agrobacterium mediated transformation technology using the binary vector PV-BVGT08. This vector contained between the left and right border region the following sequences: a coding region composed of a chloroplast transit peptide coding sequence from the  Arabidopsis thaliana  EPSPS (designated ctp2) joined to the CP4-EPSPS coding sequence and under the regulation of the Figwort mosaic virus promoter (pFMV) and the E9-3′ transcriptional termination sequence from  Pisum sativum.    
     In a preferred embodiment of the invention the 3706 bp, 664 bp, 288 bp, 751 bp and 1042 bp DNA fragments show at least 95%, preferably at least 99%, more preferably at least 99,9%, identity with the nucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 17, respectively. 95% identity, for example, means that 95% of the nucleotides of a given sequence are identical with the compared sequence. For this purpose, the sequences may be aligned and compared using the BLAST programme. Most preferably, the 3706 bp DNA fragment has the nucleotide sequence of SEQ ID NO: 6, the 664 bp DNA fragment has the nucleotide sequence of SEQ ID NO: 13, the 288 bp DNA fragment has the nucleotide sequence of SEQ ID NO: 11, the 751 bp DNA fragment has the nucleotide sequence of SEQ ID NO: 12, and/or the 1042 15p DNA fragment has the nucleotide sequence of SEQ ID NO: 17. 
     The present invention also refers to seed deposited with the NCIMB, Aberdeen (Scotland, U.K.), and having the accession number NCIMB 41158 or NCIMB 41159. Such seed can be used to obtain a glyphosate tolerant sugar beet plant. The seed can be sawn and the growing plant will be glyphosate tolerant. 
     The invention also relates to a cell, tissue or part of a glyphosate tolerant sugar beet plant. 
     Another aspect of the present invention is a method for the identification of a glyphosate tolerant sugar beet plant, characterized in that the method comprises the step(s) of a) amplifying a DNA fragment of between 630-700 bp, preferably 664 bp, from the genomic DNA of said sugar beet plant, parts or seeds thereof, using polymerase chain reaction with a first primer having the nucleotide sequence of SEQ ID NO: 1 and a second primer having the nucleotide sequence of SEQ ID NO: 2, and/or b) amplifying a DNA fragment of 3500-3900, preferably 3706 bp, from the genomic DNA of said sugar beet plant, parts or seeds thereof, using polymerase chain reaction with a first primer having the nucleotide sequence of SEQ ID NO: 3 and a second primer having the nucleotide sequence of SEQ ID NO: 4, and/or c) amplifying a DNA fragment of 270-300 bp, preferably 288 bp, from the genomic DNA of said sugar beet plant, parts or seeds thereof, using polymerase chain reaction with a first primer having the nucleotide sequence of SEQ ID NO: 7 and a second primer having the nucleotide sequence of SEQ ID NO: 8, and/or d) amplifying a DNA fragment of 710-790 bp, preferably 751 bp, from the genomic DNA of said sugar beet plant, parts or seeds thereof, using polymerase chain reaction with a first primer having the nucleotide sequence of SEQ ID NO: 9 and a second primer having the nucleotide sequence of SEQ ID NO: 10, and/or e) amplifying a DNA fragment of 990-1100 bp, preferably 1042 bp, from the genomic DNA of said sugar beet plant, parts or seeds thereof, using polymerase chain reaction with a first primer having the nucleotide sequence of SEQ ID NO: 14 and a second primer having the nucleotide sequence of SEQ ID NO: 16. 
     The method allows easy detection of a transgenic glyphosate tolerant sugar beet plant with standard molecular biology techniques. 
     The invention further relates to a test kit for the identification of a transgenic glyphosate tolerant sugar beet plant or its cells, tissue or parts. The kit comprises at least one primer pair with a first and a second primer for polymerase chain reaction, which allow to identify specifically event H7-1, its cells, tissue or parts. 
     Preferably, the first primer has the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 7 or SEQ ID NO: 9 or SEQ ID NO: 14, and the second primer has the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 16. 
     According to a further embodiment of the present invention, the first and second primer each recognize a nucleotide sequence which forms part of the nucleotide sequence of SEQ ID NO: 5. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The following figures serve to elucidate the invention. 
       It shows: 
         FIG. 1  Map of the binary vector PV-BVGT08 
         FIG. 2  Identification of H7-1 by PCR analysis. DNA samples from 18 plants have been analyzed. Neg. control=DNA from non-transformed sugarbeet; pos. control=DNA from H7-1 original transformant. 
         FIG. 3  Identification of event H7-1 by Multiplex-PCR and discrimination between transgenic event H7-1 and non-transgenic plants. DNA samples from 54 plants have been analyzed. 
         FIG. 4  The pFMV-ctp2-CP4-EPSPS-E9-3′-insert with cleavage sites of restriction enzymes HindIII, XbaI, ClaI, PstI, and BamHI. 
         FIG. 5  Insert/copy number analysis of event H7-1. For the Southern blot analysis 10 μg of H7-1 genomic DNA were digested with PstI, HindIII, XbaI, ClaI and BamHI (lane 3 to 7). Non-transformed genomic DNA as a negative control was digested with BamHI (lane 8). Plasmid PV-BVGT08 as a positive control was digested by BamHI. Lanes 2 and 9 represent size markers. The blot was probed with a  32 P-labelled CP4-EPSPS coding region. The probe is an internal sequence of the CP4-EPSPS gene covering basepairs 447-1555. 
         FIG. 6  Southern blot analysis of event H7-1 to evaluate ctp2-CP4-EPSPS coding region intactness. 10 μg of H7-1 genomic DNA, non-transgenic control DNA and non-transgenic control DNA mixed with PV-BVGT08 were digested with XbaI and HindIII/BamHI. The blot was probed with a  32 P-labelled CP4-EPSPS-PCR fragment. The probe represents the sequence of PV-BVGT08, bp 447-1555. 
         FIG. 7  Southern blot analysis of event H7-1 to evaluate promoter region intactness. 10 μg of H7-1 genomic DNA, non-transgenic control DNA and non-transgenic control DNA mixed with PV-BVGT08 were digested with HindIII, XbaI and SacI/XhoI. The blot was probed with a  32 P-labelled promoter fragment (HindIII)(=PV-BVGT08 sequence, bp 7972-8583) or with the complete promotor-ctp2-CP4-EPSPS-E9-3′-cassette (PmeI/XhoI)(=PV-BVGT08 sequence, bp 7935-2389). 
         FIG. 8  Southern blot analysis of event H7-1 to evaluate polyadenylation region intactness. 10 μg of H7-1 genomic DNA, non-transgenic control DNA and non-transgenic control DNA mixed with PV-BVGT08 were digested with EcoRI/PstI, XbaI, HindIII, and PstI. The blot was probed with a  32 P-labelled E9-3′ polyadenylation fragment (BamHI/XhoI)(=PV-BVGT08 sequence, bp 1702-2389). 
         FIG. 9  Fragments used as probes to evaluate absence of backbone vector DNA in event H7-1. 
         FIG. 10  Southern blot analysis of event H7-1 to evaluate absence of backbone vector DNA in event H7-1. 10 μg of H7-1 genomic DNA, non-transgenic control DNA and non-transgenic control DNA mixed with PV-BVGT08 were digested with XbaI. The blots were probed with  32 P-labelled probes encompassing the entire backbone of PV-BVGT08 (probe 1-4). One blot was probed with a labelled CP4-EPSPS fragment. 
         FIG. 11  Comparison between the PCR fragments and the PV-BVGT08 sequences on the left border region. 
         FIG. 12  Comparison between the PCR fragments and the PV-BVGT08 sequences on the right border region. 
         FIG. 13  Analysis of the genomic DNA outside the right junction of the insert. Approximately 50 ng of either event H7-1 genomic DNA, non-transgenic control DNA or water were used for PCR reactions with the primer combinations P1, where both primers are located outside of the insert, and P3, where one primer is located within the insert and the other primer lies outside the insert. 
         FIG. 14  Analysis of the genomic DNA outside the left junction of the insert. Approximately 50 ng of H7-1 genomic DNA, non-transgenic control DNA and water were used for PCR-reactions with the primer combinations P2, where both primers are located outside of the insert, and P4, where one primer is located within the insert and the other primer lies outside the insert. 
         FIG. 15  Progeny map of H71-1 seed lots. 
         FIG. 16  Southern blot analysis of event H7-1 to evaluate whether the inserted DNA is stably integrated into the genome. 10 μg of H7-1 genomic DNAs (the original transformant H7-1-1995 and three progenies, H7-1-1996 to 1998) and non-transgenic control DNAs from different origins were digested with BamHI, XbaI, and HindIII. The blot was probed with a  32 P-labelled CP4-EPSPS probe of PV-BVGT08 (=bp 447-1555). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention is further defined, by way of illustration only, by reference to the following examples. 
     A list of abbreviations used is given below: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 ~ 
                 approximately 
               
               
                   
                 ° C. 
                 degree Celsius 
               
               
                   
                 bidest 
                 double destillated sterile water 
               
               
                   
                 bp 
                 base pair(s) 
               
               
                   
                 CTAB 
                 cetyltrimethylammonium bromide 
               
               
                   
                 DNA 
                 deoxyribonucleic acid 
               
               
                   
                 
                   E. coli 
                 
                 
                   Escherichia coli 
                 
               
               
                   
                 EDTA 
                 ethylendiaminetetraacetic acid 
               
               
                   
                 FIG. 
                 Figure 
               
               
                   
                 h 
                 hour 
               
               
                   
                 HCl 
                 hydrochloric acid 
               
               
                   
                 kb 
                 kilobase pair 
               
               
                   
                 kg 
                 kilogram 
               
               
                   
                 M, mM, 
                 molar, millimolar 
               
               
                   
                 min 
                 minutes 
               
               
                   
                 Na 2 HPO 4 /NaH 2 PO 4   
                 sodium phosphate 
               
               
                   
                 NaCl 
                 sodium chloride 
               
               
                   
                 NaOH 
                 sodium hydroxide 
               
               
                   
                 nt 
                 nucleotide 
               
               
                   
                 PCR 
                 polymerase chain reaction 
               
               
                   
                 pmol 
                 picomole 
               
               
                   
                 RNase 
                 ribonucleic acid nuclease 
               
               
                   
                 rpm 
                 revolutions per minute 
               
               
                   
                 RR 
                 Roundup Ready ® 
               
               
                   
                 RT 
                 room temperature 
               
               
                   
                 SDS 
                 sodium dodecyl sulfate 
               
               
                   
                 sec 
                 second 
               
               
                   
                 SEVAG 
                 Chloroform:Isoamyl alcohol (24:1) 
               
               
                   
                 SSC 
                 standard saline citrate 
               
               
                   
                 TE 
                 Tris-EDTA buffer 
               
               
                   
                 TRIS 
                 tris(hydroxymethyl)-aminomethane 
               
               
                   
                   
               
             
          
         
       
     
     EXAMPLE 1 
     Identification of Event H7-1 
     Sugar beet ( Beta vulgaris ) genotype 3S0057 has been genetically modified to express a CP4-5-Enol-Pyruvyl-Shikimate-3-Phosphate Synthase or CP4-EPSPS, which confers tolerance to the herbicide glyphosate and is also used as a selectable marker. This transgenic line was produced by  Agrobacterium - tumefaciens  mediated transformation technology using the binary vector PV-BVGT08. The T-DNA of the vector used for sugar beet transformation contained between the left and the right border region the following sequences: a coding region composed of a chloroplast transit peptide coding sequence from the  Arabidopsis thaliana  EPSPS (designated ctp2) joined to the CP4-EPSPS coding sequence and under the regulation of the 35S Figwort mosaic virus promoter (pFMV) and the  Pisum sativum  rbcS-E9-gene 3′-transcriptional termination sequence. 
     The following methods were used: 
     I. DNA Extraction: 
     Method 1: 
     Fresh leaf or other tissue was collected (20 to 100 mg in a 1.5 ml tube) and 400 μl extraction buffer (see below) were added. The tissue was ground with a small pestle. The mixture was vortexed for 5 sec and incubated 30 min to 60 min at room temperature. A centrifugation step at 13,000 rpm for 1 min followed. The supernatant containing the DNA was poured into a new 1.5 ml tube and mixed with 320 μl isopropanol. The mixture was incubated at room temperature for 2 min. A DNA precipitate is formed after the addition of ethanol. After centrifugation at 13,000 rpm for 5 min the ethanol was decanted. The sample was allowed to air dry. The pellet was redissolved in 400 μl H 2 O or TE-buffer (see below). 
                                                           Extraction buffer (100 ml):                                          20 ml   1   M Tris (pH 7.5)             5 ml   5   M NaCl             5 ml   0.5   M EDTA            2.5 ml   20%   SDS           67.5 ml       H 2 O                        
TE-buffer:
     10 mM Tris-HCl (pH 8.0)   1 mM EDTA
 
Method 2:
   
     Fresh plant material (20 to 100 mg) was collected in a 1.5 ml Eppendorf tube and 500 μl CTAB buffer (65° C.) (see below) were added. The mixture was incubated 1 to 1.5 h at 65° C., and subsequently centrifuged for 5 sec. 5 μl RNase A (10 mg/ml), were added. The mixture was incubated 30 min at 37° C. and subsequently centrifuged for 5 sec. 200 μl SEVAG were added. After mixing and centrifugation at 13,000 rpm for 10 min the supernatant was transferred to a new 1.5 ml tube. 1 volume isopropanol (about 400 μl) was mixed carefully with the supernatant. A centrifugation step at 13,000 rpm for 10 min followed. 600 μl 70% ethanol were added. The pellet was washed by inverting the tube several times. Again, the mixture was centrifuged at 13,000 rpm for 2 min. The ethanol was carefully discarded. The tube was inverted and drained on clean paper. The sample was allowed to air dry for 15 min. The pellet was redissolved in 50 μl H 2 O (see below). 
     CTAB buffer: 
     
         
         1.4 M NaCl 
         20 mM EDTA 
         100 mM Tris-HCl 
         2% (w/v) CTAB
 
SEVAG:
 
         Chloroform:Isoamylalcohol (24:1)
 
RNase-buffer:
 
         10 mM Tris, 15 mM NaCl, pH 7.5
 
RNase A:
 
         10 mg RNase/ml RNase-buffer 
         (5 ml bidest+50 mg RNase A, Aliquots in 1.5 ml tubes, boil tubes 30 min at 100° C., storage at −20° C.) 
       
    
     Normally, Method 1 was used. With this method it is possible to extract a high number of DNA samples per day and the DNA quality is acceptable. Method 2 was used, when the leaf material was older or if there were problems with the DNA quality. Method 2 is more complex, requires more time, and gives a lower DNA yield, but of higher quality. 
     Normally, quantitation of DNA is not performed, and typically in a PCR reaction 0.5 to 1 μl of the extracted DNA solution is used. 
     PCR reaction: 
     For the PCR reaction, a 10× buffer mix of buffer+dNTP&#39;s was prepared. The procedure is as follows: 
     
       
         
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 10× buffer 
                 final PCR reaction 
               
               
                 Stock solution 
                 volume 
                 conc. 
                 conc. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 M Tris-HCl, 
                 100 
                 μl 
                 0.1 
                 M 
                 10 
                 mM Tris-HCl, 
               
               
                   
                 pH 8.3 
                   
                   
                   
                   
                   
                 pH 8.3 
               
               
                 1 
                 M KCl 
                 500 
                 μl 
                 0.5 
                 M 
                 50 
                 mM KCl 
               
               
                 100 
                 mM dATP 
                 20 
                 μl 
                 2 
                 mM 
                 0.2 
                 mM dATP 
               
               
                 100 
                 mM dCTP 
                 20 
                 μl 
                 2 
                 mM 
                 0.2 
                 mM dCTP 
               
               
                 100 
                 mM dTTP 
                 20 
                 μl 
                 2 
                 mM 
                 0.2 
                 mM dTTP 
               
               
                 100 
                 mM dGTP 
                 20 
                 μl 
                 2 
                 mM 
                 0.2 
                 mM dGTP 
               
               
                 100 
                 mM MgCl 2   
                 150 
                 μl 
                 15 
                 mM 
                 1.5 
                 mM MgCl 2   
               
             
          
           
               
                 HPLC water 
                 170 
                 μl 
                   
                   
               
             
          
           
               
                   
                 1000 
                 μl total 
               
               
                   
               
             
          
         
       
     
                                                           PCR reaction (25 μl):                                        DNA   0.5   μl           Primer 1   1   μl (20 pmol)           Primer 2   1   μl (20 pmol)           Taq polymerase 1   0.2   μl (1 U, from Oncor                   Appligene S.A.,                   Heidelberg, Germany)           Buffer 10 × conc.   2.5   μl           Water   18.8   μl                        
III. Identification of H7-1 by PCR
 
     The Identification of H7-1 was performed by PCR using event specific primers: 
     
       
         
               
               
             
           
               
                 Upper Primer (SEQ ID NO: 1): 
                   
               
               
                 H7-207U30: 5′ TTA ATT TTT GCA GGC GAT GGT GGC TGT 
               
               
                   
               
               
                 TAT 3′ 
               
               
                   
               
               
                 Lower Primer (SEQ ID NO: 2): 
               
               
                 H7-841L30: 5′ CAT ACG CAT TAG TGA GTG GGC TGT CAG 
               
               
                   
               
               
                 GAC 3′ 
               
             
          
         
       
     
     The upper primer is located outside of the insert, and is part of the sugar beet genomic DNA. The lower primer is located within the inserted CP4-EPSPS gene. PCR conditions: 
     
       
         
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 PCR conditions: 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 94° C. 4 min 
                 STEP 1 
               
               
                   
                 95° C. 30 sec 
                 STEP 2a 
               
               
                   
                 55° C. 30 sec 
                 STEP 2b 
               
               
                   
                 72° C. 2 min 
                 STEP 2c 
               
               
                   
                 72° C. 5 min 
                 STEP 3 
               
               
                   
                  4° C. over night 
                 STEP 4 
               
               
                   
                   
                 reaction complete 
               
               
                   
                   
               
             
          
         
       
     
     Steps 2a-c were repeated 34 times. 
     The expected PCR product is a DNA fragment of 664 bp (SEQ ID NO: 13, see  FIG. 2 ). 
     IV. Identification of H7-1 by Multiplex-PCR 
     For the discrimination of non-transgenic and transgenic plants, whether homozygous or hemi/heterozygous, a Multiplex-PCR with three different primers was performed. Following primers were used: 
     
       
         
               
               
               
             
           
               
                 H72 (SEQ ID NO: 14): 
                 5′ GCTCTGACACAACCGGTAAATGCATT 
                   
               
               
                   
                 GGCC 3′ 
               
               
                   
               
               
                 H7S2 (SEQ ID NO: 15): 
                 5′ GACCCATAGTTTGATTTTAAGCACGA 
               
               
                   
               
               
                   
                 CATG 3′ 
               
               
                   
               
               
                 H7R2 (SEQ ID NO: 16): 
                 5′ GCAGATTCTGCTAACTTGCGCCATCG 
               
               
                   
               
               
                   
                 GAG 3′ 
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 PCR conditions: 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 94° C. 2 min 
                 STEP 1 
               
               
                   
                 94° C. 1 sec 
                 STEP 2a 
               
               
                   
                 60° C. 45 sec 
                 STEP 2b 
               
               
                   
                 72° C. 90 sec 
                 STEP 2c 
               
               
                   
                 72° C. 5 min 
                 STEP 3 
               
               
                   
                  4° C. over night 
                 STEP 4 
               
               
                   
                   
                 reaction complete 
               
               
                   
                   
               
             
          
         
       
     
     Steps 2a-c were repeated 34 times. 
     Non-transgenic plants show only one PCR fragment of about 350 bp. Homozygous transgenic plants show one fragment of about 1.042 kb. Heterozygous plants show both fragments (see  FIG. 3 ). 
     EXAMPLE 2 
     Characterization of Event H7-1 
     Molecular analysis was performed to characterize the integrated DNA present in the event H7-1. Specifically, the insert number (number of integration sites within the sugar beet genome), the copy number (the number of DNA fragments within one locus), the integrity of the inserted coding region and its regulatory elements, the pFMV promoter and E9-3′ transcriptional termination sequence, the absence of backbone sequences of the vector used for transformation and the stable inheritance of the insert were determined. Further, the sequences flanking the DNA insert were identified. 
     The inserted DNA of the sugar beet transformation event H7-1 was characterized by using Southern blot, PCR and Inverse-PCR techniques. Positive and negative controls (PV-BVGT08, non-transgenic plant DNA) were included and were treated in the same manner as the test substance (H7-1). 
     DNA was isolated from batch number 74903H of event H7-1 plants grown in 1997. DNA was also isolated from the original transformant H7-1/3S0057 (=6401VH) in 1995 and from three additional progenies produced in 1996, 1997, and 1998. (H7-1/64801H, H7-1/74922H, and H7-1/83002S). 
     The non-transgenic sugar beet line 3S0057 served as control. In addition, sugar beet lines 5R7150, 8K1180, and 6S0085 were used as a negative control. Those lines are common non-transgenic lines used for breeding conventional sugar beet. 
     Reference substances correspond to the plasmid PV-BVGT08 used for the transformation. The plasmid DNA and DNA from the control sugar beet line were mixed together, digested with restriction enzyme and separated by electrophoresis on agarose gels in parallel to the test substances. The plasmid served as a size marker for the expected fragment and as a positive hybridization control. The plasmid DNA was mixed with the genomic plant DNA at a concentration representing less than 1 copy of the element being analysed to demonstrate the sensitivity of the Southern blot method (˜10 μg genomic DNA and ˜28 pg PV-BVGT08 DNA). For size estimations the molecular size marker RAOUL™ (ONCOR/Appligene, catalog #160673) was used. 
     DNA Isolation: 
     Plant tissue (1 to 3 g wet weight) from batch number 74903H of event H7-1 was ground in liquid nitrogen to a fine powder using a mortar and pestle. The powder was transferred to a 50 ml Oakridge tube, and 7.5 ml of preheated (60° C.) CTAB buffer (2% CTAB, 100 mM Tris-HCl, 20 mM EDTA pH 8.0, 1.4 M NaCl and 0.2% mercapto-ethanol) was added. The samples were incubated at 65° C. for approximately 30 min with intermittent mixing. An equal volume (8 ml) of a mixture of RT chloroform:isoamylalcohol (24:1 v/v) was added to the samples. The suspension was mixed by inversion, and the two phases were separated by centrifugation (10 min, 9000 rpm). The aqueous phase was transferred to a new 50 ml Oakridge tube following the precipitation of the DNA by adding 5 ml isopropanol. The DNA was pelleted by centrifugation (2 min, 9000 rpm) and the supernatant was removed. The precipitated DNA was incubated with a wash solution of 76% ethanol and 10 mM ammoniumacetate for about 20 min. After centrifugation and decanting of the supernatant the DNA was vacuum dried and redissolved in TE, pH 8.0 at 4° C. overnight. 
     As an alternative method, DNA was isolated by DNeasy Plant Maxi Kit from Qiagen (Düsseldorf, Germany, catalog #68163). DNA isolation was carried out according to the manufacturer&#39;s manual. 
     As an additional alternative method, DNA was isolated by DNeasy Plant Mini Kit from Qiagen (Düsseldorf, Germany, catalog #69103). DNA isolation was carried out according to the manufacturer&#39;s manual. 
     DNA Quantification and Restriction Enzyme Digestion: 
     DNA quantification was performed using a LKB Biochrom UV/visible spectrophotometer (Amersham Pharmacia, Freiburg, Germany) or, alternatively, DNA quantification was done after agarose gel electrophoresis by scanning the DNA with the RFLPscan program (MWG-Biotech, Ebersberg, Germany). As a calibration standard the High DNA Mass Ladder from Gibco/Life Technologies (Karlsruhe, Germany) (catalog # 10496-016) was used. Restriction enzymes were purchased either from Boehringer Mannheim (Mannheim, Germany), Stratagene (Amsterdam, Netherlands) or New England Biolabs (Frankfurt, Germany) and used according to the manufacturer&#39;s manual. 
     DNA Probe Preparation: 
     PV-BVGT08 DNA was isolated from  E. coli  cultures. Probe templates homologous to the CP4-EPSPS coding region, the 35S-promoter, the E9-3′-polyadenylation region, the  35 S-ctp2-CP4-EPSPS-E9-3′-cassette, and the backbone regions were prepared by digests with the corresponding restriction enzymes following a separation by agarose gel electrophoresis or by polymerase chain reaction (PCR). The products were purified using the Gene Clean II Kit of BIO 101 (La Jolla, Calif.). Labelling of the probes (25 pg) with 32P-dCTP or 32P-dATP was achieved making use of the Megaprime™ DNA labelling system of Amersham-Pharmacia Biotech Europe (Freiburg, Germany). 
     Southern Blot Analyses: 
     The samples of DNA treated with restriction enzymes were separated by agarose gel electrophoresis for ˜15 hours at ˜35 volts. After photographing the gel, the DNA was depurinated by soaking the gel for 15 min in a 0.25 M HCl-solution, denatured by incubating the gel for 30 min in a denaturing solution of 0.5 M NaOH, 1.5 M NaCl with constant gentle agitation and finally neutralized by soaking for 2 hours in several volumes of a solution of 2 M NaCl and 1 M Tris-HCl, pH 5.5. The DNAs from the agarose gels were transferred to Hybond-N™ nylon membranes (Amersham Pharmacia Biotech Europe, Freiburg, Germany) using a PosiBlot Pressure Blotter from Stratagene according to manufacturers protocol. After soaking the filter for 15 min in 2×SSPE (20×SSPE: 3.6 M NaCl, 20 mM EDTA, 0.2 M NaH 2 PO 4 /Na 2 HPO 4  pH 7.4) the DNA was fixed to the membrane by illumination with UV-light (Transilluminator Pharmacia, Freiburg, Germany) for 0.1 min and by baking for 1 hour at 80° C. in a vacuum oven. The blots were prehybridized for 4 hours in an aqueous solution of 50% formamide, 5×SSC, 0.1% laurylsarcosine, 0.02% SDS and 2% blocking reagent (Boehringer Mannheim, Germany, catalog # 1096176). Hybridisation with the radiolabelled probe was done in fresh prehybridization solution for 16-18 hours at 42° C. After hybridisation membranes were washed for 5 min in 2×SSC at 42° C., for 20 min in 2×SSC, 1% SDS at 65° C. and for two 15 min periods in 0.2×SSC, 0.1% SDS at 68° C. Autoradiographic images of the blot were obtained by exposing the blots using Kodak Biomax MST™ film in conjunction with Kodak Biomax MS™ intensifying screens. 
     Identification of 5′ and 3′ Genomic Flanking Sequences: 
     The transgene-to-plant genomic DNA junctions were identified by using the Inverse-PCR technique. Genomic DNA was purified as described above. Approximately 1 μg of the DNA was digested in separate reactions by the restriction nucleases TaqI, AluI, NdeIII or RsaI. The digested DNA-fragments were religated by T4-ligase over night, followed by the PCR reaction. The various inverse-primer combinations were obtained using the primer analysis software OLIGO® from NBI (National Biosciences, Inc., Plymouth, Mich.). 
     Fragments resulting from this Inverse-PCR amplification were separated by gel electrophoresis, excised from the gel and purified using the Gene Clean II™ kit. The purified fragments were cloned into the vector pCR®2.1 by using the TOPO™TA cloning® Kit from Invitrogen (Groningen, Netherlands). The inserts were submitted for sequencing by MWG-Biotech (Ebersberg, Germany). Analysis of the resulting sequence data were performed using Mac Molly® Tetra DNA analysis software (Soft Gene GmbH, Bochold, Germany). 
     PCR Analysis: 
     Genomic DNA was prepared with the Plant DNAeasy Plant Mini Kit (Qiagen, Düsseldorf, Germany) according to the manufacturer&#39;s instructions. Approximately 50 ng of genomic DNA were used for PCR. The reactions were subjected to 95° C. for 30 sec, 55° C. for 30 sec, and 72° C. for 2 min for 35 cycles. PCR was done in a PTC200 cycler (Biozym, Oldendorf, Germany). PCR products were analysed by agarose gel electrophoresis. 
     1. Insert Number: 
     The insert number, the number of integration sites of transgenic DNA into the sugar beet genome, was evaluated for H7-1. In order to determine the insert number, the genomic DNA was digested with the restriction enzymes HindIII, XbaI, and BamHI. As a negative control DNA from a non transformed control plant, representing the same genetic background, was digested with Hind III. As a positive control transformation vector DNA (PV-BVGT08) was used. 
     XbaI and BamHI cleave only once in PV-BVGT08 and do not cleave inside the labelled CP4-EPSPS probe used (see  FIG. 4 ). HindIII cleaves three times in PV-BVGT08, but all three sites are located outside of the probe and on the same side, 5′, relative to the probe. Thus, each enzyme should release a single DNA fragment that would hybridize to the CP4-EPSPS probe, and would contain a part of the inserted DNA and adjacent plant genomic DNA. The number of fragments detected indicates the number of inserts present in the event. The results are shown in  FIG. 5 . 
     After digestion with the enzymes HindIII, XbaI, or BamHI only a single hybridization fragment was found, respectively. The fragments of 5.2 kb from the HindIII digest (lane 4), 4 kb from the XbaI digest (lane 5) and approximately 11 kb from the BamHI digest (lane 7) showed that the transformant H7-1 represents a single integration event ( FIG. 4 ). The strong signal in lane 1 represents the linearized PV-BVGT08 plasmid. Additional faint signals refer to small amounts of undigested PV-BVGT08 or to unspecific hybridisation background signals. 
     2. Copy Number 
     Theoretically, one integration site could consist of more than one copy of the inserted DNA. But due to the fragment sizes of the restriction analysis shown above, this was not possible. If there was more than one copy of the inserted DNA present in H7-1, additional fragments would be detected. This was proved also by a digest with the restriction enzyme PstI. PstI cleaves twice within the left and the right border sequences. One of the restriction sites is within the CP4-EPSPS coding region, so after digestion one would expect two hybridizing fragments with the CP4-EPSPS probe. One of the expected fragments corresponds to the internal fragment of about 1.2 kb. The second fragment should be a border fragment. Again, if there was more than one copy, additional fragments should be detectable. But the results show that PstI cuts the DNA as expected. The internal fragment of 1.2 kb and only one additional fragment of about 4.9 kb were detected ( FIG. 5 : lane 3,  FIG. 4 ). 
     As an additional internal control the DNA was cleaved with ClaI ( FIG. 5 : lane 6,  FIG. 4 ). As expected, a single fragment of 2.4 kb hybridized, since ClaI cleaves twice but outside left and right hand of the used CP4-EPSPS fragment. This result is also a proof of the intactness of the integrated DNA fragment and is in agreement with the results given in the following. 
     The hybridization of the plasmid PV-BVGT08 with the CP4-EPSPS fragment results in a 8.6 kb signal (lane 1) as expected (PV-BVGT08=8590 bp). A second smaller very faint band is due to the incomplete restriction of PV-BVGT08. 
     In summary the experiments show that the transformed sugar beet line H7-1 contains a single copy integration of the T-DNA of PV-BVGT08 in the plant genome. 
     3. Coding Region Intactness 
     The integrity of the CP4-EPSPS gene cassette, with respect to the individual elements (P-FMV promoter, ctp2-CP4-EPSPS coding region, and E9 3′ non-translated region), was determined by digestion with the enzymes HindIII for P-FMV, HindIII plus BamHI for ctp2-CP4-EPSPS, and EcoRI plus PstI for the E9 3′ non-translated region. Additional experiments were performed with SacI plus XhoI for the P-FMV-ctp2-CP4 EPSPS region and for the E9 3′ region. Plasmid DNA mixed with non-transgenic sugar beet DNA and non-transgenic sugar beet DNA alone were digested with the same enzymes, as positive and negative controls, respectively. 
     These enzymes cleave within the intended DNA insert, between the left and right T-DNA Borders (see the plasmid map in  FIG. 1 ), so, if the respective elements are intact, the size of the hybridized fragments should be identical in H7-1 DNA and PV-BVGT08 DNA. 
     As an additional control the DNAs were digested with XbaI. XbaI cleaves once between the promoter and ctp2-CP4-EPSPS coding region. Therefore, one would expect a 8.6 kb fragment with the PV-BVGT08 DNA and in case of the H7-1 a border fragment which differs in size compared with the PV-BVGT08 fragment. The results are shown in  FIGS. 6 ,  7 , and  8 . 
       FIG. 6 : The digest with HindIII and BamHI released the CP4-EPSPS gene and the blot was probed with a CP4-EPSPS fragment generated by PCR. The negative control (lane 6) did not show any hybridisation bands. Genomic DNA from the event H7-1 and plasmid PV-BVGT08 mixed with non-transgenic DNA both produced an approximately 1.7 kb fragment which corresponds to the expected size. The digest with XbaI resulted in the expected 8.6 kb fragment of the linearized PV-BVGT08. For event H7-1 the digest resulted in the approximately 4.0 kb border fragment (see also  FIGS. 4 and 5 ). Again, the negative control did not show any signal. 
       FIG. 7 : The digest with HindIII released the Figwort mosaic virus promoter and the blot was probed with a promoter fragment generated by PCR. The negative control (lane 5) did not show any hybridization signals. Genomic DNA from event H7-1 and plasmid PV-BVGT08 mixed with non-transgenic DNA both produced a hybridizing fragment of 0.6 kb approximate size. This fragment corresponds to the expected size (lane 4 and 6) of the promoter. 
     The digest with XbaI resulted in the expected 8.6 kb fragment of the linearized PV-BVGT08 and in the approximately 1.3 kb left border fragment (lane 1 and 3) from event H7-1. This 1.3 kb fragment is also an additional proof that the event H7-1 contains only a single copy of the transgene. Again the negative control (lane 2) did not show any signal. 
     The digest with SacI/XhoI released the promotor together with the CP4-EPSPS coding region and the polyadenylation region. Hybridization with the complete promotor-ctp2-CP4-EPSPS-polyadenylation signal cassette (PmeI/XhoI fragment) produced the expected 2.3 kb promotor-ctp2-CP4-EPSPS and the 0.7 kb polyadenylation signal fragments both with the PV-BVGT08 DNA mixed with non-transgenic DNA and with H7-1 genomic DNA (lane 9 and 11). 
       FIG. 8 : The digest with PstI and EcoRI released the E9-3′ polyadenylation signal and the blot was probed with a polyadenylation signal fragment. The negative control (lane 3) did not show any hybridization bands. Plasmid PV-BVGT08 mixed with non-transgenic DNA and genomic DNA from the event H7-1 both produced an approximately 0.6 kb fragment which corresponds to the expected size. The digest with XbaI resulted in the expected 8.6 kb fragment of the linearized PV-BVGT08 and in an approximately 4.0 kb border fragment with event H7-1. 
     The digest with PstI released the E9-3′polyadenylation signal combined with a 0.5 kb 3′portion of the CP4-EPSPS coding region. The resulting 1.2 kb fragment was detectable as expected either with the H7-1 genomic DNA and also with the PV-BVGT08 DNA. 
     The digest with HindIII resulted in the 8.0 kb fragment of the linearized PV-BVGT08 minus the promoter fragment (lane 13) and in a 5.2 kb border fragment (lane 11) with H7-1. The single 5.2 kb fragment from the HindIII digest and the single 4.0 kb fragment from the XbaI digest are also an additional proof that the event H7-1 contains only one copy of inserted DNA. Again the negative controls did not show any signal. 
     In summary the results of the blots prove, that all elements of the transferred DNA are intact and that the event H7-1 contains a single intact CTP2-CP4-EPSPS coding region with its regulatory elements, the pFMV promoter and E9-3′ transcriptional termination sequence. 
     4. Analysis for the Detection of Backbone Fragments 
     The backbone region of a Ti-plasmid is defined as the region outside of the T-DNA bounded with left and right border sequences, which consists of the ori genes and selection genes for bacterial replication and bacterial selection and which is normally not transferred to the plant genome by Agrobacterium mediated transformation. To confirm the absence of the backbone vector DNA in the event H7-1, genomic DNA from H7-1, from a non transformed control and genomic DNA from H7-1 mixed with PV-BVGT08 DNA was digested with the restriction enzyme XbaI and probed with three overlapping PCR generated probes, that encompassed the entire backbone sequence. A fourth probe consists of the whole backbone in one fragment. 
     The probes used represent the backbone sequence (see  FIG. 9 ):
         1: bp 2730-5370   2: bp 5278-6419   3: bp 6302-7851   4: bp 2730-7851       

       FIG. 10  shows the result of the Southern blot analysis. Lanes 6, 10, 14, and 18: digest of H7-1 genomic DNA probed with the backbone fragments of the whole backbone did not show any hybridization bands. Only lanes 4, 8, 12, 16, and 20: H7-1 genomic DNA mixed with PV-BVGT08 DNA showed bands of 8.6 kb, as expected. The bands represent the linearized PV-BVGT08 DNA. 
     Lanes 2 and 4: H7-1 genomic DNA, and H7-1 genomic DNA mixed with PV-BVGT08, and hybridized with the CP4-EPSPS fragment, showed hybridizing signals. The 4 kb band in lane 2 represents the right border fragment, the two bands of lane 4 represent again the 4.0 kb right border fragment and the 8.6 kb linearized PV-BVGT08 plasmid. Both bands have the same intensity. This is a clear indication that the concentration of the added PV-BVGT08 DNA is comparable with the concentration of the CP4-EPSPS element in the H7-1 DNA. The concentration of the used plasmid DNA is equivalent to 0.5 copies. If there were backbone sequences integrated in the H7-1 genome, clear signals should be detectable. 
     These results prove that H7-1 does not contain any detectable backbone sequence of the plasmid used for the transformation. This result was also supported by the data of the analysis of the 5′ and 3′ genomic flanking regions (see below). 
     5. Identification of 5′ and 3′ Genomic Flanking Sequences 
     Agrobacterium mediated transformation normally leads to the integration of all sequences between the left and right border into the plant genome. The 5′ and 3′ ends of the integrated plasmid DNA should be within or near the left or the right border sequences, respectively. Therefore an Inverse-PCR technique was used to identify those regions. The cloned PCR products were sequenced and the sequence data were compared to PV-BVGT08 sequence. 
       FIG. 11  shows the alignment for the sequence from the cloned Inverse PCR fragment (D1U.RPT) (=genome H7-1, upper sequence), obtained with primers for analysis of the left border region, versus the PV-BVGT08 sequence (lower sequence). The comparison of both sequences showed that the homology stopped exactly within the border sequence. 
       FIG. 12  shows the alignment for the sequence from the cloned Inverse-PCR fragment (B3UNI.RPT) (=genome H7-1, upper sequence), obtained with primers for analysis of the right border region, versus the PV-BVGT08 sequence (lower sequence). The comparison of both sequences showed that the homology stopped already 18 nucleotides in front of border sequence. 
     In total, this is a clear indication, that the sequence between the left and right borders of the Ti-plasmid PV-BVGT08 is integrated correctly. The sequence stopped within or immediately in front of the borders. These data support the results of the backbone analysis, that no sequences of the backbone outside the border regions were integrated into the H7-1 genome. 
     To determine whether the flanking sequences on the right or left side of the insert in sugar beet event H7-1 are intact plant genomic sequences, inverse PCR analysis with primer combinations P1, P2, P3 and P4 were performed. 
     The primers of primer combinations P1 and P2 are located outside of the insert. If the DNA of the insertion locus within event H7-1 is identical with DNA of a non-transformed control, the PCR should result in two PCR fragments representing synthesis from the two primer combinations. The primers of primer combinations P3 and P4 are designed in a way such that one of the respective primers is located inside the CP4-EPSPS insert and the other primer is located outside the insert, within the plant genomic DNA. So the PCR should produce fragments from the H7-1 event DNA only. 
     Sequence data from Inverse-PCR-technique combined with data from the PV-BVGT08 vector results in a sequence which includes the H7-1 insert (PV-BVGT08 sequence), the right and left junction regions and additional sugar beet genomic DNA (SEQ ID NO: 5). 
     For the identification of the transgene-to-plant genomic DNA junctions (identification of event specificity) and for the genomic DNA regions to the left and right sides of the insert following primer combinations were used: 
     P1 combination (primer for analyzing genomic DNA outside the right border region, SEQ ID NO: 18, SEQ ID NO: 19): 
     
       
         
               
               
               
             
               
               
             
           
               
                 Upper primer: 
                 5′ CGG TAA ATG CAT TGG CCT TTG TT 
                   
               
               
                   
               
               
                 Lower primer: 
                 5′ CAC CCA GAT CCC AAT AAA ACC GTA AT 
               
               
                   
               
             
          
           
               
                 Expected PCR-product 241 bp (SEQ ID NO: 20) 
                   
               
             
          
         
       
     
     P2 combination (primer for analyzing genomic DNA outside the left border region, SEQ ID NO: 21, SEQ ID NO: 22): 
     
       
         
               
               
               
             
               
               
             
           
               
                 Upper Primer: 
                 5′ AAA TGG TTG TAG ATA AAT AAG GAA 
                   
               
               
                   
               
               
                   
                 ATC A 
               
               
                   
               
               
                 Lower primer: 
                 5′ ACA TGT TTG AGC ACT CTT CTT GT 
               
               
                   
               
             
          
           
               
                 Expected PCR-product 377 bp (SEQ ID NO: 23) 
                   
               
             
          
         
       
     
     P3 combination (primer for analyzing the transgene-to-plant genomic DNA junction, SEQ ID NO: 7, SEQ ID NO: 8): 
     
       
         
               
               
               
             
               
               
             
           
               
                 Upper Primer: 
                 5′ ATG CAT TGG CCT TTG TTT TTG AT 
                   
               
               
                   
               
               
                 Lower Primer: 
                 5′ TGT CGT TTC CCG CCT TCA G 
               
               
                   
               
             
          
           
               
                 Expected PCR-product 288 bp (SEQ ID NO: 11) 
                   
               
             
          
         
       
     
     P4 combination (primer for analyzing the transgene-to-plant genomic DNA junction, SEQ ID NO: 9, SEQ ID NO: 10): 
     
       
         
               
               
               
             
               
               
             
           
               
                 Upper Primer: 
                 5′ CGC TGC GGA CAT CTA CAT TTT TGA 
                   
               
               
                   
               
               
                   
                 AT 
               
               
                   
               
               
                 Lower primer: 
                 5′ AGT TAA CTT TCC ACT TAT CGG GGC 
               
               
                   
               
               
                   
                 ACT G 
               
               
                   
               
             
          
           
               
                 Expected PCR-product 751 bp (SEQ ID NO: 12) 
                   
               
             
          
         
       
     
     PCR Experiments with Event H7-1 DNA and with DNA from a non-transgenic control plant, using the primer combination P3, which has one primer being located within the H7-1 insert, yields a fragment with the event H7-1 DNA only. In contrast, PCR experiments using the primer combination P1, homologous to sequences outside the insert, yield fragments with both event H7-1 and non-transgenic control DNA. See  FIG. 13  for these results. 
     The results indicate that the sequence next to the right junction of the insert is present in the transgenic event H7-1 DNA and in the DNA from non-transgenic plants. It can be concluded that this DNA outside the event H7-1 insert is non-transgenic genomic DNA. 
     PCR experiments conducted with event H7-1 DNA and DNA from a non-transgenic control plant using primer combination P4, which has one of the primers located inside the CP4-EPSPS insert, yields a fragment with the event H7-1 DNA only. In contrast, PCR experiments using the primer combination P2, homologous to sequences outside the insert, yield fragments with both event H7-1 and non-transgenic control DNA. See  FIG. 14  for these results. 
     The results indicate that the sequence next to the left junction of the insert is present both in the transgenic event H7-1 DNA and in the DNA from non-transgenic plants. It can be concluded that the DNA outside the left junction is non-transgenic genomic DNA. 
     In summary, it can be stated that the sequences outside of the sugar beet event H7-1 insert are identical with sequences present in non-transgenic plants. It can be concluded that these sequences are plant genomic sequences present in the parental line used for transformation and in other conventional sugar beet lines. 
     6. Stability Over Generations 
     To demonstrate the stability of the integrated DNA the original transformation event H7-1 was compared with three progenies (64801H, 74922H, and 83002S; see  FIG. 15 ) of this line resulting from self pollination with non-transgenic sugar beet lines. The original transformed line and the progenies were produced in 1995, 1996, 1997, and 1998. 
     AS controls four different non-transgenic sugar beet lines were analysed (3S0057, 5R7150, 8K1180, 6S0085). All DNAS were digested with XbaI, HindIII, and BamHI, respectively, and hybridized with a labelled CP4-EPSPS fragment. To demonstrate that the T-DNA is stably integrated in the plant genome, all lanes of the H7-1 progenies digested with the same restriction enzyme should show a band of exact the same size. 
     The DNAs from the progenies of H7-1 lanes 3 to 6 show the expected fragments: DNA digested with BamHI resulted in bands of approximately 11 kb, digests with XbaI produced fragments of 4.0 kb and HindIII restriction produced bands of 5.2 kb. All bands from the same restriction but from different years were identical in their size. All non-transgenic lines did not show any signal ( FIG. 16 ). 
     These results demonstrate that the introduced sequence is stably integrated into the genomic DNA and stably inherited.