J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 1

Appendix 2

Evidence for the Absence of Vector Backbone DNA

INDEX

Section Page

Index 1

Introduction 2

Results 3

Materials and Methods 19

Conclusions 22

References 23

Certification 24
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 2

INTRODUCTION

Three methods were used to ensure that the events do not contain vector backbone DNA. The first 

method was based on the presence of the negative selectable isopentenyl isomerase (ipt) marker gene 

in the vector backbone. During transformation, inadvertent transfer of backbone DNA comprising the ipt

gene expression cassette from Agrobacterium to plant cells would trigger ipt gene expression and, 

consequently, the formation of the cytokinin-type hormone isopentenyladenosine. Shoots from 

transformed ipt gene-expressing cells develop a “cytokinin-overproduction” phenotype that causes

stunted phenotype, abnormal leaves, and an inability to root. Only shoots that were phenotypicallyindistinguishable from controls were selected and allowed to develop into whole plants. This screening 

step helped to guarantee that transformed plants lack a functionally-active backbone marker gene, but 

could not detect the presence of other backbone DNA in the events. The second method was used on 

events that had passed the first screening method. This method confirmed the absence of backbone 

DNA with Southern blot hybridization. The third method was designed to amplify fragments indicative of 

junctions between DNA insert border regions and flanking backbone DNA or regions within the 

backbone DNA that flank the DNA insert. 
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 3

RESULTS

Richael et al. (2008) previously showed the efficacy of the ipt gene lowers the frequency of 

transformation events carrying inadvertent vector backbone DNA in addition to the actual DNA insert.

Upon transformation, shoots displaying a cytokinin-overproduction phenotype (expressing the ipt gene) 

were simply ignored, and only those indistinguishable from wild-type were transferred and analyzed by 

PCR for the presence of a DNA insert. Transformed shoots were propagated in tissue culture to generate 

“events”, and three plants of each event were planted in the greenhouse, together with untransformed 

controls, for molecular analyses.

EcoRI-digested DNAs of the 10 events, their untransformed counterparts, and various positive controls

were hybridized with a total of 6 probes that span overlapping segments of the vector backbone (Figure 

1 and 2, below, and Table 1 and Figure 15 in Materials and Methods). As shown in Figure 2, some gaps 

exist between probes 4 and 5, and probes 5 and 6. The section of vector backbone between probes 4 

and 5 contains the terminator for ubiquitin (from S. tuberosum); thus the probes were not designed to 

detect this section that contains potato genomic DNA. The non-overlapping segment between probes 5 

and 6 also contains DNA belonging to the Ubi7 promoter and the ubiquitin monomer (UBQmoni), also 

from potato. The positive controls were obtained by transforming potato with pSIM1278 and selecting 

for plants containing backbone (G23 contains most of the backbone, and TT4 and T130 carry the entire 

backbone). In addition to the 10 events considered for deregulation, two events, E67 and G9, were 

included in most analyses. The lack of bands corresponding to the 6 probes and the 3 positive controls

(TT4, T130, and G23) as shown in Figures 3-10 verify that the 10 events do not contain vector backbone 

DNA. When evaluating the gels, the presence of any band indicates hybridization with the probes, yet 

will vary in size from the positive controls, hence they may not line up on the gel.

In some cases, Southern blots shown in this appendix will use the same probes and enzyme digests as 

found in other studies (for example, see Appendix 1. Characterization of Inserted DNA). The 

observations may differ, depending upon the conditions under which the gels were run. Therefore all 

bands described in the tables representing Southern blot data in this appendix refer to the gels 

presented within this appendix.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 4

Figure 1. Vector of pSIM1278

The vector backbone region, on the left, starts at position 9,957-bp and ends at 19,468-bp (= 9,512-bp). The backbone DNA 

consists mainly of bacterial DNA and is intended only to support maintenance of the DNA insert prior to plant transformation. 

The DNA insert region, including flanking Border sequences, is on the right from 19,469-bp to 19,660 and from 1-bp to 9,956, 

which is a total of 10,148-bp. The DNA insert consists of T-DNA border-like sequences and potato genomic DNA which are 

intended for stable integration into the plant genome.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 5

Figure 2. Probes for the backbone of pSIM1278 (9512 bp)

Table 1. Genetic Elements of the pSIM1278 Backbone

Genetic Element Origin Intended Function Other 

effects on 

plant 

Genbank Accession 

Number

Start-End 

Point in 

pSIM1278

(bp)

Reference

SacII restriction site S. tuberosum Site for connection of Ubi7 

promoter with LB flanking 

sequence.

None AJ272136.1 19,219-

19,224

Polyubiquitin

promoter (Ubi7)

including the coding 

sequence for a 76-

amino-acid potato 

ubiquitin monomer 

(UBQmon)

S. tuberosum

var. Ranger 

Russet

Drives expression of the ipt

backbone marker gene

None U26831.1 17,479-

19,218

Isopentenyl 

transferase (ipt) 

gene

Agrobacterium

tumefaciens

condensation of AMP and 

isopentenylpyrophosphate 

to form isopentenyl-AMP, a 

cytokinin

Cytokinin 

formation

NC_002377.1 16,744-

17,466

Smigocki 

and 

Owens 

1988

Terminator of the 

ubiquitin-3 gene 

(tUbi3)

S. tuberosum Terminate ipt gene 

transcription

None GP755544.1 16,038-

16,392

Garbarino 

and 

Belknap 

1994

Neomycin 

phosphotransferase 

III (nptIII) gene

E. coli Aminoglycoside 

phosphotransferase

None FJ362602.1 15,048-

15,842

Courvalin 

et al. 1977

Origin of replication 

for pBR322 (pBR322 

ori)

E. coli Start position for plasmid 

replication in bacterial cells

None J01784.1 14,477-

14,757

*

(pBR322 bom) E. coli pBR322 region for 

replication in E. coli

None J01749.1 14,077-

14,337

*

pVS1 replicon 

( pVS1Rep)

Pseudomonas 

fluorescens 

plasmid pVS1

pVS1 region for replication 

in Agrobacterium

None AJ537514.1 (4,501-

5,501)

12,667-

13,667

*

pVS1 partitioning 

protein StaA (PVS1 

Sta)

Pseudomonas 

fluorescens 

plasmid pVS1

pVS1 stability None AJ537514.1 (6,095-

7,095)

11,074-

12,074

*

overdrive Agrobacterium

tumefaciens

Enhances cleavage at the 

Right Border site

None K00549.1 (103-132) 9,963-

9,992

*

*: see: Cambia (2012) - http://www.cambia.org/daisy/cambia/585.html - (General structure map of pCAMBIA vectors)
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 6

For both the Southern analysis and PCR testing, H50 was analyzed separately from the other events. 

The event H50 was not originally selected for deregulation, but was added later when the event H23 

was disqualified for containing backbone. We show the results for all other events (E12, E24, F10, F37, 

J3, J55, J78, G11, and H37) and probes 1-4 in Figures 3-6. None of the untransformed controls nor 

events showed any band associated with backbone as shown in the lane for TT4. In Figure 7, illustrating 

hybridization with probe 5, we show a band indicating backbone in event H23 (a faint band, different in 

size from the positive controls), and H23 was then dropped from consideration for deregulation. In 

Figure 8, all lines, including controls, have bands in common with probe 6, indicating that the native 

promoters have homology with that probe. However, backbone would only be confirmed in an event if 

there were unique bands associated with the positive controls and not found in the untransformed 

controls. None of those were observed. 

Results for testing all 6 probes with event H50 and its untransformed control are shown in Figures 9 and 

10. No hybridization occurred with probes 1-5, indicating that no backbone was present (Figures 9, 10). 

Using probe 6, we saw bands in both H50 and its control, again showing homology with that probe and 

the native promoters. However, there were no unique bands corresponding with positive controls G23 

or T130 that would represent backbone (Figure 10).

Further confirmation of absence of backbone was obtained using PCR and multiple primers (Table 3)

associated with the border regions. As shown in Figures 11 -12, using multiple primers, no bands were 

amplified, thus confirming that events and untransformed controls did not contain backbone close to 

the right border. Additional primers (Table 3) were used with PCR and the lack of bands upon 

amplification confirmed that backbone was not present in the vicinity of the left border (Figure 13-14)

for all events and the untransformed controls. 
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 7

Figure 3. DNA Gel Blot Hybridization with Vector Backbone Probe 11

 Marker

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 TT4

23.1

9.4

6.6

4.4

2.3

2.0

1

All events, except for H50, are represented on the hybridized filter. EC = Russet Burbank control, FC = 

Ranger Russet control, JC = Atlantic Control, GC = variety G control, HC = variety H control, TT4 = positive 

control carrying backbone. Lane 1 = DIG II markers with fragment sizes in kb. E67, G9, and H23 not 

included in the submission.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 8

Figure 4. DNA Gel Blot Hybridization with Vector Backbone Probe 2

1

 Marker

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 TT4

23.1

9.4

6.6

4.4

2.3

2.0

1

All events, except for H50, are represented on the hybridized filter. EC = Russet Burbank Control, FC = 

Ranger Russet Control, JC = Atlantic Control, GC = variety G Control, HC = variety H Control, TT4 = 

positive control carrying backbone. Lane 1 = DIG II markers with fragment sizes in kb. E67, G9, and H23 

not included in the submission.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 9

Figure 5. DNA Gel Blot Hybridization with Vector Backbone Probe 3

1

 Marker

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 TT4

23.1

9.4

6.6

4.4

2.3

2.0

1

All events, except for H50, are represented on the hybridized filter. EC = Russet Burbank Control, FC = 

Ranger Russet Control, JC = Atlantic Control, GC = variety G Control, HC = variety H Control, TT4 = 

positive control carrying backbone. Lane 1 = DIG II markers with fragment sizes in kb. E67, G9, and H23 

not included in the submission.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 10

Figure 6. DNA Gel Blot Hybridization with Vector Backbone Probe 4

1

 Marker

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 TT4

23.1

9.4

6.6

4.4

2.3

2.0

1

All events, except for H50, are represented on the hybridized filter. EC = Russet Burbank Control, FC = 

Ranger Russet Control, JC = Atlantic Control, GC = variety G Control, HC = variety H Control, TT4 = 

positive control carrying backbone. Lane 1 = DIG II markers with fragment sizes in kb. E67, G9, and H23 

not included in the submission.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 11

Figure 7. DNA Gel Blot Hybridization with Vector Backbone Probe 5

1

 Marker

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 TT4 T130

H23 H37 TT4 

23.1

9.4

6.6

4.4

2.3

2.0

1

All events, except for H50, are represented on the hybridized filter. EC = Russet Burbank Control, FC = 

Ranger Russet Control, JC = Atlantic Control, GC = variety G Control, HC = variety H Control, TT4 and 

T130 are positive controls carrying backbone. The H23 event is not included in this submission as it 

contains vector DNA. Lane 1 = DIG II markers with fragment sizes in kb. The insert is a section of the 

filter with a longer exposure time. E67, G9, and H23 not included in the submission.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 12

Figure 8. DNA Gel Blot Hybridization with Vector Backbone Probe 6

1

 Marker

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 TT4 T130

23.1

9.4

6.6

4.4

2.3

2.0

1

All events, except for H50, are represented on the hybridized filter. EC = Russet Burbank Control, FC = 

Ranger Russet Control, JC = Atlantic Control, GC = variety G Control, HC = variety H Control, TT4 and 

T130 are positive controls carrying backbone. Lane 1 = DIG II markers with fragment sizes in kb. The 

presence of bands common in events and untransformed controls shows that probe 6 has homology 

with the native promoters. Backbone would be indicated by bands found in the events but not in the 

untransformed controls; however, none were observed. E67, G9, and H23 not included in the 

submission.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 13

Figure 9. DNA Gel Blot Hybridization for Event H50 with Vector Backbone Probes 1-31

1These membranes carried genomic DNA from positive control events (G23 and T130), negative control 

(untransformed H, HC) and event H50, hybridized with vector probes 1 to 3. Lane 1 = DIG II markers with 

fragment sizes in kb.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 14

Figure 10. DNA Gel Blot Hybridization for Event H50 with Vector Backbone Probes 4-61

1These membranes carried genomic DNA from the positive control lines (G23 and T130), negative 

control (untransformed H, HC) and event H50, hybridized with vector probes 4 to 6. G23 does not 

contain the entire backbone; it lacks the DNA sequence associated with probe 6. Lane 1 = DIG II markers 

with fragment sizes in kb.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 15

An additional approach to identify vector backbone sequences was based on PCR methods using primers 

designed to amplify either junctions between the DNA insert border regions and flanking backbone DNA 

or regions within the backbone DNA that flank the DNA insert (see Materials and Methods). Efficacy of 

the method was confirmed by using pSIM1278 DNA as a positive control. Figures 11 and 12 show that

none of the events presented for deregulation produce PCR bands that are indicative of the presence of 

vector backbone DNA at the Right Border side of the inserted DNA.

Figure 11. Confirmation of the Absence of Backbone DNA Flanking the Right Border of the DNA insert1

1Bands on the ethidium bromide-stained gels are indicative for the presence of Right Border/backbone 

DNA-junctions. Data for all events, except for H50, are shown (see Figure 12 for H50). M = 100-bp DNA 

marker lane (invitrogen), PC = positive plant control, WT = untransformed recipient variety (“wild type”, 

the negative control), primers printed on each gel. E67, G9, and H23 not included in the submission.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 16

Figure 12. Confirmation of the Absence of Backbone DNA Flanking the Right Border of the DNA insert

in Event H501

1Bands on the ethidium bromide-stained gels are indicative for the presence of Right Border/backbone 

DNA-junctions. M = 100-bp DNA marker lane (invitrogen), PC = positive plant control, H74 not included 

in this submission, NC = negative control (untransformed recipient variety), primers printed on each gel.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 17

Similarly, results for amplifications with Left Border-associated primers, shown in Figures 13 and 

14, indicate the absence of backbone DNA beyond the Left Border.

Figure 13. Confirmation of the Absence of Backbone DNA Flanking the Left Border of the DNA insert1

1Bands on the ethidium bromide-stained gels are indicative for the presence of Left Border/backbone 

DNA-junctions. Data for all events, except for H50, are shown (see Figure 14 for H50). M = 100-bp DNA 

marker lane (invitrogen), PC = positive plant control, WT = untransformed recipient variety (“wild type”, 

negative control), primers printed on each gel. E67, G9, and H23 not included in the submission.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 18

Figure 14. Confirmation of the Absence of Backbone DNA Flanking the Left Border of the DNA insert in 

Event H501

1Bands on the ethidium bromide-stained gels are indicative for the presence of Left Border/backbone 

DNA-junctions. M = 100-bp DNA marker lane (invitrogen), PC = positive plant control, H74 = not 

included in this submission, NC = negative control (untransformed plant), primers printed on each gel.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 19

MATERIALS AND METHODS

Plant material. Plants used for DNA gel blot analysis were grown for two months in Sunshine mix-1 

(www.sungro.com) in two-gallon pots in a greenhouse that was controlled for temperature (18°C 

minimum/27°C maximum) and light (16-h photoperiod with an intensity of ~1500 μmol/m2/s).

Use of the isopentenyl isomerase (ipt) gene as vector backbone marker. Only shoots that were 

phenotypically-indistinguishable from controls (not overproducing cytokinins) were selected during the 

transformation process and allowed to develop into whole plants. 

DNA Isolation. For plant DNA isolation, 1.0 g of young potato leaves was ground into a fine powder in a 

mortar using liquid nitrogen. The ground tissue was transferred to a pre-cooled 15 ml conical tube with 

a pre-cooled spatula and stored at -80oC until ready to process. 10 ml extraction buffer (0.35 M Sorbitol, 

0.1 M Tris-HCl, pH8.0, 0.05M EDTA) was added to, and mixed with, the powder, and the resulting 

suspension was centrifuged at 3,000 rpm for 15 min at room temperature. The pellet was thoroughly 

re-suspended in 2 ml extraction buffer containing 200 μg RNase A. After incubating the resuspended 

DNA at 650C for 20 min with 2 ml of nuclear lysis buffer (0.2 M Tris-HCl pH7.5, 0.005 M EDTA pH 8.0 and 

20 mg/ml CTAB Hexadecyl Trimetyl Ammonium Bromide) and 800 μl of 5% Sarcosyl, it was mixed with 

an equal volume of chloroform : isoamyl alcohol (24:1), vortexed for about 1 min, and centrifuged at 

3000 rpm for 5 min at room temperature. The DNA was precipitated with an equal volume of isopropyl 

alcohol, washed with 70% ethanol, air dried, and dissolved in 400-700 μl 1X Tris/EDTA buffer (TE). DNA 

concentration was measured using a spectrophotometer at an OD of 260 nm, whereas quality was 

confirmed by running the DNA on a 0.8% agarose gel in 1X Tris/Acetate/EDTA (TAE) for 30-40 min at 80 

volts. In some cases, an alternative method was used to isolate DNA. The ground tissue from 0.7 g young 

leaves was mixed with 7 ml CTAB buffer (2% CTAB, 1.4 M NaCl, 0.1 M pH 8.0 Tris-HCI, 20 mM pH8.0 

EDTA, 100 μg/ml RNase) and incubated at 55o-65oC for 30 min followed by centrifugation at 3,000 rpm 

for 15 min. DNA was extracted twice with equal volumes of chloroform: isoamyl alcohol (24:1) by 

shaking for 10 min and centrifuged at 3000 rpm for 5 min at room temperature. The DNA was 

precipitated with equal volumes of ethanol and rinsed with 70% ethanol. The resulting pellet was dried 

in air and dissolved in TE buffer. The quality and concentration of DNA was determined 

spectrophotometrically and by running a sample on a 0.8% agarose gel. Control and event DNA was 

always extracted with the same method for a specific comparison.

DNA Gel Blot Preparation. Digested DNA was loaded on a large 0.7% agarose gel (170 ml) containing 

0.5X Tris-borate-EDTA (TBE) buffer and 3-5 μl ethidium bromide (10 mg/ml) and run at 25 volts for 18 

hrs. The gel was photographed using a gel documentation system from Alpha Innotech (Santa Clara, 

California), and then depurinated by submerging it into 0.25 N HCl for 20 min. After subsequent 

denaturing in 0.5 M NaOH /1.5 M NaCl for 2X15 min and neutralization in 1.5 M NaCl and 0.5 M Tris-HCl, 

pH7.5, for 2 X 15 min on a shaker at room temperature, the gel was equilibrated with 10X SSC for 10 

min. The transfer of DNA to the nylon membrane was carried out using 10X SSC according to a standard 

capillary transfer method. 

Probe Preparation. Nylon filters carrying EcoRI-digested DNA of the events were hybridized with six 

different probes, each of which represented an approximately 1-kb DNA fragment of the vector 

backbone (see Table 2 for primer sequences and Figure 3 for the linear arrangements of the probes

described in Table 2). Untransformed potato varieties were used as negative controls, and TT4, T130 

and G23 events carrying most or all of the pSIM1278 vector provided positive controls. 
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 20

Table 2. Backbone Probes

Backbone Probe 

Nr.

Size pSIM1278 

Coordinates

Forward Primer Reverse Primer

1 1,473-bp 9,957-11,429 ACTAGTTGTGAATAAGTCGCTGTG ATCGGAATCGACTAACAGAACAT

2 1,514-bp 11,398-12,911 CCGGGGCCGATGTTCTGTTAG GCTCGCCGGCAGAACTTGAG

3 1,588-bp 12,860-14,447 GCCGCGTGTTCCGTCCACAC CCTGTCGGGTTTCGCCACCT

4 1,660-bp 14,420-16,079 CAAGTCAGAGGTGGCGAAAC CTTTATGCTCATTGGGTTGAGTA

5 1,074-bp 16,396-17,469 AGTCCACCCGAAATATAAACAAC GGTATGGACCTGCATCTAATTTTC

6 834-bp 18,633-19,466 GCTCTAATATAGCGCATTTCAAG GCTTCCAGCCAGCCAACAGCTC

The labeling of PCR-derived probe was achieved using Hotmaster taq enzyme and buffer (Fisher 

BioReagents) according to Roche’s DIG labeling instructions. A standard 50 μl reaction consisted of 5 μl 

10 x Hotmaster Taq Buffer, 2-5 μl 10 uM forward primer, 2-5 μl 10 μM reverse primer, 5 μl DIG labeled 

dNTP (Roche), 10 ng plasmid template, 0.75 μl Hotmaster Taq polymerase, and water. The PCR 

amplification conditions were dependent on each DIG-labeled probe. PCR with regular dNTP instead of 

DIG labeled dNTP was used as control. Quality of the DIG labeled probe was assessed by running a small 

amount of the probe on 1% agarose DNA gel (it always ran slower than control PCR product). The probe 

was denatured before use by incubating the probe at 100oC for 5 min, placing on ice for 2 min.

Southern Blot Hybridization. The nylon membrane carrying transferred DNA was prehybridized in 40 ml 

pre-warmed DIG Easy Hybridization solution (Roche) at 42oC for 1-4 hrs in a bottle in a standard 

hybridization oven (Amerex Instruments Inc.) at 20-25 rpm. Hybridization was carried out by replacing 

the prehybridization buffer with a fresh amount of the same preheated solution, now containing 25-50 

μl denatured DIG labeled probe, and continuing the incubation at 42oC, 20-25 rpm for about 16 hrs. The 

hybridization solution could be store at -20oC and reused up to 3 times. The reused hybridization 

solution was heated at 68oC for 10 minutes before use to remove archives from previous runs.

Southern Blot Detection. The hybridization solution was removed and replaced by 100 ml washing 

solution I (2X SSC/0.1% SDS). The membrane was washed twice in washing solution I for 10 min at room 

temperature. This low stringency buffer was poured off and preheated high stringency washing solution 

II (0.5X SSC/0.1% SDS, 600C) was added immediately. The membrane was washed twice in washing 

solution II at 60-63oC for 20 min each at 25-30 rpm. This was followed by a brief rinse with 2X SSC to 

remove SDS. The membrane was rinsed with 150 ml of 1X DIG Washing Solution (Roche) in a tray for 2 

min and incubated in 1X Blocking solution (Roche) for 0.5-3 hrs on a low-speed shaker. The blocked 

membrane was incubated with DIG antibody solution (1:10,000 dilution of Anti-DIG-alkaline phosphate 

conjugate with 1X Blocking solution) for 30 min on a shaker. The membrane was washed twice (15 min 

each) with 1X DIG Washing Solution (Roche) and equilibrated with 1X detection buffer. The detection 

reaction was carried out with 2 ml CDP-Star solution (1:100 diluted stock of CDP-Star with 1X detection 

buffer) for 5 min. The membrane was wrapped in a plastic film and exposed to the Z-ray film in the 

dark. Depending on the experiment multiple exposures were taken from 30 sec to 30 min. The films 

were developed with Konica SRX-101A Z-ray film developer. The developed films were scanned to 

obtain the final images. 

PCR-Based Identification of Vector Backbone sequences. The standard 30 μl PCR reaction mixture 

consisted of 3 μl of 10X PCR buffer, 0.6 μl of 10 mM dNTP, 0.6 μl of 10 μM forward primer, 0.6 μl of 10 

μM reverse primer, 100 ng of genomic DNA template, with 0.2 μl of HotMaster Taq polymerase (Fisher 

BioReagents). The PCR was carried out under the following amplification conditions: 1 cycle of 3 min at 
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 21

95 °C followed by 40 cycles of 30 sec at 94 °C, 30 sec at 55°C, 45 sec at 68 °C, and finishing with 10 min at 

68 °C. 

Primers were designed to amplify fragments indicative of (1) junctions between the DNA insert border 

regions and flanking backbone DNA or (2) regions within the backbone DNA that flank the DNA insert. 

The primer pair JY725-JY726 amplified a 377-bp fragment comprising the junction at the Right Border 

region and flanking backbone, and primers JY915-JY749 were used to amplify a 215-bp backbone 

fragment flanking the Right Border (see Table 3 and Figures 11-12). The primer pair JY718-JY719 

amplified a 587-bp fragment comprising the junction at the Left Border region and flanking backbone, 

and primers JY931-JY932 were used to amplify a 134-bp backbone fragment flanking the Left Border 

(see Table 3 and Figures 13- 14). H50 was run separately on the gels to verify similarities to H74. 

However, H74 is not a part of this submission.

Table 3. Primers Used for Detecting Backbone

Primers Used for Detecting Backbone Adjacent to Left Border of pSIM1278

Name Sequence Position in pSIM1278 product Backbone bp

JY719 GAGCTGTTGGCTGGCTGGAAG 19445-19465, in backbone

JY718 GTTGGAAATCAATTATCACTGAG 349-371, in AGP promoter 587 bp 24

JY931 CGAGATCATCCGTGTTTCAA 19278-19297, in backbone

JY932 GATACAGGCAGCCCATCAGT 19392-19411, in backbone 134 bp 134

Primers Used for Detecting Backbone Adjacent to Right Border of pSIM1278

Name Sequence Position in pSIM1278 product Backbone bp

JY725 GCTTCCCGTATACAACATAACATG 9621-9644, in GBSS promoter

JY726 GATCTCAAACAAACATACACAGCG 9974-9997, in backbone 377 bp 41

JY915 ACTAGTTGTGAATAAGTCGCTGTG 9957-9980, in backbone

JY749 GAAGCCGACTGCACTATAGCAG 10150-10171, in backbone 215 bp 215

Figure 15. PCR Probe Positions for Backbone Detection
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 22

CONCLUSIONS

Event Ranger Russet F10 lacks any detectable vector backbone DNA.

Event Ranger Russet F37 lacks any detectable vector backbone DNA.

Event Russet Burbank E12 lacks any detectable vector backbone DNA.

Event Russet Burbank F10 lacks any detectable vector backbone DNA

Event Atlantic J3 lacks any detectable vector backbone DNA.

Event Atlantic J55 lacks any detectable vector backbone DNA.

Event Atlantic J78 lacks any detectable vector backbone DNA.

Event G11 lacks any detectable vector backbone DNA.

Event H37 lacks any detectable vector backbone DNA.

Event H50 lacks any detectable vector backbone DNA.

A robust triple confirmation process was utilized to prove the lack of vector backbone in any of the 10 

events, which are the focus of this deregulation petition. These studies confirm that the events 

presented for deregulation do not contain backbone DNA. They indicate that further review of the 

safety of the vector DNA is not needed as this DNA is not present in the InnateTM events.
J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 23

REFERENCES

Cambia (2012) pCambia Vectors. http://www.cambia.org/daisy/cambia/585.html (Accessed 7 May 

2012).

Courvalin P, Weisblum B, Davies J (1977) Aminoglycoside-modifying enzyme of an antibiotic-producing 

bacterium acts as a determinant of antibiotic resistance in Escherichia coli. P Natl Acad Sci USA,

74: 999-1003.

Garbarino JE, Belknap WR (1994) Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and 

expression of its promoter in transgenic plants. Plant Mol Biol, 24: 119-127.

Richael CM, Kalyaeva M, Chretien RC, Yan H, Adimulam S, Stivison A, Weeks JT, Rommens CM (2008) 

Cytokinin vectors mediate marker-free and backbone-free plant transformation. Transgenic Res 

17: 905-917. 

Smigocki AC, Owens LD (1988) Cytokinin gene fused with a strong promoter enhances shoot 

organogenesis and zeatin levels in transformed plant cells. P Natl Acad Sci USA, 85: 5131-5135.