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 1 9

Conclusions 2 2

Reference s 2 3

Certification 2 4J.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 ) mark er gene in the vector backbone. During transformati on, i nadvertent 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. S hoots from
transf ormed ipt gene

- expressing cells develop a “cytokinin

- overproduction” phenotype that causes stunted phenotype, abnormal leaves, and an inability to root. Only shoots that were phenotypically

indistinguishable from controls were selected and allowed to devel op 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 pass ed 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 reg ions 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 t he efficacy of the ipt gene lower s 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 . Trans formed 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.

Eco RI

- digested DNA s of the 10 events, their untransformed co unterparts, 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 1 5 in Materials and Methods )

. As shown in Figure 2 , some gaps exist betw een 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

- over lapping segment between probes 5
and 6 also contain s 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 back bone (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. T he 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 prob es, yet will vary in size from the po sitive 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

observa tions 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 p SIM1278

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 f or 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 (9 512 bp)

Table 1. Genetic Elements of the pSIM1278 B ackbone

Genetic Element Origin Intended Function Other
effects on plant Genbank Accession

Number Sta rt

- 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

P olyubiquitin promoter ( Ubi7 ) including the coding sequence for a 76

- amino

- a cid potato ubiquitin monomer ( UBQmon ) S

. tuberosum

var. Ranger

Russet Drives expression of the ipt

backbone marker gene None U26831.1 17,479

- 19,21 8

Isopentenyl transferase ( ipt )

gene Agrobacterium

tumefaciens condensation of AMP and

isopentenylpyr ophosphate 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

199 4 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

r eplicati on 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 re plication

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

tumefac iens 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 n or events showed any band associated with backbone as shown in the la ne for TT4. In Figure 7 , illustrating hybridization with probe 5

, we show a band indicating b ackbone 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

prom oters 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 . N one of those were observed.

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

10

. N o hybridization occurred with probes 1

- 5, indicating that no backbone was present ( Figure s 9, 10 )

. Using probe 6, we saw bands in both H50 and its control, again sh owing homology with that probe and
the native promoters . H owever , 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 a nd multiple primers ( Table 3 )
associated with the border regions. As shown in Figure s 1 1

- 1 2 , using multiple primers, no bands were amplified, thus confirming that events and untransformed controls did not contain backbone close to the right border. Addi tional primers ( Table 3 ) were used with PCR and the lack of bands upon amplification confirmed that backbone was not present in the vicini ty of the left border ( Figure 1 3

- 1 4 )
for all events and the untransformed controls

.J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 7 Figure 3

. DNA G el B lot H ybr idization with V ector B ackbone P robe 1 1

Marker

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 contr ol, TT4 = positive control carrying backbone. Lane 1 = DIG II markers with fragment sizes in k b. 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 G el B lot H ybridization with V ector B ackbone P robe 2 1

Marker

1 All events , except f or 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 fragmen t sizes in k b. 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 G el B lot H ybridization with V ector B ackbone P robe 3 1

Marker

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 k b. 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 G el B lot H ybridization with V ector B ackbone P robe 4 1

Marker

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 k b. 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 G el B lot H ybridization with V ector B ackbone P robe 5 1

Marker

1 A ll 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 H 23 event is not included in this submission as it

contains vector DNA. Lane 1 = DIG II markers with fragment sizes in k b. The inse r t 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

. DN A G el B lot H ybridization with V ector B ackbone P robe 6 1

Marker

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 = v ariety H Control, TT4 and T130 are positive controls carrying backbone. Lane 1 = DIG II markers with fragment sizes in k b. The presence of bands common in events and untransformed controls shows that probe 6 has homology
with the native promoters . Backbo ne 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 G el B lot H ybridization for E vent H50 with V ector B ackbone P robes 1

- 3 1

1 These 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 k b.J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 14 Figure 10

. DNA G el B lot H ybridization for Event H50 with V ector B ackbone P robes 4

- 6 1

1 These 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

c ontain the entire backbone; it lacks the DNA sequence associated with probe 6. Lane 1 = DIG II markers with fragment sizes in k b.J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 15

A n 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 a nd flanking backbone DNA

or regions within the ba ckbone DNA that flank the DNA insert (see Materials and Methods ). Efficacy of the method was confirmed by using pSIM1278 DNA as a positive control. Fig ures 1 1 and 1 2 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 1 1

. Confirmation of the Absence of Backbone DNA Flanki ng the Right Border of the DNA insert 1

1 Bands 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 1 2 for H50 ). M = 100

- bp DNA
marker lan e (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 1 2

. Confirmation of the Absence of Backbone DNA F lanking the Right Border of the DNA insert
in Event H50 1

1 Bands 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, r esults for amplifications with Left Border

- associated primers, shown in Figures 1 3 and

1 4

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

Figure 1 3

. Confirmation of the Absence of Backbone DNA Flanking the Left Border of the DNA insert 1

1 Bands on the ethidium bromide

- stained gels are indicative for the presence of Left Border/backbone

DNA

- junctions. Data for al l events, except for H50, are shown (see Figure 1 4 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 inclu ded in the submission.J.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 18

Figure 1 4

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

Event H50 1

1 Bands on the ethidium bromide

- stained gels are indicative for the presence of Lef t Border/backbone

DNA

- junct ions. 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 iso pentenyl 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 Iso lation. 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

- 80 o C 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

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

DNA at 65 0 C 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 Bro mide) and 800 μl of 5% Sarcosyl, it was mixed with an equal volume of chloroform : isoamyl alcohol (24:1)

, vortex ed 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 μ g/ml RNase) and incubate d at 55 o

- 65 o C 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 preci pitated 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. C ontrol 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 acc ording to a standard capillary transfer method

.

Probe Preparation. Nylon filters carrying Eco RI

- 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 (se e 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 pro vided po sitive 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 GCTCGCCGGCAGA ACTTGAG

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 GCTCTAATAT AGCGCATTTCAAG 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 regul ar 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 incub ating the probe at 100 o C for 5 min, placing on ice for 2 min

.

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

pre

- warmed DIG Easy Hybridization solution (Roche) at 42 o C for 1

- 4 hrs in a bottle in a stand ard 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 t he incubation at 42 o C, 20

- 25 rpm for about 16 hrs. The hybridization solution could be store at

- 20 o C and reused up to 3 times. The reused hybridization
solution was heated at 68 o C 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 wash ed 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, 60 0 C) was added immediately. The membrane was washed twice in washing
solution II at 60

- 63 o C 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 CD P

- 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 w ere 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 condit ions: 1 cycle of 3 min atJ.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 an d 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 Figure s 1 1

- 1 2 )

. The primer pair JY718

- JY719 amplified a 58 7

- bp fragment comp rising the junction at the Left Border region and flanking backbone, and primers JY931

- JY932 were us ed to amplify a 134

- bp backbone fragment flanking the Left Border (see Table 3 and Figure s 1 3

- 1 4 ). H50 was run separately on the gels to verify similarities to H74. However, H74 is not a part of this submission.

Table 3 . Primers U sed for D etecting B ackb one Primers U sed for D etecting B ackbone A djacent to Left Border of pSIM1278

Name Sequence Position in pSIM1278 product Backbone bp

JY719 GAGCTGTTGGCTGGCTGGAAG 19445

- 19465, in backbone

587 bp 24 JY718 GTTGGAAATCAATTATCACTGAG 349

- 371, in AGP promoter

JY931 CGAGATCATCCGTGTTTCAA 19278

- 19297, in backbone

134 bp 134 JY932 GATACAGGCAGCCCATCAGT 19392

- 19411, in backbone

Primers U sed for D etecting B ackbone A djacent to Right Border of pSIM1278

Name Sequence Position in pSIM1278 product Backb one bp

JY725 GCTTCCCGTATACAACATAACATG 9621

- 9644, in GBSS promoter

377 bp 41 JY726 GATCTCAAACAAACATACACAGCG 9974

- 9997, in backbone

JY915 ACTAGTTGTGAATAAGTCGCTGTG 9957

- 9980, in backbone

215 bp 215 JY749 GAAGCCGACTGCACTATAGCAG 10150

- 10171, in backbone

Figure 1 5

. PCR Probe Positions for Backbone Detecti onJ.R. Simplot Company Evidence for the Absence of Vector Backbone DNA 22 CONCLUSION S

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 detect able 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 vec tor 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 bac kbone 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. The y 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 REFERENCE S

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

- p roducing

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 t ransgenic 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.

Smigo cki 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 U SA

, 85: 5131

- 5135.