J.R. Simplot Company Evidence for Stability of the Inserted DNA 1

Appendix 3

Evidence for Stability of the Inserted DNA

INDEX

Section Page

Index 1

Introduction 2

Abbreviations and Definitions 2

Results 3

Discussion 45

Materials and Methods 46

Conclusions 49

References 50

Certification 51
J.R. Simplot Company Evidence for Stability of the Inserted DNA 2

INTRODUCTION

The stability of the DNA inserts was evaluated in the original transformants and again in propagated 

plant material using both DNA gel blot hybridization and a trait evaluation assay. These studies were 

carried out to ensure that the desired genetic changes in events remained stable over multiple clonal 

cycles and expressed the traits consistently over generations. Inconsistent trait expression

generationally might be triggered by rare recombination events or could also be caused by methylation.

Because potatoes are clonally propagated, standard assessments for sexually propagated crops were 

not directly applicable. Tubers rather than seeds were used to define subsequent generations since 

tubers are what is commercially planted. Results of DNA blot hybridization demonstrate consistent 

bands were present in three generatiuons of plants, indicating stability of the traits. Further evidence 

for stability was obtained by confirming trait efficacy in three generations of plants (tubers) using a

catechol assay for polyphenoly oxidase activity by visually evaluating the darkening of tuber flesh.

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.

ABBREVIATIONS and DEFINITIONS

G0 Initial generation of tubers utilized from tissue culture

G1 First generation field grown plant (after tissue culture)

G2 Second generation field grown plant

G3 Third generation field grown plant

wt “wild type”, this term is used to refer to the untransformed control of a given variety

MII Molecular size marker used in lane 1 of each 0.7% agarose gel (approximate range 2.0-23.1 kb)

MVII Molecular size marker used in lane 2 of each 0.7% agarose gel (approximate range 1.2-7.7 kb)
J.R. Simplot Company Evidence for Stability of the Inserted DNA 3

RESULTS

DNA Insert Stability in Ranger Russet Events F10 and F37

The GBS and AGP probes (see Figure 38 in Materials and Methods) were hybridized with DNA of the 

Ranger Russet controls and events F10 and F37 and evaluated over 3 generations. Hybridization was 

used to visualize three common bands present in the unmodified control Ranger Russet variety (wt) and

the F10 and F37 events. The common bands were visualized on 0.7% agarose gels with sizes of 7.4 kb, 

7.7 kb, and 9.4 kb for the GBS probe and 1.2 kb, 4.9 kb, and 6.2 kb for the AGP probe (Figures 1, 2, 3, 4, 

5, and 6).

Two additional bands hybridized with the GBS probe were observed in F10 and F37, one 2.3 kb band 

indicative of an internal DNA insert fragment and the other one a junction fragment (7.1 kb for F10 and 

7.7 kb for F37). These were observed in all G0, G1, G2, and G3 samples, indicating that the inserts of the 

original transformant (G0) remained stable into the first, second, and third vegetative generations 

(Figures 1, 2, and 3). The AGP probe visualized both an IB (internal band) of 2.3 kb and a JB (junction 

band) of 6.5 kb for F37 in G0, G1, G2, and G3 (Figures 4, 5, and 6). Additionally the AGP probe 

hybridized with a 2.6 kb fragment for all F10 samples (Figures 4, 5, and 6).

Additional evidence for DNA insert stability and black spot bruise efficacy in Ranger Russet events F10 

and F37 was obtained with a catechol assay for Ppo activity. The results with G2 tubers showed all 

Ranger Russet F10 and F37 tubers remained uncolored, indicative of resistance to black spot bruise 

(Figure 7) through Ppo5 gene silencing. 

Overall, hyridization (Figures 1, 2, 3, 4, 5, and 6) with the GBS and AGP probes shows that events F10 

and F37 had consistent bands over multiple generations by comparing G0 with G1, G2 and G3 samples. 

Also, the visual assay for PPO with catechol confirmed stability of Ppo5 gene silencing (Figure 7) from 

the initial generation through G2.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 4

Figure 1. F10 and F37: DNA Insert Stability in G0 and G1 Samples as Visualized with a GBS Probe

Ranger Russet untransformed controls (wt), F10, and F37 all show consistency with 7.1 kb, 7.7 kb, and 

9.4 kb size bands in the G0 and G1 plants. Additionally, F10 and F37 share a band the size of 2.3 kb in 

both the G0 and G1 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 5

Figure 2. F10 and F37: DNA Insert Stability in G2 Samples as Visualized with a GBS Probe

M M wt wt wt F10 F10 F10 F37 F37 F37 Line

II VII G0 G2 G2 G0 G2 G2 G0 G2 G2 Generation

23.1

9.4

8.6

7.4

6.6

4.9

4.4

3.6

2.8

2.3

2.0

1.51

1.48

1.2

0.99

6.1

1.95

1.88

Ranger Russet untransformed controls (wt), F10, and F37 all show consistency with 7.1 kb, 7.7 kb, and 

9.4 kb size bands in the G0 and G2 plants. Additionally, F10 and F37 share a band the size of 2.3 kb in 

both the G0 and G2 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 6

Figure 3. F10 and F37: DNA Insert Stability in G3 Samples as Visualized with a GBS Probe

Ranger Russet untransformed controls (wt), F10, and F37 all show consistency with 7.1 kb, 7.7 kb, and 

9.4 kb size bands in the G0 and G3 plants. Additionally, F10 and F37 share a band the size of 2.3 kb in 

both the G0 and G3 plants.

M M wt wt wt F10 F10 F10 F37 F37 F37 

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3 

2.8

2.3

2.0

1.5

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9

1.2
J.R. Simplot Company Evidence for Stability of the Inserted DNA 7

Figure 4. F10 and F37: DNA Insert Stability in G0 and G1 Samples as Visualized with an AGP Probe

Ranger Russet untransformed controls (wt), F10, and F37 all show consistency with 1.2 kb, 4.9 kb, and 

6.5 kb size bands in the G0 and G1 plants. F10 and F37 share a band the size of 2.3 kb in both the G0 and 

the G1 plants. F10 also has a band the size of 2.5 kb which is consistent in the G0 and G1 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 8

Figure 5. F10 and F37: DNA Insert Stability in G2 Samples as Visualized with an AGP Probe

M M wt wt wt F10 F10 F10 F37 F37 F37 Line

II VII G0 G2 G2 G0 G2 G2 G0 G2 G2 Generation

23.1

9.4

8.6

7.4

6.6

4.9

4.4

3.6

2.8

2.3

2.0

1.51

1.48

1.2

0.99

6.1

1.95

1.88

Ranger Russet untransformed controls (wt), F10, and F37 all show consistency with 1.2 kb, 4.9 kb, and 

6.5 kb size bands in the G0 and G2 plants. F10 and F37 share a band the size of 2.3 kb in both the G0 and 

the G2 plants. F10 also has a band the size of 2.5 kb which is consistent in the G0 and G2 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 9

Figure 6. F10 and F37: DNA Insert Stability in G3 Samples as Visualized with an AGP Probe

Ranger Russet untransformed controls (wt), F10, and F37 all show consistency with 1.2 kb, 4.9 kb, and 

6.5 kb size bands in the G0 and G3 plants. F10 and F37 share a band the size of 2.3 kb in both the G0 and 

the G3 plants. F10 also has a band the size of 2.5 kb which is consistent in the G0 and G3 plants.

M M wt wt wt F10 F10 F10 F37 F37 F37 

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3 

2.8

2.3

2.0

1.5

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9

1.0
J.R. Simplot Company Evidence for Stability of the Inserted DNA 10

Figure 7. Assay for Sustained Ppo5 Gene Silencing in G2 Tubers of F10 and F37
J.R. Simplot Company Evidence for Stability of the Inserted DNA 11

DNA Stability in Russet Burbank Events E12 and E24

Hybridization of DNA of Russet Burbank E12 and E24 plants visualizes two common bands with the GBS 

probe (7.5 and 8.5 kb) (Figures 8, 9, and 10) and three with the AGP probe (1.5, 4.4 and 5.2 kb)in all 

lanes (Figures 11, 12, and 13). These bands are indicative of the unmodified genome. Two additional 

bands in DNA of all event material, one 2.3 kb band indicative of an internal DNA insert fragment and 

the other one representing a DNA insert junction fragment (4.5 kb for E12 with GBS, ~23 kb for E24 with 

GBS, 2.2 kb for E12 with AGP, and ~20 kb for E24 with AGP), indicate that the inserts of the original 

transformant (G0) remained stable into the first, second, and third vegetative generations G1, and G2,

and G3 (Figures 8, 9, and 10).

Additional evidence for DNA insert stability in events E12 and E24 was obtained with the catechol assay 

for Ppo activity, using tubers from the second generation. As shown in Figure 14, the G2 tubers did not 

brown, except for the untransformed control material. This demonstrated that the events for each 

variety had retained their ability to silence the Ppo5 gene in tubers, after two years of field trials.

Preparation of the field trial material had taken the events through repeated tissue culture and 

vegetative generations without affecting the stability of the inserted genetic material. Note: Slight 

differences in flesh color of the event tubers is from natural variation and is not related to Ppo silencing.

Overall, hyridization (Figures 8, 9, 10, 11, 12, and 13) with the GBS and AGP probes shows that events 

E12 and E24 had consistent bands over multiple generations by comparing tubers of G0 with G1, G2 and 

G3. Also, the visual assay for PPO with catechol (Figure 14) confirmed stability of Ppo5 gene silencing 

from the initial generation through G2. 

J.R. Simplot Company Evidence for Stability of the Inserted DNA 13

Figure 9. E12 and E24: DNA Insert Stability in G2 Samples as Visualized with a GBS Probe

M M wt wt wt E12 E12 E12 E24 E24 E24 Line

II VII G0 G2 G2 G0 G2 G2 G0 G2 G2 Generation

23.1

9.4

8.6

7.4

6.6

4.9

4.4

3.6

2.8

2.3

2.0

1.51

1.48

1.2

0.99

6.1

1.95

1.88

Russet Burbank untransformed controls (wt), E12, and E24 all show consistency with 7.5 kb and 8.5 kb

size bands in the G0 and G2 plants. E12 and E24 share a band the size of 2.3 kb in both the G0 and the 

G2 plants. E12 has a 4.5 kb band and E24 has a ~23 kb band in the G0 and G2 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 14

Figure 10. E12 and E24: DNA Insert Stability in G3 Samples as Visualized with a GBS Probe

Russet Burbank untransformed controls (wt), E12, and E24 all show consistency with 7.5 kb and 8.5 kb

size bands in the G0 and G3 plants. E12 and E24 share a band the size of 2.3 kb in both the G0 and the 

G3 plants. E12 has a 4.5 kb band and E24 has a ~23 kb band in the G0 and G3 plants.

M M wt wt wt E12 E12 E12 E24 E24 E24 

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3 

2.8

2.3

2.0

1.5

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9

1.0

0.7

J.R. Simplot Company Evidence for Stability of the Inserted DNA 16

Figure 12. E12 and E24: DNA Insert Stability in G2 Samples as Visualized with an AGP Probe

M M wt wt wt E12 E12 E12 E24 E24 E24 Line

II VII G0 G2 G2 G0 G2 G2 G0 G2 G2 Generation

23.1

9.4

8.6

7.4

6.6

4.9

4.4

3.6

2.8

2.3

2.0

1.51

1.48

1.2

0.99

6.1

1.95

1.88

Russet Burbank untransformed controls (wt), E12, and E24 all show consistency with 1.5, 4.4, and 5.2 kb 

kb size bands in the G0 and G2 plants. E12 and E24 share a band the size of 2.3 kb in both the G0 and 

the G2 plants. E12 has a 2.2 kb band (slightly below the 2.2 kb band) and E24 has a ~20 kb band in the 

G0 and G2 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 17

Figure 13. E12 and E24: DNA Insert Stability in Samples as Visualized with an AGP Probe

Russet Burbank untransformed controls (wt), E12, and E24 all show consistency with 1.5, 4.4, and 5.2 kb 

(very light bands) size bands in the G0 and G3 plants. E12 and E24 share a band the size of 2.3 kb in both 

the G0 and the G3 plants. E12 has a 2.2 kb band (slightly below the 2.2 kb band) and E24 has a ~20 kb 

band in the G0 and G3 plants.

M M wt wt wt E12 E12 E12 E24 E24 E24 

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3 

2.8

2.3

2.0

1.5

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9
J.R. Simplot Company Evidence for Stability of the Inserted DNA 18

Figure 14. Assay for Sustained Ppo5 Gene Silencing in G2 Tubers of E12 and E24
J.R. Simplot Company Evidence for Stability of the Inserted DNA 19

DNA Insert Stability in Events J3, J55 and J78

Hybridization of DNA of Atlantic, J3, J55 and J78 plants visualizes three common bands with the GBS 

probe (7.0, 7.8, and 8.6 kb) and four with the AGP probe (1.4, 2.0, 5.0, and 7.2 kb) in all lanes (Figures 

15, 16, 17, 18, 19, and 20). These bands are indicative of DNA fragments of the unmodified genome.

Three additional bands in DNA of J3, one 2.3 kb band indicative of an internal DNA insert fragment and 

two others representing DNA insert junction fragments (5.8 and ~12 kb with GBS and 1.6 and 5.7 kb 

with AGP), indicate that the inserts of the original transformant (G0) remained stable into the first, 

second, and third vegetative generations (Figures 15, 16, 17, 18, 19, and 20). Similarly, J55 contains the 

internal 2.3 kb internal band and additional bands (2.3 and 4.1 kb with GBS and 1.95, 2.3 and 4.8 kb with 

AGP) that are identical in G0, G1, G2, and G3 samples (Figures 15, 16, 17, 18, 19, and 20). Event J78 also 

contains the internal 2.3 kb DNA insert fragment in all generations and a junction fragment that only 

hybridizes with the AGP probe (~9 kb) (Figures 18, 19, and 20). There is no junction fragment with the 

GBS probe because the J78 insert had a DNA insert deletion of the Gbss promoter at the Left Border site 

(as described in the Characterization of Inserted DNA study).

The Atlantic intrangenic G2-tubers could not be assayed for Ppo activity because cut tuber surfaces of 

this untransformed variety do not develop a brown precipitant when exposed to catechol. It is possible 

that they do not produce the enzyme that catalyzes the oxidation of polyphenols. Instead, additional 

evidence for DNA insert stability in events J3, J55 and J78 was obtained with a PCR test using primers 

HD121F and HD121R (see Figure 39 in Materials and Methods), which demonstrated that all Samples

analyzed contained the same amplified 0.8 kb product (part of the Asn1/ Ppo5 gene) and showing 

stability of the DNA insert in G2 Samples (Figures 21 and 22). Although G0 Samples were not available 

for this test, the presence of part of the Asn1/ Ppo5 gene indicates insertion stability in G2 Samples.

Overall, hybridization (Figures 15, 16, 17, 18, 19, and 20) with the GBS and AGP probes shows that 

events J3, J55, and J78 had consistent bands over multiple generations by comparing samples of G0 with 

G1, G2 and G3. Also, PCR testing (Figures 21 and 22) showed amplification of the same 788 bp DNA 

insert fragment in all G2 events, that was not found in the control Atlantic samples, indicating that the 

DNA insert was stable through at least the second generation.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 20

Figure 15. Atlantic J3, J55, J78: DNA Insert Stability in G0 and G1 Samples as Visualized with a GBS 

Probe

Atlantic untransformed controls (wt), J3, J55, and J78 all show consistency with 7.1 kb, 7.8 kb, 8.6 kb size 

bands in the G0 and G1 plants. J3 and J78 share a band the size of 2.3 kb in both the G0 and the G1 

plants. Additionally, J3 has 5.8 kb and ~12 kb bands in the G0 and G1 plants. J55 has bands of 2.3 kb and 

4.1 kb in the G0 and G1 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 21

Figure 16. Atlantic J3, J55, J78: DNA Insert Stability in G2 Samples as Visualized with a GBS Probe

M M wt wt wt J3 J3 J3 J55 J55 J55 J78 J78 J78 Line

II VII G0 G2 G2 G0 G2 G2 G0 G2 G2 G0 G2 G2 Generation

23.1

9.4

8.6

7.4

6.6

4.9

4.4

3.6

2.8

2.3

2.0

1.51

1.48

1.2

0.99

6.1

1.95

1.88

Atlantic untransformed controls (wt), J3, J55, and J78 all show consistency with 7.1 kb, 7.8 kb, 8.6 kb size 

bands in the G0 and G2 plants. J3 and J78 share a band the size of 2.3 kb in both the G0 and the G2

plants. Additionally, J3 has 5.8 kb and ~12 kb bands in the G0 and G2 plants. J55 has bands of 2.3 kb and 

4.1 kb in the G0 and G2 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 22

Figure 17. Atlantic J3, J55, J78: DNA Insert Stability in G3 Samples as Visualized with a GBS Probe1

1

Tubers were unavailable for this test so leaves from third generation plants were used.

Atlantic untransformed controls (wt), J3, J55, and J78 all show consistency with 7.1 kb, 7.8 kb, 8.6 kb size 

bands in the G0 and G3 plants. J3 and J78 share a band the size of 2.3 kb in both the G0 and the G3

plants. Additionally, J3 has 5.8 kb and ~12 kb bands in the G0 and G3 plants. J55 has bands of 2.3 kb and 

4.1 kb in the G0 and G3 plants.

M M J3 J3 J3 J55 J55 J55 J78 J78 J78 WT WT WT

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3 G0 G3 G3

2.8

2.3

2.0

1.5

1.2

1.0

0.7

0.5

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9
J.R. Simplot Company Evidence for Stability of the Inserted DNA 23

Figure 18. Atlantic J3, J55, J78: DNA Insert Stability in G0 and G1 Samples as Visualized with an AGP 

Probe

Atlantic untransformed controls (wt), J3, J55, and J78 all show consistency with 1.4 kb, 2.0 kb, 5.0 kb, 

and 7.2 kb size bands in the G0 and G1 plants. J3 also has bands at 1.6 and 5.7 kb in G0 and G1 plants. J3 

and J78 share a band the size of 2.3 kb in both the G0 and the G1 plants. Additionally, J55 has bands the 

size of 1.95, 2.3, and 4.8 kb in G0 and G1 plants. J78 has a band the size of ~9 kb in G0 and G1 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 24

Figure 19. Atlantic J3, J55, J78: DNA Insert Stability in G2 Samples as Visualized with an AGP 

Probe

M M wt wt wt J3 J3 J3 J55 J55 J55 J78 J78 J78 Line

II VII G0 G2 G2 G0 G2 G2 G0 G2 G2 G0 G2 G2 Generation

23.1

9.4 8.6

7.4

6.6

4.9

4.4

3.6

2.8

2.3

2.0

1.51

1.48

1.2

0.99

6.1

1.95

1.88

Atlantic untransformed controls (wt), J3, J55, and J78 all show consistency with 1.4 kb, 2.0 kb, 5.0 kb, 

and 7.2 kb size bands in the G0 and G2 plants. J3 also has bands at 1.6 and 5.7 kb in G0 and G2 plants. J3 

and J78 share a band the size of 2.3 kb in both the G0 and the G2 plants. Additionally, J55 has bands the 

size of 1.95, 2.3, and 4.8 kb in G0 and G2 plants. J78 has a band the size of ~9 kb in G0 and G2 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 25

Figure 20. Atlantic J3, J55, J78: DNA Insert Stability in G3 Samples as Visualized with an AGP Probe1

1

Tubers were unavailable for this test so leaves from third generation plants were used.

Atlantic untransformed controls (wt), J3, J55, and J78 all show consistency with 1.4 kb, 2.0 kb, 5.0 kb, 

and 7.2 kb size bands in the G0 and G2 plants. J3 also has bands at 1.6 and 5.7 kb in G0 and G2 plants. J3 

and J78 share a band the size of 2.2 kb in both the G0 and the G2 plants. Additionally, J55 has bands the 

size of 1.95, 2.3, and 4.8 kb in G0 and G2 plants. J78 has a band the size of ~9 kb in G0 and G2 plants.

M M J3 J3 J3 J55 J55 J55 J78 J78 J78 WT WT WT

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3 G0 G3 G3

2.8

2.3

2.0

1.5

1.2

1.0

0.7

0.5

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9
J.R. Simplot Company Evidence for Stability of the Inserted DNA 26

Figure 21. PCR-Based Confirmation of Insert Stability in G2 Tubers of Events J3, J55 and J78

The first lane of each gel contains Atlantic controls labeled JC, followed by a 1 kb DNA ladder 

(Invitrogen). The subsequent 8 lanes contain DNA from non-systematically chosen G2 tubers from 

Hancock, Parma, Cody, and Aberdeen. Headings above each gel correspond to the site location where 

the tubers were grown. A consistent band of 788 bp of DNA insert was amplified from the G2 tubers, 

but not controls, indicating stable integration of the DNA insert.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 27

Figure 22. PCR-Based Confirmation of Insert Stability in G2 Tubers of Events J3, J55 and J78

The first lane of each gel contains Atlantic controls labeled JC, followed by a 1 kb DNA ladder 

(Invitrogen). The subsequent 8 lanes contain DNA from non-systematically chosen G2 tubers from 

Othello, Lakeview, and Larimore. Headings above each gel correspond to the site location where the 

tubers were grown. A consistent band of 788 bp of DNA insert was amplified from the G2 tubers, but 

not controls, indicating stable integration of the DNA insert.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 28

DNA Insert Stability in Event G11

Hybridization of DNA of the G variety and G11 plants visualizes two common bands with the GBS probe 

(8.2 and ~12 kb) and four with the AGP probe (1.45, 2.1, 5.3, and 8.6 kb) in all lanes. These bands are 

indicative of DNA fragments of the unmodified genome. One additional internal DNA insert band of 2.2

kb that hybridizes with both the GBS and AGP probes indicates that the insert of the original 

transformant (G0) remained stable into the first, second, and third vegetative generations G1,G2, and 

G3 (Figures 23, 24, 25, 26, 27, and 28). An additional 5.2 kb band only hybridizing with the AGP probe 

represented a DNA insert junction fragment and confirmed the insert stability.

Additional evidence for DNA insert stability in event G11 was obtained with the catechol assay for Ppo 

activity. As shown in Figure 29, all event tubers remained uncolored, whereas the untransformed 

control material developed a deep brown color. This demonstrated that the G11 event had retained 

their ability to silence the Ppo5 gene in tubers, after two years of field trials. Preparation of the field trial 

material had taken the events through repeated tissue culture and vegetative generations without 

affecting the stability of the inserted genetic material.

Overall, hyridization (Figures 23, 24, 25, 26, 27, and 28) with the GBS and AGP probes shows that event 

G11 had consistent bands over multiple generations by comparing samples of G0 with G1, G2 and G3. 

Also, the visual assay for PPO with catechol (Figure 29) confirmed stability of Ppo5 gene silencing from 

the initial generation through G2.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 29

Figure 23. G11: DNA Insert Stability in G0 and G1 Samples Visualized with a GBS Probe1

1GC = control G; G1-1 and G1-2 refer to leaves from 2 independent G1 plants

G variety untransformed controls (wt) and G11 show consistency with 8.2 kb and ~12 kb size bands in 

the G0 and G1 plants. In addition G11 has an additional band of 2.2 kb in both the G0 and G1 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 30

Figure 24. G11: DNA Insert Stability in G2 Samples as Visualized with a GBS Probe1

1GC = G Control

G variety untransformed controls (wt) and G11 show consistency with 8.2 kb and ~12 kb size bands in 

the G0 and G2 plants. In addition G11 has an additional band of 2.2 kb in both the G0 and G2 plants.

23.1

9.4

8.6

7.4

6.6

4.9

4.4

3.6

2.8

2.3

2.0

1.51

1.48

6.1

1.95

1.88

 M M GC GC GC G11 G11 G11 

 II VII G0 G2 G2 G0 G2 G2 
J.R. Simplot Company Evidence for Stability of the Inserted DNA 31

Figure 25. G11: DNA Insert Stability in G3 Samples as Visualized with a GBS Probe

1GC = control G

G variety untransformed controls (wt) and G11 show consistency with 8.2 kb and ~12 kb size bands in 

the G0 and G3 plants. In addition G11 has an additional band of 2.2 kb in both the G0 and G3 plants.

M M wt wt wt G11 G11 G11 

II VII G0 G3 G3 G0 G3 G3 

2.8

2.3

2.0

1.5

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9
J.R. Simplot Company Evidence for Stability of the Inserted DNA 32

Figure 26. G11: DNA Insert Stability in G0 and G1 Samples Visualized with an AGP Probe1

1GC = control G, G1-1 and G1-2 refer to samples from 2 independent G1 plants

G variety untransformed controls (wt) and G11 show consistency with 1.45 kb, 2.1 kb, 5.3 kb, and 8.6 kb 

size bands in the G0 and G1 plants. G11 has two additional size bands of 2.2 kb and 5.2 kb in both the G0 

and G1 plants. 
J.R. Simplot Company Evidence for Stability of the Inserted DNA 33

Figure 27. G11: DNA Insert Stability in G2 Samples as Visualized with an AGP Probe1

1GC = control G 

G2-1 and G2-2 refer to samples from 2 independent G2 plants

G variety untransformed controls (wt) and G11 show consistency with 1.45 kb, 2.1 kb, 5.3 kb, and 8.6 kb 

size bands in the G0 and G2 plants. G11 has two additional size bands of 2.2 kb and 5.2 kb in both the G0 

and G2 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 34

Figure 28. G11: DNA Insert Stability in G3 Samples as Visualized with an AGP Probe

G variety untransformed controls (wt) and G11 show consistency with 1.45 kb, 2.1 kb, 5.3 kb, and 8.6 kb 

size bands in the G0 and G3 plants. G11 has two additional size bands of 2.2 kb and 5.2 kb in both the G0 

and G3 plants.

M M wt wt wt G11 G11 G11 

II VII G0 G3 G3 G0 G3 G3 

2.8

2.3

2.0

1.5

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9
J.R. Simplot Company Evidence for Stability of the Inserted DNA 35

Figure 29. Assay for Sustained Ppo5 Gene Silencing in G2 Tubers of Event G11
J.R. Simplot Company Evidence for Stability of the Inserted DNA 36

DNA Insert Stability in Events H37 and H50

Hybridization of DNA of the H variety as well as H37 and H50 plants visualizes two common bands with 

the GBS probe (7.8 and 10.0 kb) and five with the AGP probe (1.4, 2.1, 5.4, 6.7, and 8.0) in all lanes. 

These bands are indicative of DNA fragments of the unmodified genome. Three additional GBShybridizing bands in DNA of H37, one 2.3 kb band indicative of an internal DNA insert fragment and two 

others representing DNA insert junction fragments (3.0 and 4.5 kb), indicate that the inserts of the 

original transformants (G0) remained stable into the first, second, and third vegetative generations G1,

G2, and G3 (Figures 30, 31, 32, 33, 34, and 35). Similarly, there are three additional bands consistently 

visualized with the AGP probe in H37 DNA (2.0, 3.2 and 3.6 kb).

Event H50 also contains the 2.3 kb internal DNA insert fragment in all generations. Additionally, it carries 

a single junction fragment hybridizing with the GBS probe (6.5 kb) and two with the AGP probe (1.9 and 

3.8 kb), as was expected based on the DNA insert organization of this event. 

Additional evidence for DNA insert stability in events H37 and H50 was obtained with the catechol assay 

for Ppo activity. As shown in Figures 36 and 37, all event tubers remained uncolored, compared with the 

dark-colored untransformed control material. This demonstrated that these events for this variety had 

retained their ability to silence the Ppo5 gene in G2 tubers, after two years of field trials. Preparation of 

the field trial material had taken the events through repeated tissue culture and vegetative generations 

without affecting the stability of the inserted genetic material.

Overall, hyridization (Figures 30, 31, 32, 33, 34, and 35) with the GBS and AGP probes shows that events 

H37 and H50 had consistent bands over multiple generations by comparing samples of G0 with G1, G2 

and G3. Also, the visual assay for PPO with catechol (Figures 36 and 37) confirmed stability of Ppo5 

gene silencing from the initial generation through G2.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 37

Figure 30. H37 and H50: DNA Insert Stability in G0 and G1 Samples Visualized with a GBS Probe

 MII MVII WT WT WT H37 H37* H37 H50 H50 H50 

 G0 G1 G1 G0 G1 G1 G0 G1 G1 

*Background smear in the lane marked “H37 G1” at 1.7 kb is not a band.

H variety untransformed controls (wt), H37, and H50 show consistency with 7.8 kb and 10.0 kb size 

bands in the G0 and G1 plants. H37 and H50 have one additional band of 2.3 kb in size in G0 and G1 

plants. Additionally, H37 has two bands of 3.0 kb and 4.5 kb in size. H50 has one additional band with a 

size of 6.5 kb in G0 and G1 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 38

Figure 31. H37 and H50: DNA Insert Stability in G0 and G2 Samples Visualized with a GBS Probe

 MII MVII WT WT WT H37 H37 H37 H50 H50 H50 

 G0 G2 G2 G0 G2 G2 G0 G2 G2 

M M wt wt wt H37 H37 H37 H74 H74 H74 Line

II VII G0 G2 G2 G0 G2 G2 G0 G2 G2 Generation

23.1

9.4 8.6

7.4

6.6

4.9

4.4

3.6

2.8

2.3

2.0

1.51

1.48

1.2

0.99

6.1

1.95

1.88

H variety untransformed controls (wt), H37, and H50 show consistency with 7.8 kb and 10.0 kb size 

bands in the G0 and G2 plants. H37 and H50 have one additional band of 2.3 kb in size in G0 and G2 

plants. Additionally, H37 has two bands of 3.0 kb and 4.5 kb in size. H50 has one additional band with a 

size of 6.5 kb in G0 and G2 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 39

Figure 32. H37 and H50: DNA Insert Stability in G3 Samples as Visualized with a GBS Probe

H variety untransformed controls (wt), H37, and H50 show consistency with 7.8 kb and 10.0 kb size 

bands in the G0 and G3 plants. H37 and H50 have one additional band of 2.3 kb in size in G0 and G3 

plants. Additionally, H37 has two bands of 3.0 kb and 4.5 kb in size. H50 has one additional band with a 

size of 6.5 kb in G0 and G3 plants.

M M wt wt wt H37 H37 H37 H50 H50 H50 

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3 

2.8

2.3

2.0

1.5

23.1

9.4

8.6

7.4

6.6

6.1

4.9

4.4

3.6

1.9
J.R. Simplot Company Evidence for Stability of the Inserted DNA 40

Figure 33. H37 and H50: DNA Insert Stability in G0 and G1 Samples Visualized with an AGP Probe

 MII MVII WT WT WT H37 H37 H37 H50 H50 H50 

 G0 G1 G1 G0 G1 G1 G0 G1 G1 

H variety untransformed controls (wt), H37, and H50 show consistency with 1.4 kb, 2.1 kb, 5.4 kb, 6.7

kb, and 8.0 kb size bands in the G0 and G1 plants. H37 contains three additional bands of 2.0 kb, 3.2 kb, 

and 3.6 kb in size. H50 has two additional bands of 1.9 kb and 3.8 kb in size in G0 and G1 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 41

Figure 34. H37 and H50: DNA Insert Stability in G0 and G2 Samples Visualized with an AGP Probe

 MII MVII WT WT WT H37 H37 H37 H50 H50 H50 

 G0 G2 G2 G0 G2 G2 G0 G2 G2 

M M wt wt wt H37 H37 H37 H74 H74 H74 Line

II VII G0 G2 G2 G0 G2 G2 G0 G2 G2 Generation

23.1

9.4 8.6

7.4

6.6

4.9

4.4

3.6

2.8

2.3

2.0

1.51

1.48

1.2

0.99

6.1

1.95

1.88

H variety untransformed controls (wt), H37, and H50 show consistency with 1.4 kb, 2.1 kb, 5.4 kb, 6.7

kb, and 8.0 kb size bands in the G0 and G2 plants. H37 contains three additional bands of 2.0 kb, 3.2 kb, 

and 3.6 kb in size. H50 has two additional bands of 1.9 kb and 3.8 kb in size in G0 and G2 plants.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 42

Figure 35. H37 and H50: DNA Insert Stability in G3 Samples as Visualized with an AGP Probe

H variety untransformed controls (wt), H37, and H50 show consistency with 1.4 kb, 2.1 kb, 5.4 kb, 6.7

kb, and 8.0 kb size bands in the G0 and G3 plants. H37 contains three additional bands of 2.0 kb, 3.2 kb, 

and 3.6 kb in size. H50 has two additional bands of 1.9 kb and 3.8 kb in size in G0 and G3 plants.

M M wt wt wt H37 H37 H37 H50 H50 H50 

II VII G0 G3 G3 G0 G3 G3 G0 G3 G3 

2.8

2.3

2.0

1.5

1.2

23.1

9.4

8.6

7.4

6.6

6.1

4.9

3.6

1.9

4.6
J.R. Simplot Company Evidence for Stability of the Inserted DNA 43

Figures 36. Assay for Sustained Ppo5 Gene Silencing in G2 Tubers of Events H37 and H50
J.R. Simplot Company Evidence for Stability of the Inserted DNA 44

It should be mentioned here that this assessment did identify insert instability in another event, which is 

not part of this petition. The G2 tubers of this event displayed instability at a low frequency (Figure 37). 

Of the 50 tubers tested, one tuber developed some color when subjected to catechol. This color 

development is probably indicative of a partial reversion to Ppo5 gene expression. Importantly, this 

indicates that the visual catechol test is effective for detecting gene instability in potato events 

transformed with this construct. Given the demonstrated sensitivity of the test to identify low 

frequencies of instability, it will be used to monitor trait stability in commercial potato seed (except for 

Atlantic, where the PCR-based stability test will be used).

Figure 37. Assay for Ppo5 Gene Silencing in G2 Tubers of a G Event That is Not Considered for 

Deregulation
J.R. Simplot Company Evidence for Stability of the Inserted DNA 45

DISCUSSION

Given the demonstrated DNA insert stability in the 10 vegetatively propagated events described in this 

petition, it is unlikely for instability to arise during subsequent cycles of vegetative propagation. 

Continued trait efficacy could be readily confirmed by performing the catechol assay for Ppo activity on 

non-systematically chosen tubers. This assay is indicative of not only black spot bruise tolerance but also 

low asparagine formation because Ppo5 and Asn1 gene silencing is mediated by the same silencing 

cassette. In the unlikely case that a specific line would display trait instability, individual tubers would

revert back, either partially or fully, to the phenotype of their untransformed counterparts. Such 

reversion events will not be associated with the expression of phenotypes associated with pests or 

noxious weeds. For the Atlantic events, the PCR detection assay can be used instead of the catechol 

assay.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 46

MATERIALS and METHODS

Materials. DNA insert stability was demonstrated in the originally-transformed material (G0) by 

extracting and evaluating DNA from leaves of plants that had been propagated in vitro and never 

planted in soil. For generation-1 (G1) analyses, two propagated plants from each event and one plant 

from each control were planted in the greenhouse; one of the tubers harvested from each plant was 

planted to obtain leaves from G1 plants that were used to isolate DNA and evaluate the G1 generation. 

Tubers from this generation were planted again, and leaves of the resulting G2 plants allowed a 

characterization of that generation. For event F37, G2 tubers rather than leaves were used for the 

analyses. For all G3 samples, the genomic DNA was extracted from tubers. 

In summary, all G0, G1, and G2 DNA was from leaves except that G2 material from F37 was from tubers, 

and G3 material all came from tubers. Extracted DNA from leaves or tubers were digested with EcoRV 

and hybridized with two probes (GBS and AGP). 

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 -80°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-suspended in 2 ml extraction buffer containing 200 μg RNase A. After incubating the resuspended 

DNA at 65oC 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. It should be mentioned here 

that control and event DNA were always extracted with the same method for a specific comparison. For 

event F37, G2 tubers rather than leaves were used for DNA extraction using the same protocol.

DNA Gel (Southern) Blot Analysis. The probes used in the Southern blot analysis are represented in 

Figure 38. 10 μg of DNA was digested overnight in 400 μl final volume with at least 5 μl (10 units/μl) 

restriction enzyme (Invitrogen) at 37°C. Digested DNA was concentrated by ethanol precipitation (40 μl 

of 3M NaOAc, pH5.3 and 1 ml ethanol) at -80oC for 10 min followed by a wash with 70% ethanol. The 

DNA pellet was dissolved in 20 μl 1X TE followed by addition of 2 μl DNA gel loading buffer, which 

consists of 40% sucrose and 0.35% Orange G (Sigma) in water.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 47

Figure 38. Probes Used for the Southern Analysis

Membrane Preparation. Digested DNA was loaded on a large 0.7% agarose gel (170 ml) containing 0.5X 

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 2 X 15 min and neutralization in 1.5 M NaCl and 0.5 M Tris-HCl, pH7.5, for 2X15 min on a 

shaker at room temperature and 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 (Sambrook 

and Russell 2001). 

PCR-based stability test. For Atlantic, insert stability was evaluated with PCR using tuber-extracted DNA 

as template and the primer set HD121F (5’-CCTCTCGTAAATTCCGACAAA, at position 2113-2133 of 

the DNA insert, which is the 3’ end of AGP promoter) and HD121R (5’-TCTCAACTTGCGAACCTGAC, at 

position 2882-2901 of the spacer in the first inverted repeat) to amplify a 789-bp DNA insert fragment 

containing part of the Asn1/Ppo5 gene. Figure 39 below shows the location of the amplified fragment 

on the DNA insert.

Figure 39. PCR Primer Positions for Confirmation of Stability in Atlantic Events

Catechol assay for Ppo activity. The second stability assessment of the events was based on their black 

spot bruise susceptibility. The events including Ranger Russet and Russet Burbank were planted in plots 

with three replications in the spring for the 2nd year field trials (2010) in Parma, Larimore, Aberdeen, and 

Othello (Table 1). They were grown and harvested using standard commercial production practices. For 

each event, twelve or thirteen tubers from each site (four or five from each rep) for a total of 50 G2-

tubers from all four sites were non-systematically selected from harvested tubers (see the following 

Table 1). Similarly, G and H plants were planted in Rhinelander, WI and 50 G2-tubers were nonsystematically selected from harvested tubers (Table 1). One slice of each tuber was then assayed for 

the absence of the functional PPO enzyme by adding 1.0 mL of 25 mM catechol in 50 mM MOPS buffer, 

pH 6.5, onto each freshly cut tuber slice surface and reviewing for color development.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 48

Table 1. Number of Tubers from Each Location Assayed for PPO Activity with Catechol

Location Event 

F10

Event 

F37

Event 

E12

Event 

E24

Event 

G11

Event 

H37

Event 

H50

Parma, ID 13 13 13 13 * * *

Larimore, ND 12 12 12 12 * * *

Aberdeen, ID 13 12 12 12 * * *

Othello, WA 12 13 13 13 * * *

Rhinelander, WI * * * * 50 50 50

Total Number of Tubers 50 50 50 50 50 50 50

* None from this location
J.R. Simplot Company Evidence for Stability of the Inserted DNA 49

CONCLUSIONS

Overall Conclusion: Our data provides evidence that the genetic insertions remain stable, which was 

confirmed in the genotype and phenotype of the 10 events that are the subject of this submission, 

meaning that their inserts are stable in the original transformants as well as in G1, G2, and G3 plant 

materials. Because potato is a vegetatively propagated crop, material from tissue culture and tubers 

were used in stability testing. 

Ranger Russet Events F10 and F37: Hybridization with the GBS and AGP probes shows that events F10 

and F37 had consistent bands over multiple generations by comparing samples of G0 with G1, G2 and 

G3. Also, the visual assay for PPO with catechol confirmed stability of Ppo5 gene silencing from the 

initial generation through G2. 

Russet Burbank Events E12 and E24: Hybridization with the GBS and AGP probes shows that events E12 

and E24 had consistent bands over multiple generations by comparing samples of G0 with G1, G2 and 

G3. Also, the visual assay for PPO with catechol confirmed stability of Ppo5 gene silencing from the 

initial generation through G2.

Atlantic Events J3, J55, and J78: Hybridization with the GBS and AGP probes shows that events J3, J55, 

and J78 had consistent bands over multiple generations by comparing samples of G0 with G1, G2 and 

G3. Also, PCR testing showed amplification of the same 788 bp DNA insert fragment in all G2 events, 

that was not found in the control Atlantic samples, indicating that the DNA insert was stable through at 

least the second generation.

G Event G11: Hybridization with the GBS and AGP probes shows that event G11 had consistent bands 

over multiple generations by comparing samples of G0 with G1, G2 and G3. Also, the visual assay for 

PPO with catechol confirmed stability of Ppo5 gene silencing from the initial generation through G2.

H Events H37 and H50: Hybridization with the GBS and AGP probes shows that events H37 and H50 had 

consistent bands over multiple generations by comparing samples of G0 with G1, G2 and G3. Also, the 

visual assay for PPO with catechol confirmed stability of Ppo5 gene silencing from the initial generation 

through G2.

Thus, the stability of the inserts has been confirmed and trait stability monitoring will remain part of the 

commercial strategy for release of planting material for these events. Importantly, the stability of the 

inserts will not alter the pest characteristics of these events compared to conventional potato varieties 

and will not alter the environmental impact of potato production compared to conventional varieties.
J.R. Simplot Company Evidence for Stability of the Inserted DNA 50

REFERENCES

Sambrook J, Russell DW (2001) Southern Hybridization. In: Molecular Cloning: A Laboratory Manual. (Ed. 

Argentine J) Cold Springs Harbor Laboratory Press, Cold Springs Harbor, NY (pp. 6.33-6.38).