J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 1

Appendix 5

Efficacy and Tissue-Specificity of Gene Silencing

INDEX

Section Page

Index 1

Introduction 2

Results 3

Summary 31

Materials and Methods 33

Conclusions 35

References 37

Certification 38
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 2

INTRODUCTION

Expression of the four genes targeted for gene silencing by transformation with pSIM1278 was tested in 

tubers, stolons, leaves, stems, roots, and flowers of the potatoes. The purpose of these studies was to 

show the effectiveness of gene silencing based on RNA expression as measured through Northern blot 

analyses. It’s important to note that interpretation of the Northern blots requires judgment and the

reported results represent a qualitative approach whereby the genes were considered downregulated. 

All down-regulated genes indicated in the summary (Table 1) appeared reduced when compared with 

the respective controls, without differentiation between reduction levels, as appropriate for a 

qualitative method. When both greenhouse and field tubers were analyzed, a judgment was made after 

reviewing both gels.

Considering the potato events in this study, expression of the targeted genes would provide evidence 

that the desired phenotype with low asparagine, reduced black spot bruise, and lowered reducing 

sugars had been achieved. It could provide information relevant to the plant pest risk assessment, for 

example, all InnateTM potatoes in the study had reduced or similar expression to the controls. Thus, the 

altered expression is unlikely to affect weediness or other plant pest characteristics. Also, with these 

events, the expression studies pertain to endogenous proteins rather than new proteins that could 

require a safety evaluation, such as those associated with herbicide tolerance or insect resistance. 

Gene silencing methods were employed to lower the activity of the Asn1, Ppo5, PhL, and R1 native

proteins, and transcript levels rather than protein amounts were evaluated to link new phenotypic traits 

to changes at the molecular level. For this purpose, RNA was isolated from various tissues, and the RNA 

was used to carry out Northern blot analyses. 

The promoters used to drive expression of the two inverted repeats, the Agp promoter and the Gbss 

promoter, have been well characterized and are known to be highly active in tubers and stolons while

inducing some transcription in photosynthetically-active tissues and roots (Nakata et al. 1994; Visser et 

al. 1991). Therefore, silencing effects mediated by expression of the inverted repeats were predicted to 

be strong in tubers and stolons, and less pronounced in leaves, stems, roots, and flowers. The silencing 

effects in tissues other than tubers and stolons may have been further accentuated by the anticipated 

mobility of small RNAs produced by the silencing cassettes (Molnar et al. 2010). 

Northern blot analyses were carried out to determine the actual effects of the transformations on 

expression levels of the four targeted genes in a tissue-specific manner for each event. 

Most studies were carried out on tissues from greenhouse-grown material including tubers, stolons, 

roots, stems, leaves, and flowers. Additionally, some field-grown tubers were analyzed as well because 

tubers are the target tissues for down-regulation of the four genes.
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 3

RESULTS

Transcript Levels in Tubers

The transcript levels of target genes in various tissues of untransformed plants and the events were 

determined by Northern blot analysis.

In tubers of untransformed controls as viewed by Northern blot analyses, transcript levels were higher 

compared with the events for the Asn1, Ppo5, PhL, and R1 genes. A comparison of northern blots 

indicated that the Asn1, Ppo5, and PhL were expressed at similar levels in tubers from greenhouse 

(Figures 1A, 2A, 3A and 4A) and field (Figures 1B, 2B, 3B and 4B). One exception was observed with 

Ppo5 silencing, in which greenhouse-grown tubers had higher expression levels in both controls and 

events than in tubers from the field (Figure 2B). In contrast, the R1 gene was only partially silenced, with 

slight differences observed between events and controls in either greenhouse-grown or tubers from the 

field.

Strongly reduced transcript levels for the Asn1 and Ppo5 genes in tubers of all events were intentional 

and are associated with low-acrylamide potential and black spot bruise tolerance (Figures 1 and 2).

However, Ppo5 gene transcripts in event H50 were slightly higher in greenhouse samples than in tubers 

from the field. This finding may be due to the fact that the H50 tubers from the greenhouse were not 

fully mature; some lingering activity of Ppo genes other than Ppo5, which are only expressed in 

immature tubers, may have caused residual transcript levels.

Transcript levels for the PhL gene were partially reduced in the tubers of most events (Figure 3), except 

F10, F37, J78, and G11. This reduced PhL was intentional, and is linked to reduced amounts of glucose 

and fructose. It was expected that J78 and G11 would not have reduced transcripts of PhL, yet F10 and 

F37 were expected to show silencing of that gene. R1 transcripts were partially reduced (“whispered”) 

in tubers of most lines to help limit the degradation of starch into sugars (Figure 4). Results varied 

between greenhouse and field grown tubers, yet the overall conclusion was that some silencing of R1

occurred in most events.

Events J78 and G11 lack a functional PhL/R1 promoter silencing cassette and, consequently, display no 

reductions in transcript levels for the PhL and R1 genes (Figures 3 and 4). The lack of silencing of PhL in 

F10 and F37, along with slight reductions of R1 in most events, show that generally promoter silencing 

of these genes did not down-regulate genes as much as expected.
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 4

Figure 1. Expression of the Asn1 Gene in Tubers of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown (A) and field-grown (B) tuber tissues of events 

and controls and hybridized with the Asn1 probe (upper panel) and the internal benchmark 18s rRNA 

probe (middle panel). The predicted size of the Asn1 transcript is 2.0-kb. The lower panel shows the 

amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67 and G9 are not considered for deregulation.

A. Greenhouse-grown tuber

 

 

B. Field-grown tubers

 EC E12 E24 FC F10 F37 GC G9 G11 HC H37 H50 JC J3 J55 J78

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G, HC = 

control H, M = RNA marker

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H37 H50 H74

6.9 kb

4.7 kb

2.6 kb

1.8kb

1.5 kb

1.0 kb

575 bp

438 bp

314 bp

18S rRNA

Total RNA

Asn1

M EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H37 H50 
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 5

Figure 2. Expression of the Ppo5 Gene in Tubers of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown (A) and field-grown (B) tuber tissues of events 

and controls and hybridized with the Ppo5 probe (upper panel) and the internal benchmark 18s rRNA 

probe (middle panel). The predicted size of the Ppo5 transcript is 1.95-kb (see Genbank Accession 

U22921). The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB). EC, 

FC, JC, GC and HC are control samples from the untransformed conventional varieties. Individual event 

samples are labeled above each lane. The events E67 and G9 are not considered for deregulation.

A. Greenhouse-grown tubers

B. Field grown-tubers

 EC E12 E24 FC F10 F37 GC G9 G11 HC H37 H50 JC J3 J55 J78

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G, HC = 

control H, M = RNA marker

M EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H37 H50 EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H37 H50 H74

6.9 kb

4.7 kb

2.6 kb

1.8 kb

1.5 kb

1.0 kb

575 bp

438 bp

314 bp

18S rRNA

Total RNA

Ppo5

18s rRNA

Total RNA

Ppo5
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 6

Figure 3. Expression of the PhL Gene in Tubers of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown (A) and field-grown (B) tuber tissues of events 

and controls and hybridized with the PhL probe (upper panel) and the internal benchmark 18s rRNA 

probe (middle panel). The predicted size of the PhL transcript is 4.9-kb (see Genbank Accession X52385). 

The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, 

GC and HC are control samples from the untransformed conventional varieties. Individual event samples 

are labeled above each lane. The events E67 and G9 are not considered for deregulation.

A. Greenhouse-grown tubers

B. Field grown-tubers

 EC E12 E24 FC F10 F37 GC G9 G11 HC H37 H50 JC J3 J55 J78

 

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G, HC = 

control H, M = RNA marker

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H37 H50 H74

6.9 kb

4.7 kb

2.6 kb

1.8 kb

1.5 kb

1.0 kb

575 bp

438 bp

314 bp

18S rRNA

Total RNA

EC E12 E24 E67FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H37 H50 H50 M

PhL
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 7

Figure 4. Expression of the R1 Gene in Tubers of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown (A) and field-grown (B) tuber tissues of events 

and controls and hybridized with the R1 probe (upper panel) and the internal benchmark 18s rRNA 

probe (middle panel). The predicted size of the R1 transcript is 4.8-kb (see Genbank Accession Y09533). 

The lower panel shows the amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, 

GC and HC are control samples from the untransformed conventional varieties. Individual event samples 

are labeled above each lane. The events E67 and G9 are not considered for deregulation.

A. Greenhouse-grown tubers

 

B. Field grown-Tubers

 EC E12 E24 FC F10 F37 GC G9 G11 HC H37 H50 JC J 3 J55 J78

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G, HC = 

control H, M = RNA marker

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H37 H50 H50 M

R1

18s rRNA

Total RNA

R1
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 8

Target Gene Expression in Stolons

As shown in Figures 5, 6, 7, and 8, the Asn1, Ppo5, and R1 genes were expressed at low levels in stolons 

of untransformed plants. In contrast, high transcript levels in the controls were associated with the PhL

gene.

The transcript levels for the Asn1 gene were lower in stolons from all events, although slight for H37, 

compared with control stolons from greenhouse-grown plants (Figure 5). This modification was 

expected and is not linked to any undesired or unanticipated new phenotype.

Transcript levels of Ppo5 were lowered in events E24, G11, and H50 (Figure 6). For Ranger Russet and 

Atlantics, transcript levels in controls and events were at such low levels that silencing was not 

detectable.

Expression of the PhL gene was also down-regulated in the stolons of most events (F10, F37, E12, E24, 

J55, H37, and H50). However, PhL gene expression was unchanged for J78 and G11 stolons, which do 

not contain a functionally-active PhL/R1 promoter silencing cassette (Figure 7). Expression also did not 

appear to be silenced in stolons of J3.

The amounts of transcript for the R1 gene were slightly reduced (“whispered”) in stolons of most events

(Figure 8). This molecular change contributed to lowering of reducing sugars in tubers. However, events 

J78 and G11 lack the PhL/R1 promoter silencing cassette and, consequently, displayed no reductions in 

transcript levels for the associated genes in stolons.
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 9

Figure 5. Expression of the Asn1 Gene in Stolon Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the Asn1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the Asn1 transcript is 2.0-kb. The lower panel shows the amounts of total RNA as 

visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control samples from the 

untransformed conventional varieties. Individual event samples are labeled above each lane. The 

events E67, G9 and H23 are not considered for deregulation. Event H50 was processed separately with 

an H control.

 EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

Asn1

Total RNA

18s rRNA
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 10

Figure 6. Expression of the Ppo5 Gene in Stolon Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the Ppo5 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the Ppo5 transcript is 1.95-kb (see Genbank Accession U22921). The lower panel shows 

the amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67, G9 and H23 are not considered for deregulation. Event H50 was processed 

separately with an H control.

Total RNA
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 11

Figure 7. Expression of the PhL Gene in Stolon Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the PhL probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the PhL transcript is 4.9-kb (see Genbank Accession X52385). The lower panel shows 

the amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67 and G9 are not considered for deregulation. Event H50 was processed 

separately with an H control.

 

 EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

PhL

Total RNA

18s rRNA
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 12

Figure 8. Expression of the R1 Gene in Stolon Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the R1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the R1 transcript is 4.8-kb (see Genbank Accession Y09533). The lower panel shows the 

amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67, G9 and H23 are not considered for deregulation. Event H50 was processed 

separately with an H control.

 EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

R1

Total RNA

18s rRNA
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 13

Transcript Levels in Other Tissues

The Northern expression data for tissues other than tubers and stolons are summarized as below:

(1) In leaf tissues, transcript levels for the Asn1 gene were similar for the events and their 

untransformed counterparts, except for F10, which contained lower levels than Ranger Russet 

(Figure 9). Transcript levels for the Ppo5 gene expression were in all cases undetectable under 

our experimental conditions (Figure 10). This was expected because the Ppo5 gene would be 

most active in tubers and expression was not expected in leaves. Levels for the PhL gene were 

consistently high among the original varieties and their transformed derivatives (Figure 11). 

Transcript levels for the R1 gene were unaltered in most events when compared to their 

controls, except for G11, H37, and H50, which contained lower levels than their respective G

and H controls (Figure 12). 

(2) In stem tissues, Asn1 gene transcript levels were similar for most events and their controls, 

except that F37, E24, J3, and H50 contained less transcript than the control varieties (Figure 13). 

Interestingly, transcript levels for the Ppo5 gene were reduced in all events submitted for 

deregulation, except for G11 (Figure 14). PhL gene expression was similar in all events and their 

controls (Figure 15), and R1 gene expression was reduced in G11 only (Figure 16). 

(3) In root tissues, transcript levels for the Asn1 gene were reduced in six events (F10, F37, E12, J3, 

J55, and H50), and remained unchanged for E24, J78, G11, and H37 (Figure 17). Transcript levels 

for the Ppo5 gene were reduced in F10, F37, E12, H37, and H50 (Figure 18). This indicates that 

the promoters used to drive silencing are partially functional in underground tissues. In Atlantic 

and G varieties, low levels of transcripts were observed in both controls and events, suggesting 

that Ppo5 gene activity may be naturally lower in roots of those varieties. No significant 

reductions in expression levels were observed for the PhL gene, and for the R1 gene F10, F37, 

and H50 were lower (Figures 19 and 20). 

(4) In floral tissues, Asn1 gene transcript levels were lower for the 8 events (the H variety does not 

flower, so no data were obtained for H37 and H50) than for the original varieties (Figure 21). 

Transcripts were not detectable for the Ppo5 gene (Figure 22). Expression levels of the PhL gene 

for all events were not reduced compared with their untransformed controls (Figure 23). For 

the R1 gene, expression levels in F37 were lower than the Ranger Russet control, but all other 

events were similar to controls (Figure 24). 

It can be concluded that transcript levels of the four targeted genes were occasionally down-regulated 

in tissues other than tubers and stolons. In these cases, the reductions were partial and were not 

associated with agronomic differences in the events when compared to untransformed controls in field 

trials.
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 14

Target Gene Expression in Leaf Tissue

Transcript levels in leaf tissues were determined by using greenhouse-grown plants, assuming that 

performance in the greenhouse will predict what occurs in the field. Indeed, there is extensive 

evidence in the scientific literature (Anand et al. 2003; Kuipers et al. 1992; Mikschofsky et al. 2011)

to support that hypothesis.

In leaf tissues, transcript levels for the Asn1 gene were similar for the events and their 

untransformed counterparts, except for F10, which contained lower levels than Ranger Russet 

(Figure 9). Transcript levels for the Ppo5 gene expression were in all cases undetectable under our 

experimental conditions (Figure 10). This was expected because the Ppo5 gene would be most 

active in tubers and expression was not expected in leaves. Levels for the PhL gene were 

consistently high among the original varieties and their transformed derivatives (Figure 11). 

Transcript levels for the R1 gene were unaltered in most events when compared to their controls, 

except for G11, H37, and H50, which contained lower levels than their respective G and H controls 

(Figure 12). 

.
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 15

Figure 9. Expression of the Asn1 Gene in Leaf Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the Asn1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the Asn1 transcript is 2.0-kb. The lower panel shows the amounts of total RNA as 

visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control samples from the 

untransformed conventional varieties. Individual event samples are labeled above each lane. The events 

E67, G9 and H23 are not considered for deregulation. Event H50 was processed separately with an H 

control.

 M EC E12 E24 E67 FC F10 F37 JC J 3 J55 J78 GC G9 G11 HC H23 H37 HC H50

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G, HC = 

control H, M = RNA marker

 

M EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

18S rRNA

Total RNA

Asn1

Asn1
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 16

Figure 10. Expression of the Ppo5 Gene in Leaf Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the Ppo5 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the Ppo5 transcript is 1.95-kb (see Genbank Accession U22921). The lower panel shows 

the amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67, G9 and H23 are not considered for deregulation. Event H50 was processed 

separately with an H control.

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G, HC = 

control H, M = RNA marker

M EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 17

Figure 11. Expression of the PhL Gene in Leaf Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the PhL probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the PhL transcript is 4.9-kb (see Genbank Accession X52385). The lower panel shows 

the amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67, G9, and H23 are not considered for deregulation. Event H50 was processed 

separately with an H control.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 M HC H50

18S rRNA

Total RNA

PhL
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 18

Figure 12. Expression of the R1 Gene in Leaf Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the R1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the R1 transcript is 4.8-kb (see Genbank Accession Y09533). The lower panel shows the 

amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67, G9 and H23 are not considered for deregulation. Event H50 was processed

separately with an H control.

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G, HC = 

control H, M = RNA marker

M EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

18S rRNA

Total RNA

R1
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 19

Target Gene Expression in Stems

In stem tissues, Asn1 gene transcript levels were similar for most events and their controls, except that 

F37, E24, J3, and H50 contained less transcript than the control varieties (Figure 13). Interestingly, 

transcript levels for the Ppo5 gene were reduced in all events submitted for deregulation, except for 

G11 (Figure 14). PhL gene expression was similar in all events and their controls (Figure 15), and R1

gene expression was reduced in G11 only (Figure 16). 

Figure 13. Expression of the Asn1 Gene in Stem Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the Asn1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the Asn1 transcript is 2.0-kb. The lower panel shows the amounts of total RNA as 

visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control samples from the 

untransformed conventional varieties. Individual event samples are labeled above each lane. The events 

E67, G9 and H23 are not considered for deregulation. Event H50 was processed separately with an H 

control.

 EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

18S rRNA

Total RNA

Asn1
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 20

Figure 14. Expression of the Ppo5 Gene in Stem Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the Ppo5 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the Ppo5 transcript is 1.95-kb (see Genbank Accession U22921). The lower panel shows 

the amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67, G9 and H23 are not considered for deregulation. Event H50 was processed 

separately with an H control.

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G, HC = 

control H, M = RNA marker

M EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 HC H50

18S rRNA

Total RNA

Ppo5
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 21

Figure 15. Expression of the PhL Gene in Stem Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the PhL probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the PhL transcript is 4.9-kb (see Genbank Accession X52385). The lower panel shows 

the amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67, G9 and H23 are not considered for deregulation. Event H50 was processed 

separately with an H control.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 M HC H50

18S rRNA

Total RNA

PhL
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 22

Figure 16. Expression of the R1 Gene in Stem Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the R1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the R1 transcript is 4.8-kb (see Genbank Accession Y09533). The lower panel shows the 

amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67, G9 and H23 are not considered for deregulation. Event H50 was processed 

separately with an H control.

EC= control Russet Burbank, FC = control Ranger Russet, JC = control Atlantic, GC = control G, HC = 

control H, M = RNA marker

M EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

18S rRNA

Total RNA

R1
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 23

Target Gene Expression in Roots

In root tissues, transcript levels for the Asn1 gene were reduced in six events (F10, F37, E12, J3, J55, and 

H50), and remained unchanged for E24, J78, G11, and H37 (Figure 17). Transcript levels for the Ppo5 

gene were reduced in F10, F37, E12, H37, and H50 (Figure 18). This indicates that the promoters used to 

drive silencing are partially functional in underground tissues. In Atlantic and G varieties, low levels of 

transcripts were observed in both controls and events, suggesting that Ppo5 gene activity may be 

naturally lower in roots of those varieties. No significant reductions in expression levels were observed 

for the PhL gene, and for the R1 gene F10, F37, and H50 were lower (Figures 19 and 20). 

Figure 17. Expression of the Asn1 Gene in Root Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the Asn1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the Asn1 transcript is 2.0-kb. The lower panel shows the amounts of total RNA as 

visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control samples from the 

untransformed conventional varieties. Individual event samples are labeled above each lane. The events

E67, G9 and H23 are not considered for deregulation. Event H50 was processed separately with an H 

control.

 EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

Asn1

Total RNA

18S rRNA
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 24

Figure 18. Expression of the Ppo5 Gene in Root Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the Ppo5 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the Ppo5 transcript is 1.95-kb (see Genbank Accession U22921). The lower panel shows 

the amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67, G9 and H23 are not considered for deregulation. Event H50 was processed 

separately with an H control.

 EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

Ppo5

Total RNA

18S rRNA
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 25

Figure 19. Expression of the PhL Gene in Root Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the PhL probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the PhL transcript is 4.9-kb (see Genbank Accession X52385). The lower panel shows 

the amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67, G9 and H23 are not considered for deregulation. Event H50 was processed 

separately with an H control.

 EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

PhL

Total RNA

18S rRNA
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 26

Figure 20. Expression of the R1 Gene in Root Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the R1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the R1 transcript is 4.8-kb (see Genbank Accession Y09533). The lower panel shows the 

amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, GC and HC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67, G9 and H23 are not considered for deregulation. Event H50 was processed 

separately with an H control.

 EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 HC H23 H37 HC H50

R1

Total RNA

18S rRNA
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 27

Target Gene Expression in Floral Tissue

In floral tissues, Asn1 gene transcript levels were lower for the 8 events (the H variety does not flower, 

so no data were obtained for H37 and H50) than for the original varieties (Figure 21). Transcripts were 

not detectable for the Ppo5 gene (Figure 22). Expression levels of the PhL gene for all events were not 

reduced compared with their untransformed controls (Figure 23). For the R1 gene, expression levels in 

F37 were lower than the Ranger Russet control, but all other events were similar to controls (Figure 24). 

Figure 21. Expression of the Asn1 Gene in Floral Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the Asn1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the Asn1 transcript is 2.0-kb. The lower panel shows the amounts of total RNA as 

visualized with ethidium bromide (EB). EC, FC, JC, and GC are control samples from the untransformed 

conventional varieties. Individual event samples are labeled above each lane. The events E67 and G9 are 

not considered for deregulation.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 

18S rRNA

Total RNA

Asn1
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 28

Figure 22. Expression of the Ppo5 Gene in Floral Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the Ppo5 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the Ppo5 transcript is 1.95-kb (see Genbank Accession U22921). The lower panel shows 

the amo unts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, and GC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67 and G9 are not considered for deregulation.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 

18S rRNA

Total RNA

Ppo5
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 29

Figure 23. Expression of the PhL Gene in Floral Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the PhL probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the PhL transcript is 4.9-kb (see Genbank Accession X52385). The lower panel shows 

the amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, and GC are control 

samples from the untransformed conventional varieties. Individual event samples are labeled above 

each lane. The events E67 and G9 are not considered for deregulation. 

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 

18S rRNA

Total RNA

PhL
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 30

Figure 24. Expression of the R1 Gene in Floral Tissue of Events and Controls

Total RNA (20 μg) was isolated from greenhouse-grown tissues of events and controls and hybridized 

with the R1 probe (upper panel) and the internal benchmark 18s rRNA probe (middle panel). The 

predicted size of the R1 transcript is 4.8-kb (see Genbank Accession Y09533). The lower panel shows the 

amounts of total RNA as visualized with ethidium bromide (EB). EC, FC, JC, and GC are control samples 

from the untransformed conventional varieties. Individual event samples are labeled above each lane. 

The events E67 and G9 are not considered for deregulation.

EC E12 E24 E67 FC F10 F37 JC J3 J55 J78 GC G9 G11 

18S rRNA

Total RNA

R1
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 31

SUMMARY

It was concluded that transcript levels of the four targeted genes were mostly down-regulated in tubers 

of the events that carry at least one intact DNA insert, as would be expected from using tuber/stolonspecific promoters. For some events, E24 and H50, all four genes were also down-regulated in stolons. 

The two events J78 and G11, which contain a truncated DNA insert with a functionally active Asn1/Ppo5

silencing cassette, produced tubers that contained reduced transcript levels for the Asn1 and Ppo5

genes only, since they did not contain the PhL/R1 silencing cassette. Overall, more down-regulation of 

the four targeted genes occurred in tubers and stolons than in the roots, stems, leaves, or flowers (Table 

1).

The selected events were more strongly affected in the expression levels of the Asn1 and Ppo5 genes 

than in those of the R1 and PhL genes. This result coincided with our specific interest in (1) preventing 

the formation of Ppo protein and free ASN to the greatest extent possible, and (2) only partially blocking 

the conversion of starch into glucose and fructose. 

There were occasional changes in transcript levels in tissues other than tubers and stolons,

demonstrating some leakiness of the tuber/stolon-specific promoter (Table 1). Such event-specific 

variations may have come about because of position effects. For instance, it is possible that the driving 

promoters within the DNA insert are enhanced in activity by genetic elements in adjacent plant DNA. An 

alternative explanation is that small RNAs produced in tubers through expression of the silencing 

cassettes migrate into other tissues, especially roots and stems (Molnar et al. 2010). In most cases of 

altered expression levels in tissues other than tubers and stolons, the differences were found to be 

minor. Unanticipated reductions in gene expression in non-tuber tissues were not associated with 

agronomic differences in the field and are, therefore, not expected to have any impact on the safety of 

the plants or their pest potential.
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 32

Table 1. Summary of Down-Regulated Genes in Different Tissues

Event Tubers1Stolons1Roots1Stems1Leaves1Flowers1, 2

F10 A P R A L R A P R P A A

F37 A P R A L R A P R A P A R

E12 A P L R A L R A P P A

E24 A P L R A P L R A P A

J3 A P L R A R A A P A

J55 A P L R A L R A P A

J78 A P A P A

G11 A P A P R R A

H373A P L R L R P P R

H503A P L R A P L R A P R A P R

1

A = Asn1, P = Ppo5, L = PhL, R = R1. Letters in table indicate down-regulated gene expression by tissue.

2

The partially down-regulated Asn1 gene expression in flowers might alter the amino acid composition of the event 

flowers. Such effects will be limited to a reduction in ASN and an increase in GLN. Since ASN and GLN are similar 

non-essential amino acids, changes in the levels of these compounds is not expected to affect the quality of petal, 

nectar, and pollen as feed for insects or other organisms.

3

Events derived from variety H do not produce flowers.
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 33

MATERIALS AND METHODS

Plant material. Gene expression levels were determined by carrying out RNA gel blot analyses on 

tubers, stolons, roots, stems, leaves, and flowers of greenhouse-grown plants of all events and controls. 

Generation 2 (G2) tubers from the field (Othello, 2010 for Ranger Russet, Russet Burbank and Atlantic, 

and Rhinelander, 2010 for the G and H varieties) were subjected to this analysis as well. Three 

replications per event were used in the analysis.

RNA Isolation. RNA was extracted from 1 g of tuber, stolon and root tissue using Plant RNA reagent 

(Invitrogen, Carlsbad, California), whereas Trizol reagent (Invitrogen) was used to extract RNA from 

leaves, flowers and stems. The concentration of isolated RNA was measured spectrophotometrically at 

260 nm, and RNA quality was confirmed by running a sample on a 1% agarose gel with 1X MOPS buffer 

(200 mM MOPS, 50 mM NaOAc , 20 mM EDTA , pH7.0) for 30-60 min at 90 volts. 

RNA transfer. 20-25 g of RNA was denatured in RNA loading dye (Sigma) for 10-min at 650C followed 

by incubation on ice for 5-min. Denatured RNA was loaded onto an RNA gel containing 1% agarose, 1X 

MOPS, 0.1-0.25 g/ml ethidium bromide and 2% formaldehyde (5.5 ml 36.5% stock in 100 ml solution). 

The gel was run at 80-85 volts for 2-3 hrs and then photographed using the gel documentation system 

from Alpha Innotech (Santa Clara, California). The gel was soaked twice in 10X SSC for 15-min to remove 

the formaldehyde. RNA was transferred from the gel to a positively charged nylon membrane (Roche, 

Indianapolis) by capillary blotting with 10X SSC for 16-18 hrs, and the transferred RNA was stabilized 

onto the filter by UV cross-linking (UVP, Upland, California). The RNA-containing membrane was stored 

at 40C.

Preparation of DIG labeled Probe. A PCR based method was used to prepare DIG labeled probes for 

four target transcripts and an internal control of 18s rRNA. The primers used for PCR amplification and 

the lengths of probes are listed in Table 2. A typical 50 μl labeling reaction consisted of 5 μl HotMaster 

Taq Buffer (Fisher BioReagents), 2-5 μl of 10M forward primer, 2-5l of 10 μM reverse primer, 5μl 

DIG-labeled dNTP (Roche, Indianapolis), 5-30 ng plasmid template, 0.50-0.75 μl HotMaster Taq 

polymerase, and dH2O for a total volume of 50 μl. PCR conditions were specific for each DIG-labeled 

probe. The DIG-labeled probe was checked on 1% agarose gel and always ran slower than the control 

PCR product. The probe was denatured before use by incubating for 5 min at 1000C and then 

transferring to ice. 

Hybridization. Nylon membranes containing transferred RNA were pre-hybridized in 40 ml pre-warmed 

DIG Easy Hybridization solution (Roche, Indianapolis) for at least 1-4 hrs at 20-25 rpm in a hybridization 

oven set at 42

O

C (Amerex Instruments). The hybridization solution was replaced by a mix of 40 ml fresh 

pre-warmed hybridization solution and 25-50μl of denatured DIG-labeled probe, and the membrane was 

incubated in this mix for 3-16 hrs at 42OC. The hybridization solution can be store at -200C and reused. 

The reused hybridization solution was heated at 680C for 10 minutes before use.
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 34

Table 2. Primers and Probes Used for Expression Analysis of Potato Genes

Probe 

name

Hybridization 

target

Primer sequences Probe size (bp)

ASN1 Asn1 transcript F: GGTTGATGACTGATGTCCCCTTTG

R: TAGTTAGCTCCTTATTGTGAGCTC

1115

PPO1Ppo5 transcript F: ATCTTCCACTCCTAAGCCCTCTCAAC

R: CCGCCAAAGAACATCCGAGG

864

PHL PhL transcript F: CAATTCCAGATTCATCCATTTCAC

R: CATCCCCAGGGTAGAGTATGTAAC

968

R1 R1 transcript F: ACCAGGGATTCCTAACCTCAAC

R: CAGGTATATCACTCTTTGTTAC

852

18s 18s rRNA F: GCATTTGCCAAGGATGTTTT

R: GTACAAAGGGCAGGGACGTA

694

1

The PPO probe was derived from a cDNA of the Ppo5 gene of S. tuberosum, which is highly expressed in 

tubers. The probe also hybridized with Ppo genes expressed in other tissues. The sequence in pSIM1278 

used to silence the Ppo5 gene consists of the untranslated trailer of a Ppo gene from S. verrucosum.

Detection. The hybridization solution was removed and replaced by 100 ml washing solution I (2X 

SSC/0.1% SDS). Nylon membranes were washed twice in washing solution I for 10 min at RT in the 

hybridization oven at 25-30 rpm. This low stringency buffer was poured off and preheated high 

stringency washing solution II (0.5X SSC/ 0.1% SDS) was immediately added. The membrane was washed 

twice in washing solution II at 600C for 20 min each at 25-30 rpm. This was followed by a wash for 20 

min with washing solution III (0.1X SSC/ 0.1% SDS) at 650C and a brief rinse with 2X SSC to remove SDS. 

The membrane was rinsed with 100 ml 1X DIG Washing Solution (Roche, Indianapolis) in a tray at room 

temperature for 2 min. and incubated in 1X Blocking solution (Roche, Indianapolis) for 30 min. to 3 hrs 

on a shaker. The blocked membrane was incubated with DIG antibody solution (1:10,000 dilution of 

Anti-DIG-alkaline phosphate conjugate with 1X Blocking solution, Roche, Indianapolis) for 30 min on a 

shaker. The membrane was washed twice (15 min each) with 1X DIG Washing Solution 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 Z-ray film in the dark. Depending on the experiment multiple exposures were taken 

from 30 sec to 10 min). The films were developed with Konica SRX-101A X-ray film developer. The x-ray 

films were scanned to obtain electronic images. 
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 35

CONCLUSIONS

The targeted four genes (Asn1, Ppo5, PhL, and R1) are partially silenced in tubers of most events, except 

for J78 and G11 which do not contain the cassette for silencing the PhL/R1 genes (Table 1). In addition 

the PhL gene did not appear down-regulated in tubers of the Ranger Russet events, F10 or F37. The 

Asn1 and Ppo5 genes are down-regulated in stolons, roots and stems for some of the events. The Asn1

gene was down regulated in flowers of all events with the exception of the H variety that does not 

produce flowers. These results indicate that the promoters used for gene silencing are not entirely 

tissue-specific. 

Considering the potato events in this study, expression of the targeted genes would provide evidence 

that the desired phenotype with low asparagine, reduced black spot bruise, and lowered reducing 

sugars had been achieved. It could provide information relevant to the plant pest risk assessment, for 

example, all InnateTM potatoes in the study had reduced or similar expression to the controls. Thus, the 

altered expression is unlikely to affect weediness or other plant pest characteristics. Also, with these 

events, the expression studies pertain to endogenous proteins rather than new proteins that could 

require a safety evaluation, such as those associated with herbicide tolerance or insect resistance. The 

altered gene expression levels did not affect the agronomic characteristics or tuber composition of the 

event compared to the conventional untransformed variety. 

Supporting data for these conclusions are available in Appendix 6. Field Performance and Tuber 

Evaluations and Appendix 9. Compositional Analyses.

Event Ranger Russet F10: The targeted genes are at least partially silenced in tubers, except for PhL, and 

all but Ppo5 are silenced in stolons. The Asn1, Ppo5, and R1 genes are down-regulated in roots, the Ppo5

gene in stems, and the Asn1 gene in leaves and flowers (Table 1). 

Event Ranger Russet F37: The targeted four genes are at least partially silenced in tubers, except for 

PhL,, and all but Ppo5 are silenced in stolons. Some of these are also down-regulated in expression, at 

least partially, in roots (Asn1, Ppo5, and R1), stems (Asn1 and Ppo5), and flowers (Asn1 and R1) (Table 

1). 

Event Russet Burbank E12: The targeted four genes are at least partially silenced in tubers, and all but 

Ppo5 are silenced in stolons . Some of these are also down-regulated in expression, at least partially, in 

roots (Asn1 and Ppo5), stems (Ppo5), and flowers (Asn1) (Table 1). 

Event Russet Burbank E24: The targeted four genes are at least partially silenced in tubers and stolons. 

Some of these are also down-regulated in expression, at least partially, in stems (Asn1 and Ppo5) and 

flowers (Asn1) (Table 1). 

Event Atlantic J3: The targeted four genes are at least partially silenced in tubers, and Asn1 and R1 are 

silenced in stolons. Some of these are also down-regulated in expression, at least partially, in roots 

(Asn1), stems (Asn1 and Ppo5), and flowers (Asn1) (Table 1). 
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 36

Event Atlantic J55: The targeted four genes are at least partially silenced in tubers, and all but Ppo5 are 

silenced in stolons. Some of these are also down-regulated in expression, at least partially, in roots 

(Asn1), stems (Ppo5), and flowers (Asn1) (Table 1). 

Event Atlantic J78: The Asn1 and Ppo5 genes are silenced in tubers, the Asn1 gene is silenced in stolons, 

the Ppo5 gene is silenced in stems, and the Asn1 gene is silenced in flowers (Table 1). Silencing was not 

observed for the PhL/R1 genes, as expected, since J78 does not contain the cassette for silencing the 

PhL/R1 genes.

Event G11: The Asn1 and Ppo5 genes are silenced in tubers and stolons; the R1 gene is partially silenced 

in leaves and stems; and the Asn1 gene is silenced also in flowers (Table 1). Silencing in tubers and 

stolons was not observed for the PhL/R1 genes, as expected since G11 does not contain the cassette for 

silencing the PhL/R1 genes. The lower expression of R1 in leaves and stems was not expected. Such 

unanticipated reductions in gene expression in non-tuber tissues were not associated with agronomic 

differences in the field and are, therefore, not expected to have any impact on the safety of G11 plants 

or their pest potential.

Event H37: The targeted four genes are at least partially silenced in tubers, and PhL and R1 are silenced 

in stolons. Some of these are also down-regulated in expression, at least partially, in roots (Ppo5), stems 

(Ppo5), and R1 is silenced in leaves (Table 1). 

Event H50: The targeted four genes are at least partially silenced in tubers and stolons. Some of these 

are also down-regulated in expression, at least partially, in roots (Asn1, Ppo5, and R1), stems (Asn1 and 

Ppo5), and R1 is silenced in leaves (Table 1). 
J.R. Simplot Company Efficacy and Tissue-Specificity of Gene Silencing 37

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