J.R. Simplot Company Black Spot Bruise Tolerance 1

Appendix 7

Black Spot Bruise Tolerance

INDEX

Section Page

Index 1

Introduction 2

Results 3

Discussion 5

Materials and Methods 6

Conclusions 7

References 8

Certification 9
J.R. Simplot Company Black Spot Bruise Tolerance 2

INTRODUCTION

Black spot bruise is a discoloration affecting bruised tubers that represents one of the most important 

quality issues for the potato industry. The condition is a result of leakage of polyphenol oxidase (Ppo) 

from damaged plastids into the cytoplasm. In the cytoplasm, the enzyme oxidizes polyphenol, which 

then form dark precipitants. One of the two silencing cassettes of the pSIM1278 DNA insert contains 

two copies of a fragment of the Ppo5 gene from Solanum verrucosum positioned as an inverted repeat 

between regulatory elements. Expression of this inverted repeat triggers silencing of the potato Ppo5

gene. The variety Atlantic is different from other potatoes in that it doesn’t express a Ppo5 gene in high 

enough levels to blacken wounded tubers, and therefore was omitted from these tests.

Field-grown tubers from the seven events derived from the black spot bruise susceptible varieties; 

Ranger Russet, Russet Burbank, G, and H, were assayed in two ways for black spot bruise tolerance. 

Tuber discoloration is caused by the activity of polyphenol oxidase, which oxidizes phenols including 

catechol to produce compounds that rapidly polymerize to produce pigments. An indirect method to 

test for black spot bruise tolerance is based on pipetting 1-ml catechol (25 mM in 50 mM MOPS, pH6.5) 

onto the cut surfaces of tubers and monitoring for the Ppo-dependent development of a dark brown 

precipitate. Tubers for Ranger Russet and Russet Burbank controls and events were harvested from 

three different plants that grew in Canyon County, ID, in 2009, and G and H controls and events were 

obtained from independent plants from Oneida County, WI. The assay was not applied to Atlantic (and 

the Atlantic events J3, J55, and J78) because Atlantic does not brown like the other varities when 

exposed to catechol, and shows some tolerance to black spot bruise.

The second method of assaying for bruise tolerance was by treating nine tubers of each event to 10 

revolutions in a bruise barrel (rotating drum), incubating at room temperature for 3 days, followed by 

peeling mechanically, and then counting the number of both blackspot bruise and lineshatter (shatter 

bruise) spots. Tubers were from Canyon County, ID, for Ranger Russet and Russet Burbank controls and 

events; and G and H varieties were from Oneida County, WI, or Montcalm County, MI. All were 

harvested in 2009.
J.R. Simplot Company Black Spot Bruise Tolerance 3

RESULTS

Tubers for Ranger Russet and Russet Burbank controls and events were harvested from three different 

plants that grew in Canyon County, ID, in 2009 (due to limitations of tuber material), and G and H

controls and events were obtained from independent plants from Oneida County, WI, in 2009. An 

analysis of tubers demonstrated that none of them had detectable levels of functionally-active 

polyphenol oxidase, indicating that they display black spot bruise tolerance (Figure 1).

Figure 1. Catechol assay for polyphenol oxidase activity*

*Event G9 is not included in this submission for deregulation.

Note: Slight differences in flesh color of the event tubers is from natural variation and is not related to 

Ppo silencing. Variety H naturally has pale yellow flesh.
J.R. Simplot Company Black Spot Bruise Tolerance 4

Results from the bruise barrel test (Table 1) show that tubers of three events (F10, F37, E12) were more 

resistant to black spot bruise than tubers of their untransformed control varieties (P<0.05). Numerical 

data indicated a trend for improved tolerance in the events, E24, H37, and H50, although this was not 

always statistically significant. Data for G11 were different from control in Michigan, but not in 

Wisconsin, suggesting that environmental factors may play an important role in efficacy of the 

technology for this particular genotype. For instance, certain stress factors might induce expression of 

an alternative Ppo gene in this variety.

Table 1. Bruise Barrel Test for Black Spot Bruise Tolerance

Variety, Location - 2009 No. of bruises per tuber1

Difference from control 

(P-value)

Ranger Russet Control (Canyon County, ID) 4.56 ± 1.81

F10 (Canyon County, ID) 0.89 ± 0.78 P < 0.0001

F37 (Canyon County, ID) 0.78 ± 0.83 P < 0.0001

Russet Burbank Control (Canyon County, ID) 0.50 ± 0.79

E12 (Canyon County, ID) 0.00 ± 0.00 P = 0.0107

E24 (Canyon County, ID) 0.28 ± 0.57 P = 0.3396

Atlantic Control (Canyon County, ID) NA

J3 (Canyon County, ID) NA

J55 (Canyon County, ID) NA

J78 (Canyon County, ID) NA

G Control (Oneida County, WI) 2.50 ± 1.07

G11 (Oneida County, WI) 3.44 ± 0.88 P = 0.64

G Control (Montcalm County, MI) 0.78 ± 0.83

G11 (Montcalm County, MI) 0.11 ± 0.33 P = 0.041

H Control (Oneida County, WI) 2.67 ± 1.80

H37 (Oneida County, WI) 1.87 ± 0.641 P = 0.26

H50 (Oneida County, WI) 1.56 ± 0.73 P = 0.11

H Control (Montcalm County, MI) 2.56 ± 1.67

H37 (Montcalm County, MI) 0.75 ± 0.886 P = 0.015

H50 (Montcalm County, MI) 1.78 ± 1.20 P = 0.273

1

Data represent the mean of 9 tubers  standard error.

NA= not applicable; Atlantic controls and events appeared less susceptible to black spot bruise and were not 

included in these tests.
J.R. Simplot Company Black Spot Bruise Tolerance 5

DISCUSSION

Two different methods were utilized to measure the incidence of black spot bruise in potato events

expressing the pSIM1278 construct. They demonstrate that transformation of the black spot bruise 

sensitive varieties Ranger Russet, Russet Burbank, G and H with the DNA insert of pSIM1278 results in at 

least partial tolerance against impact-induced discoloration. The Atlantic variety lacks a highly expressed

Ppo gene in tubers and shows some tolerance to black spot bruise, thus silencing with pSIM1278 would 

not affect expression as much as with other varieties. In our construct, we targeted the Ppo5 gene that 

is predominantly expressed in mature tubers and stolons (Hunt et al. 1993; Thygesen et al.1995). As 

expected based on the specificity of the Ppo5 gene and use of the Agp and Gbss promoters, which are 

highly active in tubers and stolons (Nakata et al. 1994; Visser et al. 1991), we confirmed the predicted 

reduction in black spot bruise as also shown in Appendix 5 (Efficacy and Tissue-Specificity of Gene 

Silencing). 
J.R. Simplot Company Black Spot Bruise Tolerance 6

MATERIALS AND METHODS

Materials for polyphenol oxidase assay with catechol. Tubers for Ranger Russet and Russet Burbank 

controls and events were harvested from three different plants that grew in Canyon County, ID, in 2009, 

and G and H controls and events were obtained from independent plants from Oneida County, WI, in 

2009. The assay was not applied to Atlantic (and the Atlantic events J3, J55, and J78) because cut tubers 

from this variety do not develop a brown color when exposed to catechol.

Polyphenol oxidase activity assay. 1-ml catechol (25 mM in 50 mM MOPS, pH6.5) was pipetted onto the 

cut surfaces of three randomly chosen tubers for each event and control. The Ppo-dependent 

development of a dark brown precipitate was assessed after 20 min.

Bruise barrel test. Nine tubers of each event were subjected to 10 revolutions in a bruise barrel 

(rotating drum), incubating at room temperature for 3 days, followed by peeling mechanically, and then 

counting the number of both blackspot bruise and lineshatter (shatter bruise) spots. Tubers were from 

Canyon County, ID, for Ranger Russet and Russet Burbank controls and events; and G and H varieties 

were from Oneida County, WI, or Montcalm County, MI. All were harvested in 2009.

Statistics. Data are presented as the means of the results of three experiments, and the error bars 

shown represent the standard error of the mean. Significance was determined using the Student’s twotailed t-test (p < .05).
J.R. Simplot Company Black Spot Bruise Tolerance 7

CONCLUSIONS

Russet Event F10: Both the catechol assay and barrel test confirmed that event F10 is more resistant to 

black spot bruise than its untransformed control.

Ranger Russet Event F37: Both the catechol assay and barrel test confirmed that event F37 is more 

resistant to black spot bruise than its untransformed control

Russet Burbank Event E12: Both the catechol assay and barrel test confirmed that event E12 is more 

resistant to black spot bruise than its untransformed control

Russet Burbank Event E24: Results of the catechol assay confirmed silencing of the polyphenol oxidase 

gene in tubers. In addition, the mean value for E24 with the barrel test was lower than controls, yet this

difference was not statistically significant.

Atlantic Events J3, J55, and J78: These events were not tested for black spot bruise because the Atlantic

variety showed some resistance to darkening both with the catechol assay and with impact in the barrel 

test. 

G event G11: Results of the catechol assay confirmed silencing of the polyphenol oxidase gene in 

tubers. In addition, the barrel test showed a significant reduction in tubers from one location but no 

difference from control in a second test from another location. 

H event H37: Results of the catechol assay confirmed silencing of the polyphenol oxidase gene in tubers. 

In addition, the barrel test showed a significant reduction in tubers from one location but no difference 

from control in a second test from another location. 

H event H50: Results of the catechol assay confirmed silencing of the polyphenol oxidase gene in tubers. 

In addition, mean values for H50 with the barrel test were lower than controls, yet the differences were

not statistically significant.

An evaluation of data presented in this appendix, together with those described in Appendix 5. Efficacy 

and Tissue-Specificity of Gene Silencing, Appendix 6. Field Performance and Tuber Evaluations, 

Appendix 8. Disease Susceptibility, and Appendix 9. Compositional Analyses demonstrate that the 

enhanced black spot bruise resistance in the Russet Burbank, Ranger Russet, G, and H events did not 

change the agronomic characteristics, disease susceptibility and compositional profile of potato. 
J.R. Simplot Company Black Spot Bruise Tolerance 8

REFERENCES

Hunt MD, Eannetta NT, Yu H, Newman SM, Steffens JC (1993) cDNA cloning and expression of potato 

polyphenol oxidase. Plant Mol Biol, 21: 59-68.

Nakata PA, Anderson JM, Okita TW (1994) Structure and expression of the potato ADP-glucose 

pyrophosphorylase small subunit. J Bio Chem 269: 30798-30807.

Thygesen PW, Dry IB, Robinson SP (1995) Polyphenol oxidase in potato. A multigene family that exhibits 

differential expression patterns. Plant Physiol, 109: 525-531.

Visser RGF, Stolte A, Jacobsen E (1991) Expression of a chimeric granule-bound starch synthase-GUS 

gene in transgenic potato plants. Pant Mol Bio, 691-699.