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

Reference s 8

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

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 cytopla sm. 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 repe at 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 t o blacken wounded tubers, and therefore was omitted from these tests.

Field

- grown t ubers 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 discolorat ion 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 va rities 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 temperatur e 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 varieti es 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 limita tions 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 , indicatin g 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 n atural 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 thei r untransformed control varieties (P<0.05)

. Numerical
data indicated a trend for improved tolerance in the events

, E24, H37, and H50, al though this was not always statistically significant

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

W isconsin, suggest ing 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 B arrel T est for B lack S pot B ruise Tolerance

Variety, Location

- 2009 No. of bruises per tuber 1 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

R usset 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

A tlantic 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, M I) 0.78 ± 0.83

G11 (Montcalm County, M I) 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, M I) 2.56 ± 1.67

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

H50 (Montcalm County, M I) 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 ut ilized to measure the incidence of b lack s pot b ruise 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 pSIM12 78 results in at least partial tolerance against impact

- induced disco loration. The Atlantic variety lacks a highly expressed Ppo gene in tubers and shows some tolerance to black spot bruise , thus silencing with pS IM 1278 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 pro moters, 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 ME THODS

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 fr om 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 pipette d 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. N ine 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) sp ots. 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 r esults of three experiments, and the error bars shown represent the standard error of the mean. Significance was determined using the Student’s two

tailed 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 Bu rbank 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

g ene 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 Event s J3 , J55, and J78 : Th ese event s w ere not tested for black spot bruise because th e Atlanti c variety showed some resistance to darkening both with the catechol assay and with impact in the barrel

test

.

G e vent 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 e vent H37 : Results of the catechol assay confirmed silencing of the polyphenol oxidase gene in tubers. In addition, the barrel tes t showed a significant reduction in tubers from one location but no difference
from control in a second test from another location.

H e vent 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 Si lencing

, 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 no t change the agronomic characteristics, disease susceptibility and compositional profile of potato.J.R. Simplot Company Black Spot Bruise Tolerance 8 REFERENCE S

Hunt MD, Eannetta NT, Yu H, Newman SM, Steffens JC (1993) cDNA cloning and expression of potato polyphenol oxidase. Plant Mol Biol

, 21: 59

- 6 8.

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 exhib its 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.