1. Ensayos de campo, estudios bioquímicos y moleculares en plantas transgénicas de papa de la variedad Spunta, con el fin de liberar al mercado un evento transgénico resistente al virus PVY

2. Potato virus Y (PVY) - Potyvirus
Es uno de los virus más importantes y de mayor prevalencia que afectan al cultivo de la papa. Se han descrito razas de PVY que pueden causar necrosis en hojas y en tubérculos
Es transmitido por áfidos (Myzus persicae) de manera no persistente y también puede transmitirse mecánicamente a través de la maquinaria, las herramientas, etc.
S. tuberosum cv. Spunta: > 60%

3. Strategies to develop virus resistance in plants
R gene-mediated disease resistance
Pathogen-derived resistance in transgenic plants
Post transcriptional gene silencing (PTGS)

4. Genetics constructions and plant transformationRB LB
pNOS
p35S
tNOS
nptII
CP PVY
400

5. Screening for PVY resistance (greenhouse, mechanical infection)
Alta presión de virus
ELISA para monitorear titulo de virus
40% de infección promedio en controles de Spunta
300 clones probados
4 a 8 plantas por clon
Sometidos a infección artificial: alta presión de virus
105 lines
155 lines
260 lines

6. Screening for PVY: resistance field trials

7. Screening for PVY: resistance field trials
Field trial location and year
number of lines screened
Plant number/line
Infection
Rate (%)
Number of infected plants
General Belgrano. Spring 1998 10 5
14.5 3
General Belgrano. Fall 1999 51 4 14
General Belgrano. Spring 1999 40
9.2 9
Colonia Tirolesa. Spring 1999 67 13
24.1 54
General Belgrano. Fall 2000 12 30
29.1 8
Colonia Tirolesa. Fall 2000 72
31.8 38
General Belgrano. Spring 2000 23 26
4.6 2
Colonia Tirolesa. Spring 2000
5.6 6
General Belgrano. Fall 2001 18 22
17.0
General Belgrano. Spring 2001
30.9 7
Summary of the field trial screening procedure used to identify
PVY-resistant potato lines during

8. Screening for PVY resistance field trials: SY230, SY233
Field trial SY230 & SY233
Plant number/line
Number of
Infected
plants
Infection
Rate (%)
SY230
154
SY233
441 1
SPUNTA
661
560 85
Two of the PVY-resistant lines showing a consistant behaviour in all field trials (SY230 and SY233) were multiplied and tested in a larger field assay. In this trial, 154 out of 154 SY230 plants were found to be virus-free, and only one out of 441 SY233 plants was found to contain detectable levels of virus. In the same trial, 560 out of 661 non-transformed Spunta plants used as controls were infected with PVY, implying an infection rate as high as 85%. Based on this trial, SY230 and SY233 were chosen as candidates for further characterization and possible commercial release.
SY230 and SY233 were chosen for further characterization and possible commercial release

9. Screening for PVY resistant potato plants
Selection of kanamycin resistant lines
400 events
Mechanical infection with PVY in greenhouse
Field trial testing for natural PVY infection
Field trial testing of selected lines
Selection of SY230 and SY233
for further characterization
140 lines discarded due to poor growth
Micropropagation and greenhouse growth
155 lines discarded due to infection
83 lines discarded due to virus infection
Screening
for PVY
resistant
potato plants

10. Southern blot analysis: Transgene copy number
Probe nptII
Probe CP PVY
Xba I Eco RV
Xba I Eco RV SP SP SP SP
DNA size
Marker (bp) 9
000
6000
5000
4000
3000
2000 16 50
1000
850 RB LB
pNOS
p35S
tNOS
nptII
CP PVY
EcoRV EcoRV EcoRV XbaI

11. Transcripts analysis CP-PVY npt-II A B C D Plants infected
9,5
7,5
4,4
2,4
1,4
0,2
MW kb
Leaves Stem Root
Plants infected
Wt Wt Wt
with PVY B C Wt
YL OL S YL OL S YL OL S D
CP-PVY
npt-II

12. Protein levels in lines SY230 and SY233SP
230
233
5000 - 1 M W
2500
1000
100 50 10
PVY / Spunta 64 49 37 26 19
kDA 15
CP-PVY
NptII
SP SP Mw
Leaves Tubers 26 37 B
npt-II

13. Comparative Field Trials
Alta presión de virus
30% de infección promedio en controles de Spunta
1 clon probado
1500 plantas por clon
Sometidos a infección natural presión de virus normal

14. Comparative Field Trials

15. Comparative yield studies Average tuber weight (g)
Location
Variety
Total yield
(g/plot)
Number of
tubers/stem
Average tuber weight (g)
SY 230
3426 ± 899 5 46
 Balcarce
SY 233
4225 ± 376
5,4 59
Spunta
4265 ± 1497
6,5 45
4025 ± 1281
2,9 33
 Córdoba
4010 ± 1536
2,6 35
3040 ± 1627 30
2450 ± 490 44
 Malargüe
2100 ± 540
2,7 43
2900 ± 1000
8271 ± 2985
San Juan
8780 ± 3088
8275 ± 2323
Table 2. Comparative yield studies. Yield comparison assays between lines SY230, Sy233 and non-transformed Spunta were performed at 4 potato-producing areas of Argentina during the growth season 2005/6. Each assay comprised 4 independent plots of one row of 25 plants (5 m long) [explicar cuanto media y que comprendia cada plot]. The Balcarce and Cordoba sites are located in in the central production region of Argentina. The Malargue and San Juan sites are located close to the Andes in a virus-free, seed-tuber production area. Total yield values for each locations are presented as the mean-values resulting from the 4 independent plots and their respective standard deviations.

16. Tuber characteristics and biochemical analysis
Location
Variety
Specific
gravity
Starch %
Dry matter
Chips
colour
Glycoalkaloids
mg/100g fresh weight
Solanine
Chaconine
Total
Balcarce
SY230
1,066
11,0
18,0
4,5
2,40 ± 0,18
6,47 ± 0,37
8,87
SY233
1,068
11,3
18,6
7,5
3,18 ± 0,48
8,94 ± 1,03
12,12
Spunta
1,069
11,6
18,8 6
1,98 ± 0,27
6,05 ± 0,50
8,03
Córdoba
1,083
14,6
20,7 8
3,10 ± 0,43
7,10 ± 0,70
10,20
1,081
14,0
20,5
3,26 ± 0,67
7,63 ± 1,37
10,89
1,086
15,1
21,5
6,5
3,43 ± 0,09
9,17 ± 1,34
12,60
Malargüe
1,057
9,1
16,5
3,5
1,63 ± 0,44
4,68 ± 1,68
6,31
1,064
10,6
17,5 3
1,71 ± 0,21
5,51 ± 0,77
7,22
1,063
10,3
17,4
2,79 ± 1,79
5,93 ± 2,70
8,72
San Juan
1,080
10,7
22,8
1,32 ± 0,03
5,07 ± 0,19
6,39
11,1
22,2
2,73 ± 0,57
8,27 ± 3,26
11.00 -
2,60 ± 0,25
8,44 ± 3,16
11,04
Table 3. Tuber analysis. Biochemical and industrial parameters of potato tubers were measured in SY230, SY233 and non-transformed Spunta samples obtained from the comparative assays referred in Table 2. Values for each location are presented as the mean-values resulting from the 4 independent plots and their respective standard deviations

17. Tuber biochemical analysis, cont Carbohydrates (g/100g DW)
Location
/ Variety
Total protein (g/100g DW)
% Soluble proteins
Fat
(g/100g DW)
Crude Fiber (g/100g DW)
Ashes (g/100g DW)
Carbohydrates (g/100g DW)
San Juan
SY230
2.25  0.17
68.5  6.8
0.45  0.06
0.60  0.00
1.22  0.07
19.6  0.13
SY233
1.85  0.06
70.4  5.1
0.45  0.13
0.55  0.06
1.12  0.08
21.1  1.09
Spunta
2.42  0.61
68.3  7.6
0.48  0.10
1.23  0.23
20.8  2.37
Balcarce
2.32  0.10
64.3  6.9
0.42  0.10
0.65  0.17
1.09  0.10
16.6  2.23
2.18  0.05
61.5  11.3
0.35  0.06
0.62  0.10
1.15  0.05
16.8  2.38
2.25  0.19
62.1  20.1
0.62  0.05
1.11  0.17
16.6  3.75
Malargüe
2.25  0.37
65.8  3.9
0.40  0.00
0.58  0.05
1.11  0.08
13.8  1.05
2.45  0.06
70.0  7.4
0.48  0.05
1.05  0.13
17.4  1.18
2.50  0.14
78.1  10.0
1.08  0.03
15.6  0.63
Table 4. Tuber analysis. Biochemical parameters of potato tubers were measured in SY230, SY233 and non-transformed Spunta samples obtained from the comparative assays referred to in Table 2. Values for each location are presented as mean-values resulting from 4 samples taken from 2 independent field trials at consecutive growth seasons.

18. Conclusions
Spunta was transformed with a transgene containing the Potato virus Y (PVY) coat protein
Screening for PVY resistance under greenhouse and field conditions allowed identification of two genetically stable PVY-resistant lines: SY230 and SY233
Southern blot analysis showed that SY230 and SY233 are independent transformation events exhibiting multiple transgene insertion
Northern blot assays showed low levels of CPPVY transcripts in leaves, stem and root tissues. NptII transcripts was clearly detected
Western blot assays failed to detect transgenic CPPVY in SY230 or SY233 leaf extracts. NptII protein was detected in leaves and tubers
Biochemical and physical characteristics of SY230 and SY233 tubers are comparable to those of non-transformed Spunta

19. Silvia Cabral
María Eugenia Segretin
Alejandro Mentaberry
Fernando Bravo Almonacid
INGEBI-CONICET
Valeria Rudoy
Bjorn Welin
Maximiliano Gomez
Fabiana Stolowicz
Mariana López
Germán Serino
Cristina Dos Santos
Marcelo Criscuolo
BioSidus-TECNOPLANT
María Cecilia Bedogni
Marcelo Huarte
INTA-Balcarce