Download Diapositiva 1 - REDBIO Argentina

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
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%
Strategies to develop virus resistance in plants
R gene-mediated disease resistance
Pathogen-derived resistance in transgenic plants
Post transcriptional gene silencing (PTGS)
Genetics constructions and plant transformation
RB
pNOS
nptII
tNOS
p35S
CP PVY
tNOS
LB
400
Screening for PVY resistance
(greenhouse, mechanical infection)
260 lines
155 lines
105 lines
Screening for PVY: resistance field trials
Screening for PVY: resistance field trials
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
14.5
14
General Belgrano. Spring 1999
40
4
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
5
31.8
38
General Belgrano. Spring 2000
23
26
4.6
2
Colonia Tirolesa. Spring 2000
30
5
5.6
6
General Belgrano. Fall 2001
18
22
17.0
12
General Belgrano. Spring 2001
30
9
30.9
7
Field trial location and year
Summary of the field trial screening procedure used to identify
PVY-resistant potato lines during 1998-2001
Screening for PVY resistance field trials: SY230, SY233
Field trial SY230 & SY233
Plant number/line
Number of
Infected
plants
SY230
154
0
SY233
441
1
SPUNTA
661
560
Infection
Rate (%)
85
SY230 and SY233 were chosen for further characterization and
possible commercial release
Selection of kanamycin resistant lines
400 events
Screening
for PVY
resistant
potato plants
Micropropagation and greenhouse growth
140 lines discarded due to poor growth
Mechanical infection with PVY in greenhouse
155 lines discarded due to infection
Field trial testing for natural PVY infection
83 lines discarded due to virus infection
Field trial testing of selected lines
Selection of SY230 and SY233
for further characterization
Southern blot analysis:
Transgene copy number
Probe nptII
Probe CP PVY
Xba I
Eco RV
SP 230 233 SP 230 233
Xba I
SP 230 233
Eco RV
SP 230 233
DNA size
Marker (bp)
9000
6000
5000
4000
3000
2000
1650
1000
850
EcoRV EcoRV
RB
pNOS
nptII
tNOS
EcoRV XbaI
p35S
CP PVY
tNOS
LB
Transcripts analysis
Leaves
A
Stem
Plants infected
Root
with PVY
Wt 230 233 Wt 230 233 Wt 230 233
MW kb
9,5
7,5
4,4
2,4
1,4
0,2
CP-PVY
B
C
D
MW kb
npt-II
9,5
7,5
4,4
2,4
1,4
0,2
YL
Wt
OL
S
YL
230
OL
S
YL
233
OL
S
Protein levels in lines SY230 and SY233
A
PVY /
kDA
SP
230
233
5000
-1
MW
Spunta
2500
-1
1000
-1
100
-1
50
-1
10
-1
64
CP-PVY
49
37
26
19
15
Leaves
B
230
233
Tubers
SP
230
233
SP
Mw
37
npt-II
NptII
26
Comparative Field Trials
Comparative Field Trials
Comparative yield studies
Location
Balcarce
Córdoba
Malargüe
San Juan
Variety
Total yield
(g/plot)
Number of
tubers/stem
Average tuber
weight (g)
SY 230
3426 ± 899
5
46
SY 233
4225 ± 376
5,4
59
Spunta
4265 ± 1497
6,5
45
SY 230
4025 ± 1281
2,9
33
SY 233
4010 ± 1536
2,6
35
Spunta
3040 ± 1627
2,6
30
SY 230
2450 ± 490
2,6
44
SY 233
2100 ± 540
2,7
43
Spunta
2900 ± 1000
2,6
43
SY 230
8271 ± 2985
2,7
43
SY 233
8780 ± 3088
2,9
44
Spunta
8275 ± 2323
2,6
43
Tuber characteristics and biochemical analysis
Location
Balcarce
Córdoba
Malargüe
San Juan
Variety
Specific
gravity
Starch
%
Dry matter
%
Chips
colour
Glycoalkaloids
mg/100g fresh weight
Solanine
Chaconine
Total
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
SY230
1,083
14,6
20,7
8
3,10 ± 0,43
7,10 ± 0,70
10,20
SY233
1,081
14,0
20,5
8
3,26 ± 0,67
7,63 ± 1,37
10,89
Spunta
1,086
15,1
21,5
6,5
3,43 ± 0,09
9,17 ± 1,34
12,60
SY230
1,057
9,1
16,5
3,5
1,63 ± 0,44
4,68 ± 1,68
6,31
SY233
1,064
10,6
17,5
3
1,71 ± 0,21
5,51 ± 0,77
7,22
Spunta
1,063
10,3
17,4
3
2,79 ± 1,79
5,93 ± 2,70
8,72
SY230
1,080
10,7
22,8
6
1,32 ± 0,03
5,07 ± 0,19
6,39
SY233
1,081
11,1
22,2
6
2,73 ± 0,57
8,27 ± 3,26
11.00
Spunta
-
-
-
-
2,60 ± 0,25
8,44 ± 3,16
11,04
Tuber biochemical analysis, cont
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)
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
0.45  0.06
1.23  0.23
20.8  2.37
SY230
2.32  0.10
64.3  6.9
0.42  0.10
0.65  0.17
1.09  0.10
16.6  2.23
SY233
2.18  0.05
61.5  11.3
0.35  0.06
0.62  0.10
1.15  0.05
16.8  2.38
Spunta
2.25  0.19
62.1  20.1
0.45  0.06
0.62  0.05
1.11  0.17
16.6  3.75
SY230
2.25  0.37
65.8  3.9
0.40  0.00
0.58  0.05
1.11  0.08
13.8  1.05
SY233
2.45  0.06
70.0  7.4
0.48  0.05
0.65  0.17
1.05  0.13
17.4  1.18
Spunta
2.50  0.14
78.1  10.0
0.42  0.10
0.48  0.05
1.08  0.03
15.6  0.63
San Juan
Balcarce
Malargüe
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
Silvia Cabral
María Eugenia Segretin
Alejandro Mentaberry
Fernando Bravo Almonacid
Valeria Rudoy
Bjorn Welin
Maximiliano Gomez
Fabiana Stolowicz
Mariana López
Germán Serino
Cristina Dos Santos
Marcelo Criscuolo
INGEBI-CONICET
BioSidus-TECNOPLANT
María Cecilia Bedogni
Marcelo Huarte
INTA-Balcarce