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 LB pNOS p35S tNOS nptII CP PVY 400

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

Screening for PVY: resistance field trials

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 1998-2001

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

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

Southern blot analysis: Transgene copy number Probe nptII Probe CP PVY Xba I Eco RV Xba I Eco RV SP 230 233 SP 230 233 SP 230 233 SP 230 233 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

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 230 233 Wt 230 233 Wt 230 233 with PVY B C Wt 230 233 YL OL S YL OL S YL OL S D CP-PVY npt-II

Protein levels in lines SY230 and SY233 SP 230 233 5000 - 1 M W 2500 1000 100 50 10 PVY / Spunta 64 49 37 26 19 kDA 15 CP-PVY NptII 230 233 SP 230 233 SP Mw Leaves Tubers 26 37 B npt-II

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

Comparative Field Trials

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.

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

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.

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 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