Resistance to TSWV in Transgenic Tomato and Tobacco Varieties

Stoeva,P.1, Hristova,D.1, Nicolaeva,V.1, Batchvarova,R.1, Ivanova,L.1, Maiss,E.4, Adam,G.5, Guelemerov,S.6, Valkov,V.1, Donkova,P.1, Petrova,M.1, Gorinova,N.1, Yankulova,M.1, Inze,D.3, van Camp,W.3, Atanassov, A.1

1Institute of Genetic Engineering, 2232 Kostinbrod-2, Bulgaria;
2Institute of Plant Protection, 2232 Kostinbrod-2, Bulgaria;
3VIB, RUG, B-9000, Gent, Belgium;
4Institute of Phytopathology and Plant Protection, University of Hannover, Germany;
5Institute of Applied Botany, Department of Phytopathology, University of Hamburg, Marseilerstrasse 7, 20355 Hamburg, Germany;
6Tobacco Breeding Station, Gotze Delchev, Bulgaria

The objective of the study was to introduce by genetic engineering genes conferring resistance to TSWV in commercial tobacco and tomato genotypes. We have examined the resistance phenotype of a large number of transgenic tobacco plants originating from 12 commercial cultivars expressing the sense form of the nucleoprotein (N) gene of L3 - a Bulgarian isolate (L3) of tomato spotted wilt virus (TSWV). The analysis revealed that transgenic plants are completely protected against the homologous L3 isolate of TSWV irrespective of whether or not they contain detectable levels of translation product. The effectiveness of protection against the virus was investigated upon mechanical inoculation under greenhouse conditions and in field trials. Non-segregating resistant lines were selected and the inheritance of the resistance to TSWV was analyzed in successive generations (R3-R6). The extensive tests under controlled conditions and the two year field trials proved that the resistance to TSWV is stable in different environments and represents a normally inherited trait.

On the basis of the developed genotype independent protocol for genetic transformation of tomato the nucleoprotein (N)-gene of TSWV and Mn SOD gene from N. plumbaginifolia were introduced in different tomato cultivars and lines. Primary transgenic plants have been obtained with both constructs. All transgenic plants were selected for resistance to TSWV after mechanical inoculation with the TSWV. Resistant plants were selected among the primary transformants carrying the N gene or the Mn SOD gene.

Studies were carried out on derived R1-R3 progenies from the selected resistant primary transgenic plants carrying the Mn SOD gene and R1-R2 progenies carrying the N-gene from TSWV for: the expression of the nptII selectable marker gene by studying the rooting ability of in vitro plants on kanamycin; PCR and Southern blot analysis for NPT II selectable marker gene PAGE analysis of the expression of Mn SOD gene; immunoprotein detection of the expression of N-gene and (or) the viral accumulation by TAS or DAS-ELISA; resistance to TSWV by mechanical inoculation of transgenic plants.

The established resistance or tolerance to TSWV in the R1 progenies of transgenic plants carrying Mn SOD gene varied from 54 to 80%. In R2 and in R3 100% TSWV tolerant line were selected.

The established resistance to TSWV in the R1 progenies of transgenic plants carrying the N gene varied from 50 to 71%. In R2 progeny lines the resistance varied from 25 to 93%.

Further studies are in progress to study the inheritance of the resistant and the tolerant phenotypes.