Resistencia transgénica para el control del virus motoso del enanismo de la frambuesa (raspberry bushy dwarf virus-RBDV)

Resumo

RBDV is transmitted in association with pollen and genetic resistance has been the only effective control measure. However, recently a virus strain (R-15) has appeared which can overcome the viral resistance of most modern raspberry cultivars. A solution to this problem could be the development of transgenic resistance. The main objective of this project was to assess the resistance to RBDV infection that would be conferred by different RBDV gene sequences and to understand the mechanisms of resistance generated by these gene sequences. To assess the RBDV transgenic resistance, seven constructs containing RBDV gene sequences were introduced into Agrobacterium tumefaciens by triparental mating and the resulting cultures were used to transform leaf pieces from plants of glasshouse grown Nicotiana tabacum or in vitro-cultured N. benthamiana. Plants regenerated from kanamycin-resistant callus were allowed to set seed and seedlings of the F1 generation were tested for resistance to RBDV by manually inoculating expanded leaves and assaying for virus titre by ELISA 5 and 10 days after inoculation. More than 20 lines were obtained from transformations with each type of construct and the results showed that transformation with each construct had some effect on virus multiplication. Evidence of resistance induced by coat protein (CP) sense, CP anti-sense and polymerase gene sequences was obtained and several degrees of effectiveness of protection were identified among these constructtransgenes. Construct pJAD-10 (RBDV R-15 CP gene sequence) induced the highest level of protection and some N. benthamiana lines transformed with this construct also showed inhibition or delay in the development of the symptoms of infection by RBDV. Also, the results of challenging transgenic Nicotiana lines transformed with untranslatable CP and CP antisense sequences suggest that resistance can be generated by the expression of RBDV CP and also by mRNA CP transcripts. These constructs are now available to be inserted into raspberry germplasm to provide resistance against R-15 strains of RBDV.