Turnip mosaic virus (TuMV) has a wide host range and no resistant commercial canola variety to this virus has been reported in Iran. Thus, RNA silencing mechanism was applied to consider the possibility of improvement in resistance to TuMV in spring canola, RGS003 variety.
To obtain an effective construct for silencing, based on the bioinformatics analysis, a fragment containing 130 conserved nucleotide sequences of the TuMV coat protein gene was gained as targeting candidate to produce sense, antisense and hairpin constructs and assessed for resistance efficiency in a transient expression system in Nicotiana benthamiana by agroinfiltration. The development of symptoms after virus inoculation revealed that the highest efficiency can be obtained by hairpin construct. Therefore, the hairpin construct was applied for the transformation of canola RGS003 via cotyledonary explants using Agrobacterium tumefaciens LBA4404. In transgenic and non-transgenic canola plants, the infection and virus titer were assessed by ratio of detection via ELISA and real-time PCR. In addition, severity of disease symptoms was scored four weeks after inoculation with a TuMV isolate.
Results indicated 5-12 days delay in appearance of symptoms in transgenic plants and there was a decrease in severity of symptoms in contrast to non-transgenic plants. The increased virus concentration ratio in non-transgenic compared to transgenic plants was confirmed by qRT-PCR. The ELISA results confirmed absence of infection on five out of six transformed plants 15 dpi.
These preliminary results proved that transgenic canola plants containing hairpin of 130 nucleotide sequences of TuMV CP gene could resist against TuMV.