Figure 5.

Transgenerational tolerance sustained over a virus-free generation: (A) experimental design for the generation of progeny lines. Parental N. benthamiana plants were mock-inoculated (C0:Gus) or inoculated with PVX (P0:PVX) after which plants were allowed to set seed to provide C1 and P1 progenies, respectively. C1 and P1 plants were allowed to set seed under virus-free conditions, providing GUS:GUS and PVX:GUS progenies, respectively. A separate batch of P1 plants was inoculated with PVX to provide PVX:PVX progeny; (B) seed production in C1 (GUS:GUS) and P1 (PVX:GUS, PVX:PVX) plants. Statistically, comparisons between means were made among treatments by employing Duncan´s test. Different letters indicate significant differences between treatments; (C,D) germination rates of GUS:GUS, PVX:GUS and PVX:PVX progenies on MS medium supplemented with either 200 mM mannitol (C) or 100 mM NaCl (D); (E) representative examples of the different treatments photographed at 14 days after sowing. The accumulative germination rates were calculated at 8, 12 and 14 days after sowing. Values were obtained from two independent experiments using six progenies per treatment; (F) Western blot analysis of seedlings extracts derived from C1 and P1 progenies using antibodies against PVX CP. The lower panel shows the Ponceau S-stained membrane after blotting as control of loading. Lane C+ corresponds to extract from a PVX-infected plant diluted 1:200. Bars in each panel represent 1 cm.