Generating marker free transgenic potato cultivars with an hairpin construct of PVY coat protein

The aim of the present study was to produce marker-free transgenic potato resistant to PVY by using a construct generating self-complementary hairpin RNAs. The method has two steps: 1) establishing a robust protocol for the genetic transformation of economically important cultivars of potato by using gfp and nptII genes; 2) applying the protocol for the transfer of marker-free hairpin construct and analysis of transgenic plants by PCR. In the first step internodes were transformed by Agrobacterium tumefaciens LBA4404 with pHB2892 construct. Kanamycin, visual screening of GFP, PCR of nptII and gfp as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII were used for transgenic plant selection. Good regeneration and transformation efficiency were obtained for the cvs. Baltica and Desiree while the cvs. Agave and Delikat showed lower figures. In the second step A. tumefaciens with construct: 35SCaMV enhancer and promoter, two repeated inverted PVY-CP sequences separated by an intron and pAnos terminator was used. Regenerated plants without selection, were analysed by PCR using primers in the: 35S promoter, promoter and hairpin region, or in both repeated PVY-CP sequences. A large number of transgenic plants were identified for the most responsive cv. Baltica (17 out of 33) and Desiree (23 out of 66), but only 1 for the cv. Agave (out of 3) and none for the cv. Delikat. This simple two-step strategy might be applied to generate marker-free transgenic plants in other cvs. or plant species.