The 2011 ASHS Annual Conference

We transformed monoploid (2n=1x=12) potato clone BARD 1-3 516 with an activation tagging construct, AcDsATag-Bar_gosGFP, with green fluorescent protein (GFP), hygromycin resistance (hyg) and transposase (TPase) on the Ac element, as well as glufosinate resistance (basta or BAR) and a p35s tetramer on the Ds element. Flow cytometry revealed 33 diploid (2n=2x=24), 12 tetraploid (2n=4x=48), and five mixoploids among 50 independent transgenic plants evaluated. The diploid transgenics were crossed as female parent to a closely related wild type pollinator. Progeny were screened first by painting seedlings with 0.03% glufosinate herbicide, followed by multiplex PCR to identify putative tranposants. For 25 families, the expected 1 transgenic:1 wild type segregation was observed, indicating a single gene insertion without transposition of Ds. Six additional families segregated 3 transgenic:1 wild type, indicating two insertions without transposition. For three independent transgenics we observed 1 Ds:1 AcDs T₁ progeny, indicating active transposition but an unexpected absence of wild type plants. TAIL-PCR originating from nested primers in the Ds element indicated that 15 activation tagged lines were independent. The parental launch pad was situated on PGSC0003DMB000000017 of the Potato Genome assembly with 11 of 15 of its Ds lines transposed locally to other sites on the same scaffold and the remainder transposed to other linkage groups. The transposants abut several genes of interest including late blight resistance. We are attempting to backcross the Ds lines to the DM parent to produce activation tag seed stocks.