Genetic transformation in commercial Tasmanian cultivars of opium poppy, Papaver somniferum, and movement of transgenicpollen in the field

Abstract

We report a new transformation protocol for the pharmaceutically important opium poppy, Papaver somniferum L.; the protocol allows transformation for the first time of high yielding commercial cultivars. The method involves Agrobacterium tumefaciens infection of hypocotyl explants, followed by the production of antibiotic- or herbicide-resistant embryogenic callus and the subsequent induction of somatic embryos and plants. Key elements of the improvement are the use of buffering agents to stabilise medium pH and bottom-cooling of the cultures. Transformation was verified by PCR and Southern blot hybridisation. Transcription of transgenes was confirmed by RT–PCR and product sequencing. Expression of transgenes was detected by histochemical GUS staining, phosphinothricin acetyltransferase (PAT) enzyme assays for bar and pat genes, and western analysis of transgenic sunflower seed albumin protein. Expression of various transgenes was detected in stem, leaf, seed, capsule and latex. The pat gene was demonstrated to be stably inherited to the T₂ generation and to confer phosphinothricin (PPT) herbicide resistance. Most T₀ plants showed normal morphology, were self-fertile and the transgenes displayed the expected Mendelian segregation. The percentage of explants producing somatic embryos that developed into plantlets able to be transplanted to soil, ranged from 6–11% in two Tasmanian cultivars.

A field trial using pat transgenic plants was designed to estimate the frequency of hybridisation at various distances into buffer rows of non-transgenic poppies and to related weed species, P. somniferum spp. setigerum and P. dubium. The frequency of hybridisation to completely compatible poppy fell sharply with distance, being 2.6% at 20 cm, 2.13% at 0.5 m and falling to 0% at 2.5 and 5 m. No hybridisation could be detected to two weed species under open pollination conditions, including the compatible P. somniferum spp. setigerum, when grown as close as 20 cm, despite flowering at the same time as the transgenic plants in the presence of foraging bees.