Julie A. Chitty

RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy

We report on the silencing of codeinone reductase (COR) in the opium poppy, Papaver somniferum, using a chimeric hairpin RNA construct designed to silence all members of the multigene COR family through RNA interference (RNAi). After gene silencing, the precursor alkaloid (S)-reticuline—seven enzymatic steps upstream of codeinone—accumulated in transgenic plants at the expense of morphine, codeine, oripavine and thebaine. Methylated derivatives of reticuline also accumulated. Analysis verified loss of Cor gene transcript, appearance of 22-mer degradation products and reduction of enzyme activity. The surprising accumulation of (S)-reticuline suggests a feedback mechanism preventing intermediates from general benzylisoquinoline synthesis entering the morphine-specific branch. However transcript levels for seven other enzymes in the pathway, both before and after (S)-reticuline, were unaffected. This is the first report of gene silencing in transgenic opium poppy and of metabolic engineering to cause the high-yield accumulation of the nonnarcotic alkaloid reticuline.

Transformation of opium poppy (Papaver somniferum L.) with antisense berberine bridge enzyme gene (anti-bbe) via somatic embryogenesis results in an altered ratio of alkaloids in latex but not in roots.

The berberine bridge enzyme cDNA bbe from Papaver somniferumL. was transformed in antisense orientation into seedling explants of the industrial elite line C048-6-14-64. In this way, 84 phenotypically normal T0 plants derived from embryogenic callus cultures were produced. The selfed progeny of these 84 plants yielded several T1 plants with an altered alkaloid profile. One of these plants T1-47, and its siblings T2-1.2 and T2-1.5 are the subject of the present work. The transformation of these plants was evaluated by PCR, and northern and Southern hybridisation. The transgenic plants contained one additional copy of the transgene. The alkaloid content in latex and roots was determined with HPLC and LC-MS. We observed an increased concentration of several pathway intermediates from all biosynthetic branches, e.g., reticuline, laudanine, laudanosine, dehydroreticuline, salutaridine and (S)-scoulerine. The transformation altered the ratio of morphinan and tetrahydrobenzylisoquinoline alkaloids in latex but not the benzophenanthridine alkaloids in roots. The altered alkaloid profile is heritable at least to the T2 generation. These results are the first example of metabolic engineering of the alkaloid pathways in opium poppy and, to our knowledge, the first time that an alkaloid biosynthetic gene has been transformed into the native species, followed by regeneration into a mature plant to enable analyses of the effect of the transgene on metabolism over several generations.

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

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.

Expression of the C4 Me1 Gene from Flaveria bidentis Requires an Interaction between 5[prime] and 3[prime] Sequences.

The efficient functioning of C4 photosynthesis requires the strict compartmentation of a suite of enzymes in either mesophyll or bundle sheath cells. To determine the mechanism controlling bundle sheath cell-specific expression of the NADP-malic enzyme, we made a set of chimeric constructs using the 5[prime] and 3[prime] regions of the Flaveria bidentis Me1 gene fused to the [beta]-glucuronidase gusA reporter gene. The pattern of GUS activity in stably transformed F. bidentis plants was analyzed by histochemical and cell separation techniques. We conclude that the 5[prime] region of Me1 determines bundle sheath specificity, whereas the 3[prime] region contains an apparent enhancer-like element that confers high-level expression in leaves. The interaction of 5[prime] and 3[prime] sequences was dependent on factors that are present in the C4 plant but not found in tobacco.

Expression of the C4 Me1 Gene from Flaveria bidentis Requires an Interaction between 5 and 3 Sequences

The efficient functioning of C4 photosynthesis requires the strict compartmentation of a suite of enzymes in either mesophyll or bundle sheath cells. To determine the mechanism controlling bundle sheath cell-specific expression of the NADP-malic enzyme, we made a set of chimeric constructs using the 5’ and 3’ regions of the F/ayeria bidentis Mel gene fused to the P-glucuronidase gusA reporter gene. The pattern of GUS activity in stably transformed F. bidentis plants was analyzed by histochemical and cell separation techniques. We conclude that the 5‘ region of Mel determines bundle sheath specificity, whereas the 3’ region contains an apparent enhancer-like element that confers highleve1 expression in leaves. The interaction of 5‘ and 3’ sequences was dependent on factors that are present in the C4 plant but not found in tobacco.

Opium Poppy ( Papaver somniferum )

The genetic transformation of opium poppy, Papaver somniferum, offers the opportunity to study the mechanisms involved in the regulation of benzylisoquinoline and morphinan alkaloid biosynthesis. The development of an efficient transformation protocol for opium poppy has allowed us to transform a range of genotypes from all around the world, including previously recalcitrant high-yielding commercial Australian cultivars. The method involves Agrobacterium tumefaciens infection of hypocotyl explants, followed by the production of antibiotic or herbicide resistant embryogenic callus, the subsequent induction of somatic embryos and development into normal plants. The use of different selective agents, binary vectors, and poppy genotypes has demonstrated the robustness and reliability of this protocol in the production of many hundreds of confirmed transgenic poppies.

Pollination biology of oilseed poppy, Papaver somniferum L

Although poppies (Papaver somniferum L.) are one of the oldest cultivated plants relatively little is known of their pollination biology. We have investigated the relative importance of wind and insects in the pollination of poppies and identified potential insect pollinators. Wind pollination was found to be negligible, insect pollination was responsible for the majority of out-crossing, and self-pollination was the dominant mode of poppy fertilisation. Honeybees and flies were identified as the main potential cross-pollinators of Tasmanian poppies. Using a transgenic poppy field trial in which approximately 50% of the pollen grains produced were transgenic, we have determined the level of pollen-mediated gene flow by scoring over 50 000 seeds for the presence of a selectable marker gene. Gene flow was measured using a 10-m buffer area that surrounded the field trial. It was highest at 0.1 m with 3.26% of seeds found to be transgenic and declined over distance with 1.73% transgenic seeds at 0.5 m, 1.80% at 1 m, 0.86% at 2 m, 0.34% at 5 m, 0.12% at 9 m, and 0.18% at 10 m. These results demonstrate that under Tasmanian conditions, pollen-mediated gene flow occurs at modest levels in poppies that are in close proximity to each other and is most probably mediated by honeybees and flies.

Transgenic Flaveria bidentis

C4 plants are a group of relatively recently evolved angiosperms which are represented in a large number of both monocot and dicot genera (see Hatch 1987, 1992). C4 plants are of agricultural importance not only because they are strongly represented among tropical and subtropical crop species (maize, sorghum, sugarcane, and amaranth, for example, are C4), but also because of their acknowledged role as some of the world’orst weeds. C4 plants are so named because they initially fix atmospheric CO2 via phosphoenolpyruvate carboxylase to form the C4 acid oxaloacetate (Fig. 1; Hatch 1987). This C4 acid is converted to either aspartate or malate and transported to the bundle sheath cells, where it is decarboxylated to produce CO2, the substrate for Rubisco (Fig. 1). The C4 mechanism operates as a biochemical pump which concentrates CO2 at the site of Rubisco to levels up to ten times atmospheric. This high CO2 concentration means that Rubisco is operating at saturating CO2 in ambient air and photorespiration is almost totally suppressed. This has important implications for the efficiency of photosynthesis in air at higher temperatures and also improves the water use efficiency of C4 plants. Consequently, most C4 plants thrive under warmer and often more arid environments.