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L-Phosphinothricin (L-Pt)-resistant plants were constructed by introducing a modified phosphinothricin-N-acetyl-transferase gene (pat) via Agrobacterium-mediated gene transfer into tobacco (Nicotiana tabacum L), and via direct gene transfer into carrot (Daucus carota L). The metabolism of L-Pt was studied in these transgenic, Pt-resistant plants, as well as in the untransformed species. The degradation of L-Pt, (14)C-labeled specifically at different C-atoms, was analysed by measuring the release of (14)CO2 and by separating the labeled degradation products on thin-layer-chromatography plates. In untransformed tobacco and carrot plants, L-Pt was deaminated to form its corresponding oxo acid 4-methylphosphinico-2-oxo-butanoic acid (PPO), which subsequently was decarboxylated to form 3-methylphosphinico-propanoic acid (MPP). This compound was stable in plants. A third metabolite remained unidentified. The L-Pt was rapidly N-acetylated in herbicide-resistant tobacco and carrot plants, indicating that the degradation pathway of L-Pt into PPO and MPP was blocked. The N-acetylated product, L-N-acetyl-Pt remained stable with regard to degradation, but was found to exist in a second modified form. In addition, there was a pH-dependent, reversible change in the mobility of L-N-acetyl-Pt thin-layer during chromatography.

SummaryThe production of biodegradable polymers in transgenic plants in order to replace petrochemical compounds is an important challenge for plant biotechnology. Polyaspartate, a biodegradable substitute for polycarboxylates, is the backbone of the cyanobacterial storage material cyanophycin. Cyanophycin, a copolymer of L -aspartic acid and L -arginine, is produced via non-ribosomal polypeptide biosynthesis by the enzyme cyanophycin synthetase. A gene from Thermosynechococcus elongatus BP-1 encoding cyanophycin synthetase has been expressed constitutively in tobacco and potato. The presence of the transgene-encoded messenger RNA (mRNA) correlated with changes in leaf morphology and decelerated growth. Such transgenic plants were found to produce up to 1.1% dry weight of a polymer with cyanophycin-like properties. Aggregated material, able to bind a specific cyanophycin antibody, was detected in the cytoplasm and the nucleus of the transgenic plants.

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A system for the inducible destruction of plant tissues based on the deacetylation of the non-toxic compound N-acetyl-L-phosphinothricin (N-ac-Pt) has been developed. The argE gene product of Escherichia coli, representing a N-acetyl-L-ornithine deacetylase was identified to remove the acetyl-group from N-ac-Pt giving the cytotoxic compound L-phosphinothricin (Pt, glufosinate). Transgenic Nicotiana tabacum plants constitutively expressing the argE gene were constructed. No effect of the bacterial N-acetyl-L-ornithine deacetylase on plant growth and reproduction could be traced. However, application of N-ac-Pt on leaves of the transgenic plants led to the formation of necrotic areas due to the release of Pt. Additionally, due to the uptake of the N-ac-Pt by roots, transgenic shoots grown on medium containing N-ac-Pt bleached within 6-7 days and finally died. Untransformed controls showed no reaction to high amounts of N-ac-Pt applied, either under sterile or under unsterile conditions. In order to construct inducible male-sterile plants, the argE coding region was fused to a DNA fragment carrying sequences homologous to the tobacco TA29 promoter, known to function exclusively in the tapetum. Owing to the tapetum-specific expression of the chimeric gene the application of N-ac-Pt led to empty anthers resulting in male-sterile plants. The sanity of the female reproductive part of the male-sterile flowers could be demonstrated by cross-pollination. Without N-ac-Pt treatment the plants turned out to be completely fertile making fertility restoration in the F1 generation superfluous. The system presented is easy to handle and might be applicable to a wide range of crop plants.

The objective of the study was to test the feasibility of coexistence between genetically modified (GM) and non-GM maize under real-life agronomical conditions. GM hybrid maize with the event MON810 (Bt maize) was drilled at 30 sites in fields surrounded by near isogenic conventional maize, although only 27 sites could be finally evaluated. Field sizes of Bt maize varied between 0.3 and 23 ha, and the flowering period of the Bt and conventional maize was synchronous. At some sites, different planting dates of GM and non-GM maize or an earlier ripening conventional maize were tested in additional strips to obtain altered flowering and thereby reduce cross-pollination. The overlapping of flowering periods was successfully avoided only at two sites where non-GM maize was planted 25 or 28 days later. During harvest, samples were taken from the conventional maize in strips at distances of 0-10, 20-30, and 50-60 m to the Bt maize fields to assess the GM DNA content as a function of distance. Sampled materials included chaffed plant material intended for silage (18 sites), grains (eight sites), or crushed husks and cobs (one site). Wind effects were taken into account by sampling in all four compass directions. Quantitative PCR was used to detect the event specific MON810 DNA sequence in sampled materials. The analysis was conducted by two certified independent diagnostic testing companies selected in a pre-test. Taking averages over all compass directions and the two laboratories no samples collected beyond 10 m had levels of GM above the threshold of 0.9 %. In conclusion, the data indicate that coexistence of GM and conventional maize is possible under real-life large-scale agronomical conditions. Levels of GM DNA in harvested grain resulting from outcrossing can be managed to levels below 0.9 % by simply planting 20 m of conventional maize as a pollen barrier between adjacent fields.

SummaryThe production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness.In order to decrease the phenotypic abnormalities caused by cytosolic production of the Although all lines tested were fertile, the progeny of the highest cyanophycin-producing line showed reduced seed production compared with control plants.