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I Potrykus

Publications and source records attributed to I Potrykus.

46 records · Page 3Linked to original sources
PubMed1990

Gene transfer methods for plants and cell cultures.

Agrobacterium-mediated gene transfer provides a routine and efficient gene transfer system for a variety of plant species. As this biological vector does not, however, function with important plant species, numerous alternative approaches have been studied. Of those, direct gene transfer into protoplasts, microinjection and biolistics have been demonstrated to be effective. Others, for example, viral vectors, agroinfection, liposome injection and electrophoresis may have special merits, although transgenic plants have not been produced by these techniques yet. From methods based on pollen transformation, the pollen tube pathway, pollen maturation, incubation of dry seeds, incubation of tissues, liposome fusion with tissues, macroinjection, laser treatment and electroporation of tissues no proof of integrative transformation is available, so far, and it is difficult to envisage how these approaches will ever produce transgenic cells and plants. We discuss (a) why Agrobacterium does not function with all plants, (b) what merits and disadvantages we see for the effective methods, (c) what possibilities we foresee for some of the other approaches, and (d) why we do not expect the remaining ones to be successful.

Cells, Cultured↗
PubMed1990

Recognition efficiency of Dicotyledoneae-specific promoter and RNA processing signals in rice.

Heterologous gene expression experiments have shown that genes of Monocotyledoneae are often not transcribed in Dicotyledoneae, or produce pre-mRNA that is inefficiently or aberrantly processed. It is however not known how correctly and efficiently dicotyledon-specific gene expression signals are recognized in cells of Monocotyledoneae. Here we address this question using tobacco (Nicotiana tabacum) and rice (Oryza sativa) protoplasts transformed with the same hybrid gene constructs. Constructs including the nptII protein coding sequence fused to Cauliflower Mosaic Virus (CaMV) promoter and polyadenylation signals were used to obtain stably transformed cell lines of tobacco and rice. In one of the constructs the nptII coding region is interrupted by a modified intron-3 sequence from the soybean phaseolin gene. Although the mean number of hybrid gene copies integrated into the rice genome was on average 5- to 10-fold higher than in tobacco, the steady-state transcript level was 3 times lower. A lower level of transcript was also observed in transient expression experiments. The amount of the mature mRNA was not influenced by the presence of the intron. The phaseolin intron was processed in rice with high efficiency and an accuracy indistinguishable from that seen in tobacco.

Base Sequence↗
PubMed1990

Intermolecular homologous recombination in plants.

To study DNA topological requirements for homologous recombination in plants, we have constructed pairs of plasmids that contain nonoverlapping deletions in the neomycin phosphotransferase gene [APH(3')II], which, when intact, confers kanamycin resistance to plant cells. Protoplasts isolated from Nicotiana tabacum were cotransformed with complementary pairs of plasmids containing these truncated gene constructs. Homologous recombination or gene conversion within the homologous sequences (6 to 405 base pairs) of the protein-coding region of the truncated genes led to the restoration of the functional APH(3')II gene, rendering these cells resistant to kanamycin. Circular plasmid DNAs recombined very inefficiently, independent of the length of the homologous region. A double-strand break in one molecule only slightly increased the recombination frequency. The most favorable substrates for recombination were linear molecules. In this case, the recombination frequency was positively correlated with the length of the homologous regions. The recombination frequency of plasmids linearized at sites proximal to the deletion-homology junction was significantly higher than when linearization was distal to the homologous region. Vector homology within cotransformed plasmid sequences also increased the recombination frequency.

Blotting, Southern↗
PubMed1988

Gene targeting in plants.

Although the generation of transgenic plants is now routine, the integration of foreign genetic information has so far been at random sites in the genome. We now present evidence for directed integration into a predicted location in the host plant genome. Protoplasts of transgenic tobacco (Nicotiana tabaccum) plants carrying copies of a partial, non-functional drug-resistance gene in the nuclear DNA were used as recipients for DNA molecules containing the missing part of the gene. Molecular and genetic data confirm the integration of the foreign DNA through homologous recombination within overlapping parts of the protein coding region, resulting in the formation of an active gene in the host chromosome. This approach is referred to as gene targeting. The gene targeting frequency (the number of drug-resistant clones resulting from gene correction compared to the number of resistant clones from parallel experiments with a similar non-interrupted hybrid gene) was 0.5-4.2x10. These experiments demonstrate the possibility of producing transgenic plants with desired modifications to a specific nuclear gene.

Journal Article↗
PubMed1986

Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector.

Two bacterial antibiotic resistance genes, one coding for the neomycin phosphotransferase (NPT I) from Tn903, and the other coding for the chloramphenicol acetyltransferase from Tn9 were used as plant selectable markers. Both genes were introduced into the Nicotiana tabacum genome in a new plant expression vector, using the direct gene transfer method. The vector pDH51, used in these experiments contains a plant expression unit as a movable cassette, consisting of the strong cauliflower mosaic virus (CaMV) 35S RNA promoter and transcription terminator separated by a polylinker containing several unique restriction sites.

Acetyltransferases↗
PubMed1985

Molecular and general genetics of a hybrid foreign gene introduced into tobacco by direct gene transfer.

Two clones of N. tabacum, transformed to kanamycin resistance by direct transfer to protoplasts of a hybrid gene, consisting of the protein coding region from the bacterial gene for aminoglycoside phosphotransferase under the control of 5'/3' expression signals from cauliflower mosaic virus gene VI, in the bacterial plasmid pUC8, have been subjected to a detailed genetic crossing analysis accompanied by Southern blot analysis and enzyme activity assays of representative offspring. The genetic data obtained from large populations of R1/F1 and R2/F2 offspring as well as from more than 20 subclones of each of the original of the hybrid gene was stably integrated into chromosomal DNA of the original transformants, (b) that the gene normally was stably maintained during clonal proliferation, (c) that normally it is transmitted in a regular fashion (with exceptions) to sexual offspring, and (d) that it is inherited as a single dominant trait. Data from DNA hybridisation and enzyme assays confirm this interpretation. The functional gene is integrated together with several non-functional copies and bacterial plasmid sequences, which are inherited as one block together with the functional gene.

Brassica↗
PubMed1984

Direct gene transfer to plants.

Evidence for direct, gene-mediated stable genetic transformation of plant cells of Nicotiana tabacum is presented. A selectable hybrid gene comprising the protein coding region of the Tn5 aminoglycoside phosphotransferase type II gene under control of cauliflower mosaic virus gene VI expression signals was introduced into plant protoplasts as part of an Escherichia coli plasmid. The gene was stably integrated into plant genomic DNA and constitutively expressed in selected, drug resistant, protoplast-derived cell clones. The mode of integration of the foreign gene into the plant genome resembled that observed for DNA transfection of mammalian cells. Plants regenerated from transformed cell lines were phenotypically normal and fertile, and they maintained and expressed the foreign gene throughout the development of vegetative and generative organs. Microspores, grown in anther culture, developed into resistant and sensitive haploid plantlets. Genetic crossing analysis of one of the transformed plants revealed the presence of one dominant trait for kanamycin resistance segregating in a Mendelian fashion in the F(1) generation.

Journal Article↗