Biomedical subjects

P Christou

Publications and source records attributed to P Christou.

43 records · Page 3Linked to original sources

PubMed1989

Inheritance and expression of foreign genes in transgenic soybean plants.

DNA-coated gold particles were introduced into meristems of immature soybean seeds using electric discharge particle acceleration to produce transgenic fertile soybean plants. The lineages of integrated foreign DNA in two independently transformed plants were followed in the first (R(1)) and second (R(2)) generation of self-pollinated progeny. One plant (4615) was transformed with the Escherichia coli genes for beta-glucuronidase and neomycin phosphotransferase II; the other (3993) was transformed only with the gene for beta-glucuronidase. Segregation ratios for the introduced gene(s) were approximately 3:1 for plant 4615 and 1:1 for plant 3993 in the R(1) generation. DNA analysis showed 100% concordance between presence of the foreign gene sequences and enzyme activity. Moreover, all copies of the foreign genes are inherited as a unit in each plant. Plant 3993 segregated in a 1:1 ratio in the R(2) generation. R(1) plants derived from plant 4615, which expressed both genes, gave either 100% or 3:1 expression of both genes in the R(2) generation, demonstrating recovery of both homozygous and heterozygous R(1) plants. Our results show that foreign DNA introduced into soybean plants using electric discharge particle acceleration can be inherited in a Mendelian manner. Results also demonstrate cotransformation of tandem markers and show that both markers are inherited as closely linked genes in subsequent generations. These results indicate that whole plants can be derived from single transformed cells by a de novo organogenic pathway.

Journal Article↗

PubMed1989

Cytokinin antagonist activity of substituted phenethylamines in plant cell culture.

A series of structurally related substituted phenethylamines shows extreme toxicity toward wild-type callus tissue cultures of tobacco (Nicotiana tabacum), soybean (Glycine max), corn (Zea mays), and sunflower (Helianthus annuus L.), but tobacco crown gall cultures are resistant to the compounds. The essential components that result in toxicity of the phenethylamines include one aromatic hydroxyl and one primary aliphatic amino group. A series of attenuated crown gall cultures, generated by transformation of tobacco with various modified Agrobacterium strains, has been used to demonstrate that the resistance of crown galls to the phenethylamines is due to the expression in these tissues of isopentenyl transferase, a first step in cytokinin biosynthesis. The toxicity of the compounds to tissue cultures is very rapid, but can be overcome by prior exposure of the calli to exogenous cytokinin. Because of the relationships we have observed between cytokinins and these compounds, we propose that the substituted phenethylamines may represent a class of chemicals that can be used as specific probes to further an understanding of cytokinin metabolism in plant tissues.

Journal Article↗

PubMed1988

Stable Transformation of Soybean Callus by DNA-Coated Gold Particles.

Immature soybean (Glycine max L.) embryos from commercially important cultivars were the targets of rapidly accelerated, DNA-coated, gold particles. Protoplasts were prepared from these tissues and propagated in culture under selection conditions for the introduced neomycin phosphotransferase II gene. Kanamycin-resistant calli were obtained at a rate of approximately 10(-5). Enzyme assays and Southern blot hybridization confirmed the expression of the foreign gene and its stable integration into the soybean genome. Our results show that particle acceleration can be used for the introduction of foreign DNA into the soybean genome and indicate the technique may be useful in the recovery of engineered plants by transformation of regenerable tissues.

Journal Article↗

PubMed1988

Habituation in in vitro soybean cultures.

The habituation of soybean (Glycine max) callus can be induced rapidly, by exposing the tissue to small amounts (10(-9)molar) of compounds including 2.4-dinitrophenol and phenoxyisobutyric acid for brief periods of time. Such compounds reportedly exhibit antiauxin activity. Various soybean callus phenotypes have been isolated which continue to exhibit hormone habituation 14 months following the initiation of the experiment. Protein changes in habituated tissue under selected hormonal regimes were detected indicating changes at the level of gene expression. Habituated tissue exhibits hormonal autonomy in a manner similar to crown gall tissue, suggesting that such studies may help elucidate the mechanism of induction of crown gall disease and genetic transformation by Agrobacterium.

Journal Article↗

PubMed1987

Detection of opines by colorimetric assay.

A colorimetric procedure for confirming the presence of arginine-derived opines (nopaline and octopine) in plant tissue extracts is described. Those materials are widely used as markers of plant cell transformation and tumorigenesis mediated by the tumor-inducing plasmids of Agrobacterium tumefaciens. Nopaline and octopine are generally detected, following resolution by paper electrophoresis, by observation of the uv-fluorescent products formed upon reaction with phenanthrenequinone. We found that a further heat treatment step, compatible with paper electrophoresis, results in rapid production of a red-purple pigment. Our colorimetric assay is sensitive to 1.25-micrograms quantities of opine and eliminates problems of background fluorescence encountered with crude plant extract in the usual assay.

Arginine↗

PubMed1987

Stable transformation of soybean by electroporation and root formation from transformed callus.

Soybean protoplasts from a number of commercially important cultivars have been genetically engineered by way of electroporation using chimeric genes coding for resistance to the aminoglycoside antibiotics kanamycin and G418. Effective electroporation conditions were determined by monitoring transient expression from aminoglycoside 3'-phosphotransferase II (APHII) expression plasmids. Electroporation of protoplasts with a chimeric APHII gene and subsequent selection on media supplemented with kanamycin resulted in the recovery of calli resistant to the antibiotic. Enzyme assays for APHII activity and Southern blot hybridization confirmed the expression of the foreign DNA and its stable integration into the soybean genome. Root formation was induced from transformed calli, and these roots maintained expression of the APHII gene.

Journal Article↗

PubMed1986

Opine synthesis in wild-type plant tissue.

Opine production is associated with crown gall tissue, a neoplastic growth caused by infection of dicotyledonous plants with Agrobacterium tumefaciens. Recent publications have claimed that tissues of certain monocotyledonous plants can also be infected by Agrobacterium. Following infection, a part of the Agrobacterium Ti plasmid, T-DNA, is integrated into the chromosome of the infected plant. T-DNA, which codes for opine-synthesizing enzymes, is now used to add foreign genes to plants. A number of laboratories have used opine production in plant tissue, often after arginine feeding or preincubation as evidence for plant transformation by T-DNA vectors. In this report we provide microbiological, chromatographic, spectroscopic and chemical evidence indicating that opines can be formed in normal callus and plant tissue as a result of arginine metabolism. Therefore, researchers studying T-DNA should be aware of the capability of plant tissue to metabolize arginine to opines. Opine production following infection with T-DNA may not always be sufficient evidence to indicate transformation by the Agrobacterium Ti plasmid.

Journal Article↗