Does the world need GM foods? Yes. Interview by Sasha Nemecek.
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Animals↗
Biomedical subjects
R B Horsch
Publications and source records attributed to R B Horsch.
Commercialization of genetically engineered crops.
More abundant harvests from insect- and disease-resistant crops, vine-ripened tomatoes, or less oily potato chips or french fries are some of the benefits that will result from single gene improvements under development today. These single gene traits will be combined with the best new varieties produced by traditional plant breeding and will accelerate the pace and the scope of our ability to develop even better and more productive crops in the future. The initial group of genetic improvements were first field tested in 1987, improved upon over the past 6 years, and are finally approaching the first commercial sales over the next 3 to 4 years. The key hurdles from discovery of a promising lead to a commercial product trait include: (i) gene cloning and expression; (ii) product development; (iii) field testing; (iv) breeding into multiple elite varieties; (v) product characterization and regulatory review; (vi) public acceptance; and (vii) marketing. The expense and risk to bring transformed crops to market successfully is significantly higher than for traditionally developed new varieties. The high value of some single gene targets and the possibility for patent protection of the processes and final products provide the incentive for private investment in this area. The value to farmers, consumers, the environment and society in general is very high because the problems being solved are those that have resisted previous attempts through conventional means. Public investment in basic plant science research and private investment in product development is a powerful combination for continual improvement in lowering the cost and improving the quality of the world's food supply.
Agriculture↗
Evaluation of selectable markers for obtaining stable transformants in the gramineae.
Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 x 10(-5) to 2 x 10(-6)) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome.
Journal Article↗
Expression of mouse dihydrofolate reductase gene confers methotrexate resistance in transgenic petunia plants.
Transgenic petunia plants containing an altered (Leu22----Arg22) mouse dihydrofolate reductase gene fused to the cauliflower mosiac virus 35S (CaMV 35S) promoter and nopaline synthase (nos) polyadenylation site were obtained by transforming petunia leaf disks with an Agrobacterium tumefaciens strain carrying the chimeric gene. Transformants were directly selected for and rooted on medium containing 1 microM methotrexate (MTX). The chimeric gene was present in the regenerated plants at one to three copies and produced the expected 950-nucleotide-long transcript based on Southern and Northern hybridization analyses, respectively. Leaf pieces from the regenerated transgenic plants were able to form callus when cultured on medium containing 1 microM MTX and were able to incorporate 32P into high-molecular-weight DNA in the presence of greater than 100 microM MTX, thus demonstrating that the chimeric mouse dhfr gene was fully functional and useful as a selectable marker in plant transformation experiments. To date, this is the first report of successful expression of a vertebrate gene in transformed plant cells.
Animals↗
Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants.
Phenotypically normal petunia plants carrying chromosomal inserts of either the tomato golden mosaic virus (TGMV) A or the B component DNA, as single or tandem inserts, were obtained using an Agrobacterium tumefaciens Ti plasmid-based transformation system. Southern hybridization analysis revealed that the tandem, direct-repeat A plants contained free single and double stranded A component DNAs. No free B component DNA was detected in plants carrying tandem repeats of the B component. Progeny of self-fertilized plants appeared normal. In contrast, one-quarter of the progeny from tandem A by tandem B plant crosses showed chlorotic lesions on their leaves similar to virus symptoms. The significance of these results and the use of this method for the study of virus functions involved in TGMV replication and symptom production are discussed.
DNA Replication↗
Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens: Role of T-DNA borders in the transfer process.
We have developed a sensitive leaf disc transformation procedure for studying early and/or transient T-DNA expression during Agrobacterium tumefaciens-mediated transformation of plant cells. Using this system, we have examined the function of T-DNA border sequences on the early expression of T-DNA genes and on the stable integration of those genes in infected cells. Deletion of the right border from the T-DNA appears to permit transfer of T-DNA genes from the tumor-inducing (Ti) plasmid but greatly reduces the frequency of their stable integration. A binary vector has been constructed to permit examination of T-DNA border function in trans to the Ti plasmid. In this situation, a single T-DNA border is necessary for early expression of T-DNA genes and is sufficient for stable integration in any orientation.
Journal Article↗
Analysis of Agrobacterium tumefaciens virulence mutants in leaf discs.
The leaf disc transformation system provides a simple means to score expression of various T-DNA markers within days of infection by Agrobacterium tumefaciens as well as long-term selection for growth of transformed callus and shoots. In this report, we describe the application of this system to evaluation of marker transfer and integration in a comprehensive set of defined avirulent mutants of A. tumefaciens. We conclude that virC is not essential when the T-DNA is present on a binary vector. The most likely explanation for this is that the virC gene product is involved in generation of a T-DNA intermediate.
Arginine↗
Accumulation and assembly of soybean beta-conglycinin in seeds of transformed petunia plants.
A gene encoding the alpha'-subunit of beta-conglycinin, a seed storage protein of soybean (Glycine max), was transformed into petunia cells on a disarmed Ti-plasmid of Agrobacterium tumefaciens, and plants were regenerated. Transcripts of the introduced gene accumulated in immature embryos but not in leaves of the transformed plants. Soybean protein was first detected immunologically in proteins extracted from embryos at 10 days post pollination (d.p.p.), concurrent with the accumulation of subunits of the major petunia seed proteins. Between 10 and 16 d.p.p. the primary soybean protein detected had an apparent mol. wt. of 55 kd. The 76-kd alpha'-subunit and several smaller polypeptides accumulated between 16 and 24 d.p.p., when seeds had matured. Polypeptides <76 kd probably resulted from specific proteolytic cleavage of the alpha'-subunit. The alpha'-subunit and the smaller polypeptides assembled into multimeric proteins with sedimentation coefficients of 7-9S, similar to the sedimentation coefficients of beta-conglycinins isolated from soybean seeds. This transformation and expression system should be ideally suited for testing gene mutations to alter the amino acid composition of these seed storage proteins.
Journal Article↗
Light-regulated expression of a pea ribulose-1,5-bisphosphate carboxylase small subunit gene in transformed plant cells.
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Chloroplasts↗
Expression of bacterial genes in plant cells.
Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed.
Aminoglycosides↗