Agrobacterium tumefaciens-mediated transformation of Solanum gilo Raddi as influenced by explant type.

An efficient system for Agrobacterium tumefaciens-mediated transformation of Solanum gilo was established. The marker genes for kanamycin resistance and ß-glucuronidase expression were introduced. A comparison between cotyledon and hypocotyl explants showed that while regeneration was better from hypocotyl explants, cotyledon explants gave better transformation efficiency (46% vs. 32%). Four levels of kanamycin selection (100, 150, 200 and 250 mg/l) were tested for effect on transformation efficiency with each type of explant. Lower levels of kanamycin worked better using cotyledon explants, while higher levels of kanamycin worked better for hypocotyl explants. All nine t0 plants tested for expression of the kan (r) gene were positive. The progeny of three of these plants showed a pattern of classical Mendelian inheritance (3 to 1) for both the kan (r) and the ß-glucuronidase genes.

Expression of Escherichia coli glycogen synthase in the tubers of transgenic potatoes (Solanum tuberosum) results in a highly branched starch.

A chimeric gene containing the patatin promoter and the transit-peptide region of the small-subunit carboxylase gene was utilized to direct expression of Escherichia coli glycogen synthase (glgA) to potato (Solanum tuberosum) tuber amyloplasts. Expression of the glgA gene product in tuber amyloplasts was between 0.007 and 0.028% of total protein in independent potato lines as determined by immunoblot analysis. Tubers from four transgenic potato lines were found to have a lowered specific gravity, a 30 to 50% reduction in the percentage of starch, and a decreased amylose/amylopectin ratio. Total soluble sugar content in these selected lines was increased by approximately 80%. Analysis of the starch from these potato lines also indicated a reduced phosphorous content. A very high degree of branching of the amylopectin fraction was detected by comparison of high and low molecular weight carbohydrate chains after debranching with isoamylase and corresponding high-performance liquid chromatography analysis of the products. Brabender viscoamylograph analysis and differential scanning calorimetry of the starches obtained from these transgenic potato lines also indicate a composition and structure much different from typical potato starch. Brabender analysis yielded very low stable paste viscosity values (about 30% of control values), whereas differential scanning calorimetry values indicated reduced enthalpy and gelatinization properties. The above parameters indicate a novel potato starch based on expression of the glgA E. coli gene product in transgenic potato.

The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants.

A Bacillus thuringiensis (B.t.) cryIIIA delta-endotoxin gene was designed for optimal expression in plants. The modified cry gene has the codon usage pattern of an average dicot gene and does not contain AT-rich nucleotide sequences typical of native B.t. cry genes. We assembled the 1.8 kb cryIIIA gene in nine blocks of three oligonucleotide pairs. For two DNA blocks, the polymerase chain reaction was used to enrich for correctly ligated pairs. We compared modified cryIIIA gene with native gene expression by electroporation of dicot (carrot) and monocot (corn) protoplasts. CryIIIA-specific RNA and protein was detected in carrot and corn protoplasts only after electroporation with the rebuilt gene. Transgenic potato lines were generated containing the redesigned cryIIIA gene under the transcriptional control of a chimeric CaMV 35S/mannopine synthetase (Mac) promoter. Out of 63 transgenic potato lines, 58 controlled first-instar Colorado potato beetle (CPB) larvae in bioassays. Egg masses which produced ca. 250,000 CPB larvae were placed on replicate clones of 56 transgenic potatoes. No CPB larvae developed past the second instar on any of these plants. Plants expressing high levels of delta-endotoxin were identified by their toxicity to more resistant third-instar larvae. We show there was good correlation between insect control and the levels of delta-endotoxin RNA and protein.

Production of cyclodextrins, a novel carbohydrate, in the tubers of transgenic potato plants.

Cyclodextrins (CDs) are cyclic oligosaccharides containing six (alpha), seven (beta), or eight (gamma) glucose molecules, respectively. The cyclodextrin glycosyltransferases (CGT), which produce CDs from starch, are found only in bacteria and are used in batch fermentors with hydrolyzed starch to produce CDs commercially. Using a CGT gene from Klebsiella, we attempted to engineer the tubers of developing potatoes to produce these novel, high-value carbohydrates. A chimeric gene, consisting of (1) the patatin promoter for tuber-specific expression, (2) the small subunit of ribulose bisphosphate carboxylase (SSU) transit peptide for plastid targeting, (3) the CGT structural gene from Klebsiella and (4) the nopaline synthase 3' region, was introduced into potatoes. Both alpha and beta CDs were produced in tubers of transgenic potatoes at levels corresponding to 0.001-0.01% of the starch being converted to CDs.

A rapid screen for the detection of specific DNA sequences in plants.

We have developed a simple and quick method ("wick blot") for detecting the presence of specific DNA sequences in plants, using radiolabeled DNA probes. The method requires only small amounts of tissue, about 15-25 mg. More than a hundred samples per day can be easily extracted and blotted. It works well on various species and tissues, including leaves, embryos, and callus. The method is ideally suited for screening large numbers of putative transformants, especially populations that have not been screened by prior selection.