Glyphosate-resistant plants of `South Bay' lettuce (Lactuca sativa L.) were produced by using Agrobacterium tumefaciens containing a plasmid carrying glyphosate oxidase and EPSPS gene. An in vitro assay was performed to determine the sensitivity of `South Bay' leaf discs and seedling explants to varying glyphosate concentrations. The I50 for glyphosate leaf discs was 53.8 μm and for glyphosate seedlings 7.6 μm. There was a high correlation between the response of leaf discs and seedlings to glyphosate based on dry weight. These findings will allow identification of glyphosate-resistant transformants in an early stage of plant development, saving time and reducing the cost in generating an improved cultivar with the glyphosate resistance trait.
Antonio C. Torres, Russell T. Nagata, Robert J. Ferl, Thomas A. Bewick, and Daniel J. Cantliffe
Rod Serres, Brent McCown, Dennis McCabe, Elden Stang, Dave Russell, and Brian Martinell
Electric discharge particle acceleration was used to introduce three foreign genes into the American cranberry (Vaccinium macrocarpon Ait.). These genes were NPTII (conferring resistance to the antibiotic, kanamycin), GUS (allowing for visual verification), and B.t. (conferring resistance to lepidopteran insects). Adventitious buds were induced on stem sections prior to bombardment with DNA-coated gold pellets. Bombarded stem sections were then transferred to a selection medium containing kanamycin. The surface of the medium was overlaid with a thin layer of kanamycin solution. Approximately 35 days after blasting, proliferating cell masses and elongating shoots were observed amidst the mass of kanamycin-inhibited tissue. Seven weeks after blasting, a histochemical assay verified GUS expression in these tissues, and polymerase chain reaction was used to confirm the presence of the introduced genes.
George C.J. Fernandez
Kathryn Kamo, Alan Blowers, Franzine Smith, and Joyce Van Eck
Over one hundred Gladiolus plants of the commercially important cultivars `Jenny Lee' and `Peter Pears' have been stably transformed following particle bombardment on regenerable callus, suspension cells, or cormel slices. The phosphinothricin acetyltransferase gene which confers phosphinothricin resistance was cotransformed with either antiviral genes or the uidA reporter gene coding for β- glucuronidase expression. Transformed plants were regenerated following selection on concentrations of phosphinothricin which varied with the type of tissue used for bombardment. Integration of foreign DNA was confirmed by polymerase chain reaction and gene expression.
Alejandrina Robledo-Paz, José Luis Cabrera-Ponce, Víctor Manuel Villalobos-Arámbula, Luis Herrera-Estrella, and Alba Estela Jofre-Garfias
Microprojectile bombardment was used to introduce DNA into embryogenic callus of garlic (Allium sativum L.) and produce stably transformed garlic plants. Embryogenic calluses, derived from garlic cultivar `GT96-1', were bombarded with plasmid DNA containing genes coding for hygromycin phosphotransferase and β-glucuronidase. Putatively transformed calluses were identified in the bombarded tissue after 4 months of selection on 20 mg·L-1 hygromycin B. The transgenic nature of the selected material was demonstrated by GUS histochemical assay and Southern blot hybridization analysis, and twenty transgenic plants were regenerated.
Teryl R. Roper and Armand R. Krueger
Cranberry plants exclusively utilize ammonium forms of nitrogen. Nitrification of applied ammonium and subsequent leaching through sandy soils is a potential problem for growers. Peat, sand, and striped soils were collected in cranberry beds in central Wisconsin and soil pH was adjusted to 3.5, 4.5, or 5.5. Twenty-five grams of dry soil was placed in flasks and half the flasks were sterilized. Distilled water was added to half of the samples, and the other half received 15N-labeled ammonium. Flasks were incubated at 20°C for up to 70 days. Striped soils showed no nitrification at pH 3.5 or 4.5 during the 70 day incubation. At pH 5.5, nitrification began at 20 days and was almost complete at 70 days. Nitrification did not occur at any pH in sandy soils. This research suggests that ammonium fertilizer applied to cranberry is likely taken up before nitrification would occur.
R.N. Trigiano and R.A. May
A tissue culture laboratory exercise illustrating regeneration of whole plants from leaf segments of chrysanthemum by organogenesis is described. Using simple, common media, shoots can be generated in 5 weeks and rooted after an additional 3 weeks. Acclimatization of plants can be accomplished in a simple mistbed in the greenhouse. The exercise is adaptable to depict genotype differences among cultivars, optimization of shoot induction, effects of growth regulators, and experimental design. Callus is typically not formed during shoot formation; however, co-cultivation of leaf segments with a virulent strain of Agrobacterium tumefaciens produces callus with a strain of disarmed A. tumefaciens harboring NPTII construct affects regeneration of plants resistant to kanamycin.
George C. Elliott and Harvey J. Lang
Fungicides were applied at label rates to two commercial soilless potting media in which Iris siberica L. crowns had been potted and were subsequently grown under greenhouse conditions. Effects of fungicides on urea hydrolysis were inconsistent and generally insignificant. Ammonium oxidation was inhibited to varying degrees by Truban, Benlate, Banol, and Subdue. In a subsequent experiment, the same fungicides were added to cropped samples of the same media in vitro, followed 12 hours later by a solution containing urea and ammonium. Urea hydrolysis was essentially unaffected by fungicide treatments. Subsequent oxidation of ammonium was inhibited by Truban and Banol only in one medium. Transient accumulation of nitrite was inhibited by Truban but stimulated by Benlate in both media. When added to pure cultures of Nitrosomonas europea and Nitrobacter agilis, Truban completely inhibited oxidation of ammonium and nitrite. Benlate partially inhibited oxidation of ammonium and nitrite, while Subdue and Banal partially inhibited oxidation of ammonium but not nitrite. Chemical names used: [Methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate] (benomyl); N- (2,6-dimethylphenyl) -N- (methoxyacetyl)alanine methyl ester (metalaxyl); [2-chloro-6-(trichloromethyl)pyridine (nitrapyrin); 5-Ethoxy-3-(trichloromethyl)-1,2,4-thiadiazole (ethazol); Propyl[3-(dimethylamino)propyl]carbamate monohydrochloride (propamocarb).
Fure-Chyi Chen and Adelheid R. Kuehnle
Several procedures were tested in development of a gene transfer protocol for anthurium. Etiolated internode segments of anthurium cultivars `Rudolph' and `UH1060' were co-cultured with Agrobacterium tumefaciens LBA4404 carrying the chimeric genes neo, for antibiotic resistance, and att encoding antibacterial attacin. Assays of genomic DNA and RNA from kanamycin-resistant `Rudolph' and DNA from `UH1060' plantlets, recovered as soon as 1 year after culture on selection media, indicated the presence of introduced genes, including neo and att, and transcription of att. Western analysis confirmed the expression of attacin protein in calli induced from laminae of regenerated kanamycinresistant `Rudolph' plantlets. Use of tobacco nurse cells during co-cultivation of internodes with Agrobacterium did not increase recovery of shoots under the regeneration conditions used. Improvements in culture and antibiotic selection conditions during plant development are suggested.
A.R. Kuehnle and N. Sugii
A method was devised for infecting Anthurium andraeanum Linden ex André, an economically important ornamental monocot, with Agrobacterium tumefaciens. Tumors were obtained on plant stems 7 to 10 weeks after inoculation with oncogenic A. tumefaciens strains C58 and A281 cultured previously in an induction medium containing 200 μm acetosyringone at pH 5.5. A higher percentage of tumors were formed in vitro on etiolated internodes (32%) than on green leaf (2%) or petiole explants (3%) 4 weeks after inoculation with induced C58. All explants treated with nontumorigenic A. radiobacter or with induction medium alone failed to produce tumors. Chromatograms showed an accumulation of nopaline in internode explant tumors induced with C58. DNA amplification and hybridization studies showed that the DNA from these tumors, but not from noninoculated anthurium tissue, contained sequences homologous to the nopaline synthase gene of A. tumefaciens T-DNA. Chemical names used 3,5-dimethoxy 4-hydroxyacetophenone (acetosyringone).