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Wheat Transformation Using Cyanamide as a New Selective Agent

^a ARS, USDA, 344 Keim Hall, Univ. of Nebraska-Lincoln, Lincoln, NE 68583 USA
^b School of Public Health, Univ. of California-Berkeley, Berkeley, CA 94720 USA
^c Hechingerstr. 12, D-72144 Dusslingen, Germany
^d Institute of Biochemical Plant Pathology, GSF Research Center, Xenobiotics, D-85758 Oberschleissheim, Germany
^e ARS, USDA, WRRC, 800 Buchanan Street, Albany, CA 94710 USA

View larger version (24K): [in a new window]   Fig. 1 Schematic representation of the plasmid used for selection and transformation. Cah+ indicates the coding sequence which is under the control of the maize ubiquitin (Ubi1) promoter and the transcription termination region nos of Agrobacterium tumefaciens. A plasmid pUC8 was employed as the cloning vector

View larger version (66K): [in a new window]   Fig. 2 Selection and regeneration of transgenic wheat. A, Calli on selective medium containing 0.0375 g L-1 of cyanamide 8 wk following bombardment. B, Shoot formation from cyanamide-resistant callus tissue. C, A series of dilutions for the colorimetric assay (left to right); 0, 40, 20, 10, 5, 2, and 1 mM of cyanamide. D, Use of the colorimetric assay to test for the presence of cyanamide hydratase (left to right); nonplant sample containing no cyanamide (cuvette no. 1), nonplant sample containing 5 mM cyanamide (cuvette no. 2), transformed plant sample containing 5 mM cyanamide (cuvette no. 3), and a nontransformed plant sample containing 5 mM cyanamide (cuvette no. 4). E, Wheat plantlets sprayed with a Dormex solution (10%) 2 wk after planting; sprayed nontransformed control plant (right), sprayed 1722 plant (left). F, Wheat plants growing in the presence (3 g) of Perlka 2 wk after application, control wheat plant (left) and a plant of the 1722 line (right)

View larger version (90K): [in a new window]   Fig. 3 Southern blot analysis for T0 transgenic plants and a nontransformed control plant. Gel blot lanes contained 25 µg of genomic DNA. Hybridization was carried out with a radio-labeled Cah gene. The arrow indicates the expected position of the 1661 bp Cah gene fragment. The migration position of the molecular weight markers are shown to the left of the autoradiogram and labeled with sizes in kilobases. The left three lanes contain undigested DNA and the next three lanes contain DNA digested with EcoRI. The lanes marked TA and TB represent genomic DNA from two independently transformed lines (plant lines 1650 and 1722, respectively). Nontransformed control DNA is designated as NT and lanes labeled RC contain 25 µg of genomic nontransformed DNA plus 0.592 pg of a 0.8-kb PstI digest sequence from the pCAM plasmid which should correspond to a single copy reconstruction