In vivo performance of a dual genetic marker, manA-gfp, in transgenic bentgrass

• Published in: Genome Vol. 48; no. 4; pp. 722 - 730
• Main Authors: Fu, D, Xiao, Y, Muthukrishnan, S, Liang, G.H
• Format: Journal Article
• Language: English
• Published: Ottawa, Canada NRC Research Press 01.08.2005; Canadian Science Publishing NRC Research Press
• Subjects: Agrobacterium tumefaciens; Agrostis - drug effects; Agrostis - genetics; Agrostis - growth & development; Agrostis stolonifera var. palustris; Antibiotics; Blotting, Southern; Blotting, Western; callus; Corn; DNA, Plant - analysis; DNA, Plant - genetics; E coli; Escherichia coli; Escherichia coli - enzymology; Fluorescence; gene expression; gene transfer; Genetic markers; Genetic Markers - genetics; genetic transformation; gfp gene; Grasses; green fluorescent protein; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Herbicides; Inheritance Patterns - genetics; isomerases; manA gene; Mannose - pharmacology; Mannose-6-Phosphate Isomerase - genetics; Mannose-6-Phosphate Isomerase - metabolism; Microscopy, Fluorescence; Offspring; phosphomannose isomerase; Plant species; Plant tissues; Plants, Genetically Modified - drug effects; Plants, Genetically Modified - genetics; Plants, Genetically Modified - growth & development; Plasmids - genetics; Pollen; Proteins; recombinant proteins; reporter genes; Seeds - genetics; Seeds - growth & development; Southern blotting; Tissue Culture Techniques; Transgenic plants
• ISSN: 0831-2796; 1480-3321
• DOI: 10.1139/g05-019

A dual-marker combination, manA-gfp, comprising 2 independent expression cassettes of genes encoding an Escherichia coli phosphomannose isomerase (PMI) and a synthetic green fluorescent protein (GFP), was incorporated into the binary vector pPZP201. Agrobacterium tumefaciens-mediated transfer was used to introduce the manA-gfp into the mature-seed derived calli of Agrostis stoloifera L. 'Crenshaw'. The putative transgenic bentgrass calli were screened in Murashige and Skoog medium containing 15 g mannose/L, in conjunction with a visual examination of the GFP expression with a fluorescence stereomicroscope. Calli with GFP fluorescence grew well on the mannose selection media. A total of 24 transgenic plants derived from a single piece of callus lobe were studied for the genomic integration, expression, and function of the transgene. Genomic integration of the dual markers manA and gfp was confirmed by Southern blotting analysis, and the expression of manA also was validated by using PMI-specific antiserum. The inheritance and expression of the dual marker, manA-gfp, was demonstrated in the T1 generation. This study on the environmentally friendly markers further documented the feasibility of using alternative selection methods without using herbicide- or antibiotic-resistance markers.