The use of the phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum).
In order to meet the future requirement of using non-antibiotic resistance genes for the production of transgenic plants, we have adapted the selectable marker system PMI/mannose to be used in Agrobacterium-mediated transformation of flax (Linum usitatissimum L.) cv. Barbara. The Escherichia coli pmi gene encodes a phosphomannose isomerase (E.C. 5.1.3.8) that converts mannose-6-phosphate, an inhibitor of glycolysis, into fructose-6-phosphate (glycolysis intermediate). Its expression in transformed cells allows them to grow on mannose-selective medium. The Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the binary vector pNOV2819 that carries the pmi gene under the control of the Cestrum yellow leaf curling virus constitutive promoter was used for transformation experiments. Transgenic flax plants able to root on mannose-containing medium were obtained from hypocotyl-derived calli that had been selected on a combination of 20 g L(-1) sucrose and 10 g L(-1) mannose. Their transgenic state was confirmed by PCR and Southern blotting. Transgene expression was detected by RT-PCR in leaves, stems and roots of in vitro grown primary transformants. The mean transformation efficiency of 3.6%, that reached 6.4% in one experiment was comparable to that obtained when using the nptII selectable marker on the same cultivar. The ability of T1 seeds to germinate on mannose-containing medium confirmed the Mendelian inheritance of the pmi gene in the progeny of primary transformants. These results indicate that the PMI/mannose selection system can be successfully used for the recovery of flax transgenic plants under safe conditions for human health and the environment.
Lamblin
Frédéric
F
Laboratoire de Biologie des Ligneux et des Grandes Cultures, UPRES EA 1207, Antenne Scientifique Universitaire de Chartres, Chartres, France. frederic.lamblin@univ-orleans.fr
Aimé
Aurélie
A
Hano
Christophe
C
Roussy
Isabelle
I
Domon
Jean-Marc
JM
Van Droogenbroeck
Bart
B
Lainé
Eric
E
D001483:Base Sequence; D015415:Biomarkers; D017931:DNA Primers; D019597:Flax / Q000235:genetics*; D008359:Mannose-6-Phosphate Isomerase / Q000235:genetics*; D018520:Plant Shoots / Q000254:growth & development; D030821:Plants, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012231:Rhizobium / Q000502:physiology*; D012639:Seeds; D014169:Transformation, Bacterial
D015415:Biomarkers; D017931:DNA Primers; D008359:Mannose-6-Phosphate Isomerase