Light strongly promotes gene transfer from Agrobacterium tumefaciens to plant cells.
Light conditions during Agrobacterium-based plant transformation, the most routinely used method in plant genetic engineering, differ widely and, to our knowledge, have not been studied systematically in relation to transformation efficiency. Here, light effects were examined in two already optimized transformation procedures: coculture of Agrobacterium tumefaciens with callus from two genotypes of the crop plant Phaseolus acutifolius (tepary bean) and coculture of root segments from two ecotypes of Arabidopsis thaliana. Except for the light conditions during coculture, all steps followed established procedures. Coculture was done either under continuous darkness, under a commonly used photoperiod of 16 h light/8 h darkness or under continuous light. beta-glucuronidase (GUS) production due to the transient expression of an intron-containing uidA gene in the binary vector was used to evaluate T-DNA transfer. In all situations, uidA expression correlated highly and positively with the light period used during coculture; it was inhibited severely by darkness and enhanced more under continuous light than under a 16 h light/8 h dark photoperiod. The promotive effect of light was observed with Agrobacterium strains harboring either a nopaline-, an octopine- or an agropine/succinamopine-type non-oncogenic helper Ti plasmid. The observed positive effect of light has obvious implications for developing and improving transient and stable transformation protocols, specifically those involving dark coculture conditions.
Zambre
Mukund
M
Department of Plant Systems Biology, Vlaams Interuniversitair Instituut voor Biotechnologie, Universiteit Gent, K.L. Ledeganckstraat 35, 9000 Gent, Belgium.
Terryn
Nancy
N
De Clercq
Janniek
J
De Buck
Sylvie
S
Dillen
Willy
W
Van Montagu
Marc
M
Van Der Straeten
Dominique
D
Angenon
Geert
G
D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000528:radiation effects; D017360:Arabidopsis / Q000235:genetics / Q000528:radiation effects; D004269:DNA, Bacterial / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D008027:Light; D027805:Phaseolus / Q000235:genetics / Q000528:radiation effects; D010944:Plants / Q000235:genetics* / Q000528:radiation effects; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D004269:DNA, Bacterial; C036483:T-DNA; D005966:Glucuronidase