A novel two T-DNA binary vector allows efficient generation of marker-free transgenic plants in three elite cultivars of rice (Oryza sativa L.).
A pilot binary vector was constructed to assess the potential of the 2 T-DNA system for generating selectable marker-free progeny plants in three elite rice cultivars (ZhongZuo321, Ariete and Khao Dawk Mali 105) known to exhibit contrasting amenabilities to transformation. The first T-DNA of the vector, delimited by Agrobacterium tumefaciens borders, contains the hygromycin phosphotransferase (hpt) selectable gene and the green fluorescent protein (gfp) reporter gene while the second T-DNA, delimited by Agrobacterium rhizogenes borders, bears the phosphinothricin acetyl transferase (bar) gene, featuring the gene of interest. 82-90% of the hygromycin-resistant primary transformants exhibited tolerance to ammonium glufosinate mediated by the bar gene suggesting very high co-transformation frequency in the three cultivars. All of the regenerated plants were analyzed by Southern blot which confirmed co-integration of the T-DNAs at frequencies consistent with those of co-expression and allowed determination of copy number for each gene as well as detection of two different vector backbone fragments extending between the two T-DNAs. Hygromycin susceptible, ammonium glufosinate tolerant phenotypes represented 14.4, 17.4 and 14.3% of the plants in T1 progenies of ZZ321, Ariete and KDML105 primary transformants, respectively. We developed a statistical model for deducing from the observed copy number of each T-DNA in T0 plants and phenotypic segregations in T1 progenies the most likely constitution and linkage of the T-DNA integration locus. Statistical analysis identified in 40 out of 42 lines a most likely linkage configuration theoretically allowing genetic separation of the two T-DNA types and out segregation of the T-DNA bearing the bar gene. Overall, though improvements of the technology would be beneficial, the 2 T-DNA system appeared to be a useful approach to generate selectable marker-free rice plants with a consistent frequency among cultivars.
Breitler
Jean-Christophe
JC
UMR PIA1096, Biotrop and Crop Protection Programmes, Cirad-Amis, TA40/03, Av. Agropolis, F-34398 Montpellier, France. breitler@cirad.fr
Meynard
Donaldo
D
Van Boxtel
Jos
J
Royer
Monique
M
Bonnot
François
F
Cambillau
Laurence
L
Guiderdoni
Emmanuel
E
D000123:Acetyltransferases / Q000235:genetics; D000383:Agriculture / Q000379:methods; D000613:Aminobutyrates; D015139:Blotting, Southern; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics*; D017930:Genes, Reporter; D008040:Genetic Linkage; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D016013:Likelihood Functions; D015233:Models, Statistical; D012275:Oryza / Q000235:genetics*; D010641:Phenotype; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D030821:Plants, Genetically Modified; D012231:Rhizobium / Q000235:genetics; D013045:Species Specificity; D014170:Transformation, Genetic
D000613:Aminobutyrates; D017931:DNA Primers; D004269:DNA, Bacterial; C036483:T-DNA; D049452:Green Fluorescent Proteins; C003121:phosphinothricin; D000123:Acetyltransferases; C056243:phosphinothricin N-acetyltransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase