Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics.
We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice ( Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance ( hpt), green fluorescent protein ( gfp) and beta-glucuronidase ( gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75-95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30-40% of the T(0) plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T(1) progenies showed that 30-50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.
Sallaud
C
C
Biotrop Programme, Cirad-Amis, Avenue Agropolis, 34398 Montpellier Cedex 5, France.
Meynard
D
D
van Boxtel
J
J
Gay
C
C
Bès
M
M
Brizard
J P
JP
Larmande
P
P
Ortega
D
D
Raynal
M
M
Portefaix
M
M
Ouwerkerk
P B F
PB
Rueb
S
S
Delseny
M
M
Guiderdoni
E
E
D016960:Agrobacterium tumefaciens / Q000502:physiology; D001483:Base Sequence; D017796:Caulimovirus / Q000235:genetics; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics*; D018745:Genome, Plant; D023281:Genomics; D005966:Glucuronidase / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D012275:Oryza / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014170:Transformation, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; D008164:Luminescent Proteins; C036483:T-DNA; D049452:Green Fluorescent Proteins; D005966:Glucuronidase