Journal
THEORETICAL AND APPLIED GENETICS
Volume 106, Issue 8, Pages 1396-1408Publisher
SPRINGER
DOI: 10.1007/s00122-002-1184-x
Keywords
Functional genomics; Gene transfer; Insertional mutagenesis; Oryza sativa L.; T-DNA
Categories
Funding
- French National Plant Genomics initiative Genoplante
- EU [BIOTECH CT 97-2132]
Ask authors/readers for more resources
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 TDNA 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available