title: Dissociation (Ds) constructs, mapped Ds launch pads and a transiently-expressed transposase system suitable for localized insertional mutagenesis in rice.
abstract: We have developed a transiently-expressed transposase (TET)-mediated Dissociation (Ds) insertional mutagenesis system for generating stable insertion lines in rice which will allow localized mutagenesis of a chromosomal region. In this system, a Ds containing T-DNA construct was used to produce Ds launch pad lines. Callus tissues, from single-copy Ds/T-DNA lines, were then transiently infected with Agrobacterium harbouring an immobile Ac (iAc) construct, also containing a green fluorescent protein gene (sgfpS65T) as the visual marker. We have regenerated stable Ds insertion lines at a frequency of 9-13% using selection for Ds excision and GFP counter selection against iAc and nearly half of them were unique insertion lines. Double transformants (iAc/Ds) were also obtained and their progeny yielded approximately 10% stable insertion lines following excision and visual marker screening with 50% redundancy. In general, more than 50% of the Ds reinsertions were within 1 cM of the launch pad. We have produced a large number of single-copy Ds/T-DNA launch pads distributed over the rice chromosomes and have further refined the Ds/T-DNA construct to enrich for "clean" single-copy T-DNA insertions. The availability of single copy "clean" Ds/T-DNA launch pads will facilitate chromosomal region-directed insertion mutagenesis. This system provides an opportunity for distribution of gene tagging tasks among collaborating laboratories on the basis of chromosomal locations.
authors.affiliation: CSIRO Plant Industry, GPO Box 1600, 2601, Canberra, ACT, Australia. Narayana.upadhyaya@csiro.au
authors.forename: Narayana M
authors.initials: NM
authors.lastname: Upadhyaya
authors.forename: Qian-Hao
authors.initials: QH
authors.lastname: Zhu
authors.forename: Xue-Rong
authors.initials: XR
authors.lastname: Zhou
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Eamens
authors.forename: Mohammad S
authors.initials: MS
authors.lastname: Hoque
authors.forename: Kerrie
authors.initials: K
authors.lastname: Ramm
authors.forename: Ramannee
authors.initials: R
authors.lastname: Shivakkumar
authors.forename: Kathryn F
authors.initials: KF
authors.lastname: Smith
authors.forename: Shu-Ting
authors.initials: ST
authors.lastname: Pan
authors.forename: Suzhi
authors.initials: S
authors.lastname: Li
authors.forename: Kefan
authors.initials: K
authors.lastname: Peng
authors.forename: Song J
authors.initials: SJ
authors.lastname: Kim
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Dennis
mesh_terms: D002874:Chromosome Mapping; D032461:Chromosomes, Plant; D018983:DNA Footprinting; D018744:DNA, Plant; D016208:Databases, Factual; D018628:Gene Dosage; D017343:Genes, Plant; D017930:Genes, Reporter; D018745:Genome, Plant; D016254:Mutagenesis, Insertional; D012275:Oryza / Q000235:genetics*; D014170:Transformation, Genetic; D019076:Transgenes; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018744:DNA, Plant; D019895:Transposases