Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection.
An Agrobacterium-mediated transformation system with glyphosate selection has been developed for the large-scale production of transgenic plants. The system uses 4-day precultured immature embryos as explants. A total of 30 vectors containing the 5-enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium strain CP4 (aroA:CP4), which confers resistance to glyphosate, were introduced into wheat using this system. The aroA:CP4 gene served two roles in this study-selectable marker and gene of interest. More than 3,000 transgenic events were produced with an average transformation efficiency of 4.4%. The entire process from isolation of immature embryos to production of transgenic plantlets was 50-80 days. Transgenic events were evaluated over several generations based on genetic, agronomic and molecular criteria. Forty-six percent of the transgenic events fit a 3:1 segregation ratio. Molecular analysis confirmed that four of six lead transgenic events selected from Agrobacterium transformation contained a single insert and a single copy of the transgene. Stable expression of theAROA:CP4 gene was confirmed by ELISA through nine generations. A comparison of Agrobacterium-mediated transformation to a particle bombardment system demonstrated that the Agrobacterium system is reproducible, has a higher transformation efficiency with glyphosate selection and produces higher quality transgenic events in wheat. One of the lead events from this study, no. 33391, has been identified as a Roundup Ready wheat commercial candidate.
Hu
T
T
Monsanto Company, 700 Chesterfield Parkway West, St. Louis, MO 63017, USA. tianci.hu@monsanto.com
Metz
S
S
Chay
C
C
Zhou
H P
HP
Biest
N
N
Chen
G
G
Cheng
M
M
Feng
X
X
Radionenko
M
M
Lu
F
F
Fry
J
J
D019470:Biolistics; D015139:Blotting, Southern; D015870:Gene Expression; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006540:Herbicides / Q000494:pharmacology; D030821:Plants, Genetically Modified; D012038:Regeneration; D012231:Rhizobium / Q000235:genetics*; D012641:Selection, Genetic; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics; D014908:Triticum / Q000187:drug effects* / Q000235:genetics*; D000097797:Glyphosate
D005998:Glycine; D006540:Herbicides; D000097797:Glyphosate