title: High throughput Agrobacterium tumefaciens-mediated germline transformation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.).
abstract: Agrobacterium tumefaciens mediates high frequency of germline transformation of cotton meristem explants. The meristem transformation system we developed is rapid, high throughput and genotype-flexible. We have developed a high throughput cotton transformation system based on direct Agrobacterium inoculation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.). The explants were inoculated with a disarmed A. tumefaciens strain, AB33 harboring a 2 T-DNA binary vector pMON114908. This vector contained a gene of interest, an intron-disrupted β-glucuronidase gene in one T-DNA, and a selectable marker gene, aadA in the other T-DNA. Critical factors, such as method of co-culture, culture temperature during selection, composition of selection medium, and selection scheme were found to influence transformation frequency. The cycle time from initial inoculation to the transplanting of transgenic plants to soil was 7-8 weeks. Stable integration of transgenes and their transmission to progeny were confirmed by molecular and genetic analyses. Transgenes segregated in the expected Mendelian fashion in the T1 generation for most of the transgenic events. It was possible to recover marker-free events in the T1 generation when utilizing a binary vector that contained the selectable marker and gene of interest expression cassettes on independent T-DNAs. The procedure presented here has been used to regenerate thousands of independent transgenic events from multiple varieties with numerous constructs, and we believe it represents a major step forward in cotton transformation technology.
authors.affiliation: Monsanto Company, Agracetus Campus, 8520 University Green, P. O. Box 620999, Middleton, WI, 53562, USA, yurong.chen@monsanto.com.
authors.forename: Yurong
authors.initials: Y
authors.lastname: Chen
authors.forename: Anatoly
authors.initials: A
authors.lastname: Rivlin
authors.forename: Andrea
authors.initials: A
authors.lastname: Lange
authors.forename: Xudong
authors.initials: X
authors.lastname: Ye
authors.forename: Zarir
authors.initials: Z
authors.lastname: Vaghchhipawala
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Eisinger
authors.forename: Erik
authors.initials: E
authors.lastname: Dersch
authors.forename: Miriam
authors.initials: M
authors.lastname: Paris
authors.forename: Brian
authors.initials: B
authors.lastname: Martinell
authors.forename: Yuechun
authors.initials: Y
authors.lastname: Wan
mesh_terms: D016960:Agrobacterium tumefaciens / Q000502:physiology*; D015139:Blotting, Southern; D018920:Coculture Techniques; D003470:Culture Media / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics; D005819:Genetic Markers; D014644:Genetic Variation; D005854:Germ Cells / Q000378:metabolism*; D005947:Glucose / Q000032:analysis; D003368:Gossypium / Q000235:genetics* / Q000382:microbiology*; D040582:Inheritance Patterns / Q000235:genetics; D018519:Meristem / Q000235:genetics* / Q000382:microbiology*; D030821:Plants, Genetically Modified; D013696:Temperature; D014170:Transformation, Genetic; D019076:Transgenes
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial; D005819:Genetic Markers; C036483:T-DNA; D005947:Glucose