High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda).
Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.
Wenck
A R
AR
Forest Biotechnology Group, North Carolina State University, Raleigh 27695, USA.
Quinn
M
M
Whetten
R W
RW
Pullman
G
G
Sederoff
R
R
D000098:Acetophenones / Q000494:pharmacology; D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D001483:Base Sequence; D046508:Culture Techniques; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D008969:Molecular Sequence Data; D041603:Pinus taeda; D014170:Transformation, Genetic / Q000187:drug effects; D014774:Virulence / Q000235:genetics
D000098:Acetophenones; D004269:DNA, Bacterial; C036483:T-DNA; C051667:acetosyringone