Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens.
Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.
Bidney
D
D
Pioneer Hi-Bred International, Inc., Department of Biotechnology Research, Johnston, IA 50131.
Scelonge
C
C
Martich
J
J
Burrus
M
M
Sims
L
L
Huffman
G
G
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics; D006368:Helianthus / Q000187:drug effects / Q000235:genetics*; D007612:Kanamycin / Q000494:pharmacology; D019868:Kanamycin Kinase; D007613:Kanamycin Resistance / Q000235:genetics; D009355:Neomycin / Q000494:pharmacology; D010770:Phosphotransferases / Q000235:genetics; D010947:Plants, Toxic; D010957:Plasmids / Q000235:genetics; D014026:Nicotiana / Q000187:drug effects / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics*
D004269:DNA, Bacterial; C036483:T-DNA; D007612:Kanamycin; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D005966:Glucuronidase; D009355:Neomycin