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Plant Mol Biol. 1999 Feb;39(3):407-16.

High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda).

Author information

1
Forest Biotechnology Group, North Carolina State University, Raleigh 27695, USA.
2
NC St U, Raleigh

Abstract

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.

PMID:
10092170
DOI:
10.1023/a:1006126609534
[Indexed for MEDLINE]

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