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 c onifer 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 Agroba cterium to improve transformation. Additional copies of virulence genes wer e added to three common disarmed strains. These extra virulence genes inclu ded either a constitutively active virG or extra copies of virG and virB, b oth from pTiBo542. In experiments with Norway spruce, we increased transfor mation efficiencies 1000-fold from initial experiments where little or no t ransient 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 line s selected on kanamycin-containing medium. In loblolly pine, transient expr ession increased 10-fold utilizing modified Agrobacterium strains. Agrobact erium-mediated gene transfer is a useful technique for large-scale generati on of transgenic Norway spruce and may prove useful for other conifer speci es.