Ar. Wenck et al., High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda), PLANT MOL B, 39(3), 1999, pp. 407-416
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.