Wenck A R, Quinn M, Whetten R W, Pullman G, Sederoff R
Forest Biotechnology Group, North Carolina State University, Raleigh 27695, USA.
Plant Mol Biol. 1999 Feb;39(3):407-16. doi: 10.1023/a:1006126609534.
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.
农杆菌介导的基因转移是许多植物生物技术实验室的首选方法；然而，这种生物体在针叶树转化中的大规模应用受到外植体材料繁殖困难、选择效率和低转化频率的限制。我们分析了共培养条件和不同的无致病力农杆菌菌株以提高转化效率。向三种常见的无致病力菌株中添加了毒力基因的额外拷贝。这些额外的毒力基因包括一个组成型活性的virG或来自pTiBo542的virG和virB的额外拷贝。在挪威云杉的实验中，我们将转化效率从最初几乎检测不到或没有瞬时表达的实验提高了1000倍。从与农杆菌共培养的快速分裂的胚性悬浮培养细胞中产生了100多个表达标记基因β-葡萄糖醛酸酶（GUS）的转化株系。GUS活性用于监测瞬时表达并进一步测试在含卡那霉素培养基上选择的株系。在火炬松中,利用改良的农杆菌菌株瞬时表达提高了10倍。农杆菌介导的基因转移是大规模产生转基因挪威云杉的有用技术，并且可能对其他针叶树种也有用。
