Simple, rapid and efficient transformation of genotype Nisqually-1: a basic tool for the first sequenced model tree

Genotype Nisqually-1 is the first model woody plant with an available well-annotated genome. Nevertheless, a simple and rapid transformation of Nisqually-1 remains to be established. Here, we developed a novel shoot regeneration method for Nisqually-1 using leaf petiole and stem segment explants. Numerous shoots formed in the incision of explants within two weeks. The optimized shoot regeneration medium (SRM) contained 0.03 mg l^-1 6-benzylaminopurine, 0.02 mg l^-1 indole-3-butyric acid and 0.0008 mg l^-1 thidiazuron. Based on this, Agrobacterium-mediated genetic transformation of stem explants was examined using the vector pBI₁₂₁ that contains the β-glucuronidase (GUS) as a reporter gene. Consequently, factors affecting transformation frequency of GUS-positive shoots were optimized as follows: Agrobacteria cell suspension with an OD₆₀₀ of 0.4, 20 min infection time, 2 days of co-cultivation duration and the addition of 80 µM acetosyringone into Agrobacteria infective suspension and co-cultivation SRM. Using this optimized method, transgenic plantlets of Nisqually-1 - with an average transformation frequency of 26.7% - were obtained with 2 months. Southern blot and GUS activity staining confirmed the integration of the foreign GUS gene into Nisqually-1. This novel transformation system for Nisqually-1 was rapid, efficient, and simple to operate and will improve more genetic applications in this model tree.