Microprojectile-mediated DNA delivery to the Salicaceae family

Abstract

A transient gene expression system was developed for the Salicaceae family using microprojectile-mediated DNA delivery (Biolistic™) to cell suspensions. Using Populus nigra × Populus maximowiczii line NM1, 10 variables were optimized. Optimum transient gene expression under the 10 conditions tested was obtained with a 7- to 9-day-old cell suspension. Highest transient levels were observed with samples positioned 13.5 cm from the stopping plate and bombarded with 1.6-μm gold particles coated with 1 μg of DNA precipitated with CaCl2. Assaying for β-glucuronidase gene expression was performed 1 day after bombardment. The fate of the microparticles in the bombarded cells was studied, showing that 58.9% of cells expressing the β-glucuronidase gene had microparticles located in the nucleus or its vicinity and the remaining cells had microparticles in the cytoplasm. Cell suspensions from five different lines (P. nigra × P. maximowiczii lines NM1 and NM6, Populus deltoides × P. nigra line DN106, Populus tremula × Populus alba line 7171-B4, and Salix alba sanguined line SA-2) yielded transient gene expression. The relative strengths of β-glucuronidase expression in the lines tested were NM1 = 7171-B4 > NM6 > DN106 > SA-2. Six plasmid constructions were also tested in line NM1 for transient β-glucuronidase gene expression. The x-glucuronide histochemical assay did not reveal any differences, but the methyl umbelliferone glucuronide fluorescent assay yielded the following relative levels of transient gene expression with the different promoter sequences: 35S-35S-AMV enhancer = 35S-AMV enhancer > 35S-35S = 35S-35S-AMV enhancer with the β-glucuronidase – neomycin phosphotransferase fusion > 35S. Four transgenic cell lines of P. nigra × P. maximowiczii were characterized for kanamycin resistance and neomycin phosphotransferase II gene activity. Polymerase chain reaction and Southern hybridization analyses indicated the presence of the β-glucuronidase and neomycin phosphotransferase II genes in the genome of three of these transgenic lines. Key words: microprojectile, particle bombardment, Salicaceae, poplar, willow.