Methods for suspension culture, protoplast extraction, and transformation of high-biomass yielding perennial grass m>Arundo donaxm>
文摘
m>Arundo donaxm> L. is a promising biofuel feedstock in the Mediterranean region. Despite considerable interest in its genetic improvement, m>Arundom> tissue culture and transformation remains arduous. The authors developed methodologies for cell- and tissue culture and genetic engineering in m>Arundom>. A media screen was conducted, and a suspension culture was established using callus induced from stem axillary bud explants. DBAP medium, containing 9 µM 2,4-D and 4.4 µM BAP, was found to be the most effective medium among those tested for inducing cell suspension cultures, which resulted in a five-fold increase in tissue mass over 14 days. In contrast, CIM medium containing 13 µM 2,4-D, resulted in just a 1.4-fold increase in mass over the same period. Optimized suspension cultures were superior to previously-described solidified medium-based callus culture methods for tissue mass increase. Suspension cultures proved to be very effective for subsequent protoplast isolation. Protoplast electroporation resulted in a 3.3 ± 1.5% transformation efficiency. A dual fluorescent reporter gene vector enabled the direct comparison of the CAMV 35S promoter with the switchgrass ubi2 promoter in single cells of m>Arundom>. The switchgrass ubi2 promoter resulted in noticeably higher reporter gene expression compared with that conferred by the 35S promoter in m>Arundom>.
