title: Methods for suspension culture, protoplast extraction, and transformation of high-biomass yielding perennial grass Arundo donax.
abstract: Arundo donax L. is a promising biofuel feedstock in the Mediterranean region. Despite considerable interest in its genetic improvement, Arundo tissue culture and transformation remains arduous. The authors developed methodologies for cell- and tissue culture and genetic engineering in Arundo. 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 Arundo. The switchgrass ubi2 promoter resulted in noticeably higher reporter gene expression compared with that conferred by the 35S promoter in Arundo.
authors.affiliation: Department of Plant Sciences, University of Tennessee, Knoxville, Tennessee, USA.|Department of Biosciences, University of Milan, Milano, Italy.
authors.forename: Gaia
authors.initials: G
authors.lastname: Pigna
authors.affiliation: Department of Plant Sciences, University of Tennessee, Knoxville, Tennessee, USA.
authors.forename: Taniya
authors.initials: T
authors.lastname: Dhillon
authors.affiliation: Department of Plant Sciences, University of Tennessee, Knoxville, Tennessee, USA.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Dlugosz
authors.affiliation: Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, USA.
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Yuan
authors.affiliation: Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, USA.
authors.forename: Connor
authors.initials: C
authors.lastname: Gorman
authors.affiliation: Department of Biosciences, University of Milan, Milano, Italy.|National Research Council, Institute of Biophysics, Milano, Italy.
authors.forename: Piero
authors.initials: P
authors.lastname: Morandini
authors.affiliation: Department of Food Science, University of Tennessee, Knoxville, Tennessee, USA.|Department of Mechanical, Aerospace, and Biomedical Engineering, University of Tennessee, Knoxville, Tennessee, USA.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Lenaghan
authors.affiliation: Department of Plant Sciences, University of Tennessee, Knoxville, Tennessee, USA.
authors.forename: C Neal
authors.initials: CN
authors.lastname: Stewart
mesh_terms: D018533:Biomass; D018929:Cell Culture Techniques / Q000379:methods*; D003470:Culture Media / Q000737:chemistry; D017930:Genes, Reporter; D008897:Panicum / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics; D006109:Poaceae / Q000166:cytology* / Q000235:genetics* / Q000254:growth & development; D011092:Polyethylene Glycols / Q000737:chemistry; D011401:Promoter Regions, Genetic; D013535:Suspensions; D046509:Tissue Culture Techniques / Q000379:methods; D014170:Transformation, Genetic
chemical_list: D003470:Culture Media; D013535:Suspensions; D011092:Polyethylene Glycols
keywords: Arundo; Electroporation; Protoplast; Suspension Culture; Transformation