DEVELOPING TOOLS FOR ACCELERATING THE DOMESTICATION OF TWO PROMISING BIOFUEL CROPS: ARUNDO DONAX (GIANT REED) AND PANICUM VIRGATUM (SWITCHGRASS)

The need for energy and the unsustainability in the long term of fossil fuels encouraged the quest for more environmental-friendly energy sources. One alternative is exploiting plants to produce biofuels. The screening of many perennial grasses suggested Miscanthus (Miscanthus spp), Arundo (Arundo donax), and reed canary grass (Phalaris arundinacea) in Europe and switchgrass (Panicum virgatum) in both Europe and US as good candidates for bioenergy production. This thesis focuses on two of these plants: Arundo and switchgrass. Both plants already show a good agronomic performance, but they underwent little domestication, so there is room for their genetic improvement. On the contrary to switchgrass, breeding, genetic transformation and in vitro cultivation of Arundo are lagging behind for the sterility and the recalcitrance of this plant. We developed a method for in vitro cultivation of Arundo that is faster and more efficient than the one previously reported in literature. Calli are induced from stem segments on an MS-based medium containing 9 μM 2,4-D and then a suspension culture can be obtained using the same medium supplemented with 4.4 μM BAP, leading to a 5-fold increase in cell mass over 14 days. This medium was the best in terms of growth rate and final cell density among the four tested media. Protocols for protoplast isolation from suspension cultures and electroporation were also established, allowing a transformation Tools for improving biofuel crops 4 efficiency up to 3.3±1.5 %. The use of a novel vector with two distinct fluorescent protein reporters (GFP and RFP) driven, respectively, by the Cauliflower Mosaic Virus 35S and the ubi2 promoter of switchgrass, allowed a direct comparison of the strength of the two promoters in Arundo. The switchgrass ubi2 promoter provided a much higher expression compared with the 35S promoter. Lastly, as a further methodological advancement, we developed a method to cryopreserve mesophyll protoplast of switchgrass. Both vitrification and slow-freezing methods were tested. Slow freezing is more efficient than vitrification with a percent recovery of alive protoplast of 45.8% vs. 5.2%.