Biolistic transformation of elite genotypes of switchgrass (Panicum virgatum L.)
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With a novel elite genotype, SA37, and an improved transformation protocol, it is now possible to routinely and efficiently engineer switchgrass using biolistic transformation.
Transformation of elite switchgrass (Panicum virgatum L.) genotypes would facilitate the characterization of genes related to cell wall recalcitrance to saccharification. However, transformation of explants from switchgrass plants has remained difficult. Therefore, the objective of this study was to develop a biolistic transformation protocol for elite genotypes. Three switchgrass genotypes (ST1, ST2, and AL2) were previously selected for tissue culture responsiveness. One genotype, SA37, was selected for further use due to its improved formation of callus amenable to transformation. Various medium sets were compared and a previously published medium set provided cultures with >96 % embryogenic callus, and data on transient and stable gene expression of RFP were used to optimize biolistic parameters, and further validate the switchgrass (PvUbi1) promoter. SA37 proved to be the most transformable, whereas eight transgenic calli on average were recovered per bombardment of 20 calli (40 % efficiency) when using a three-day day preculture step, 0.6 M osmotic adjustment medium, 4,482 kPa rupture disks and 0.4 μm gold particles which traveled 9 cm before hitting the target callus tissue. Regenerability was high, especially for ST2, for which it is possible to recover on average over 400 plants per half-gram callus tissue. It is now possible to routinely and efficiently engineer elite switchgrass genotypes using biolistic transformation.
KeywordsParticle bombardment Switchgrass Panicum virgatum L. Plant transformation Genetic engineering pANIC vector set
The authors gratefully acknowledge Zeng-Yu Wang for supplying ST1 and ST2 and C. Neal Stewart Jr. and David G.J. Mann for supplying Alamo 2. April Lang assisted in the tissue culture and data collection for this project. Hussein Haleem helped with the statistical analysis of these data. This research was supported by funding from the BioEnergy Science Center, and by state and federal monies allocated to the Georgia Agricultural Experiment Stations. The BioEnergy Science Center is a US Department of Energy BioEnergy Research Center supported by the Office of Biological and Environmental Research in the Department of Energy Office of Science.
Conflict of interest
The authors declare that they have no conflict of interest.
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