Biosciences Center, National Renewable Energy Laboratory, Golden, CO, USA.
National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO, USA.
Methods Mol Biol. 2020;2096:61-79. doi: 10.1007/978-1-0716-0195-2_6.
As a robust perennial C4-type monocot plant and a native species to North America, switchgrass (Panicum virgatum) has been evaluated and designated as a strong candidate bioenergy crop by the U.S. DOE. Although genetic modifications of switchgrass have been used to successfully reduce the recalcitrance of switchgrass biomass for biofuel production, the generation of transgenic switchgrass is still a slow and laborious process. A transient protoplast system can provide an excellent platform to accelerate the selection of genes-of-interest for tailoring switchgrass biomass. However, partially due to the lack of the complete genomic information, the attempts to optimize the transient protoplast system for switchgrass remain scarce. In this chapter, we provide an improved protocol for switchgrass protoplast isolation, increased transformation efficiency using CsCl gradient ultracentrifugation-derived plasmid DNA and extended application of the transient switchgrass protoplast system to analyze protein expression using western blot.
作为一种强健的多年生 C4 型单子叶植物和北美的本土物种，柳枝稷（Panicum virgatum）已被美国能源部评估并指定为一种有潜力的生物能源作物。尽管已经对柳枝稷进行了基因改造，以成功降低其生物量对生物燃料生产的抗性，但转基因柳枝稷的产生仍然是一个缓慢而费力的过程。瞬时原生质体系统可以为选择感兴趣的基因来定制柳枝稷生物质提供一个极好的平台。然而，部分由于缺乏完整的基因组信息，优化柳枝稷瞬时原生质体系统的尝试仍然很少。在本章中，我们提供了一种改良的柳枝稷原生质体分离方案，使用 CsCl 梯度超速离心衍生的质粒 DNA 提高了转化效率，并扩展了瞬时柳枝稷原生质体系统的应用，以使用 Western blot 分析蛋白质表达。
