Background

As perennials species, Populus is an ideal model system to explore scientific questions that can not be easily addressed in annual plants such as Arabidopsis and rice (Jansson and Douglas, 2007). For instance, autumn leaf senescence (Wang et al., 2021), sex determination (Muller et al., 2020; Xue et al., 2020), wood formation (Chen et al., 2019), and seasonal dormancy (Tylewicz et al., 2018), can be more easily studied in Populus than in annual plants. As the lifespan of Populus plants usually spans decades, centuries, or even millennia (Munné-Bosch, 2008), it is necessary to establish a simple and rapid system to study gene function and signal transduction in woody plants. Mesophyll protoplasts are a powerful and versatile tool for conducting cell-based experiments to study gene function and signaling pathways (Yoo et al., 2007). They provide a good way to study important scientific questions at biochemical, molecular, cellular, genetic, genomic, transcriptomic, proteomic, and single-cell levels. Previously reported protocols for preparation and transfection of mesophyll protoplasts mainly focused on the model plant Arabidopsis (Yoo et al., 2007). However, the protocol for isolating Arabidopsis mesophyll protoplasts is not suitable for Populus protoplast preparation because of the different cell wall components (Lin et al., 2014). The protocol summarized here is a simple (fewer reagents, only four reagents are required) and efficient (less time, isolation and transfection takes approximately 7 h) procedure (Figure 1) compared with previously reported Populus protoplast preparation (Guo et al., 2012; Tan et al., 2013). A histone variant HTB9 fused with GFP is transiently expressed in P. tomentosa mesophyll protoplasts (Figure 2). This cellular system provides a helpful tool for studying gene expression in woody plants. Moreover, this protocol can also be applied for other Populus species leaves such as Populus alba, Populus trichocarpa, Populus alba × P. tremula var. glandulosa (84K), and Populus davidiana × P. bolleana, or leaves of other woody species such as Eucalyptus urophylla × E. grandis or Eucalyptus grandis × E. urophylla grown in tissue culture media.