Development of a rapid, low-cost protoplast transfection system for switchgrass (Panicum virgatum L.)

Abstract

Key messageA switchgrass protoplast system was developed, achieving a cost reduction of ~1000-fold, a threefold increase in transformation efficiency, and a fourfold reduction in required DNA quantity compared to previous methods.AbstractIn recent years, there has been a resurgence in the use of protoplast systems for rapid screening of gene silencing and genome-editing targets for siRNA, miRNA, and CRISPR technologies. In the case of switchgrass (Panicum virgatum L.), to achieve economic feasibility for biofuel production, it is necessary to develop plants with decreased cell wall recalcitrance to reduce processing costs. To achieve this goal, transgenic plants have been generated with altered cell wall chemistry; however, with limited success owing to the complexity of cell walls. Because of the considerable cost, time, and effort required to screen transgenic plants, a protoplast system that can provide data at an early stage has potential to eliminate low performing candidate genes/targets prior to the creation of transgenic plants. Despite the advantages of protoplast systems, protoplast isolation in switchgrass has proven costly, requiring expensive lab-grade enzymes and high DNA quantities. In this paper, we describe a low-cost protoplast isolation system using a mesophyll culture approach and a cell suspension culture. Results from this work show a cost reduction of ~1000-fold compared to previous methods of protoplast isolation in switchgrass, with a cost of $0.003 (USD) per reaction for mesophyll protoplasts and $0.018 for axenic cell culture-derived protoplasts. Further, the efficiency of protoplast transformation was optimized threefold over previous methods, despite a fourfold reduction in DNA quantity. The methods developed in this work remove the cost barrier previously limiting high-throughput screening of genome-editing and gene silencing targets in switchgrass, paving the way for more efficient development of transgenic plants.

The Physcomitrella patens Chloroplast Proteome Changes in Response to Protoplastation

“…They provide an invaluable experimental system for the analysis of protein subcellular localization, protein–protein interactions, and investigation of various biological processes (Zhang et al, 2011; Guo et al, 2012; Pitzschke and Persak, 2012; Burris et al, 2016). Despite the enzymatic treatment, protoplasts are considered to maintain many of the physiological activities of intact plants.…”

Methods for suspension culture, protoplast extraction, and transformation of high‐biomass yielding perennial grass Arundo donax

“…After 20 min of incubation at room temperature (RT), protoplasts were washed twice with 1 and 4 mL of W5 and collected by centrifugation at 100 × g for 6 min. Protoplasts were then resuspended in 1 mL of WI (0.6 M mannitol, 4 mM KCl, 4 mM MES, pH 5.7), transferred to a 12‐well plate (Corning Incorporated, Corning, NY, USA) and incubated at 28°C in the dark for 20 h. As a positive control, switchgrass mesophyll protoplasts were transformed using PEG as previously described . The transformation efficiency (%) was calculated by counting the number of protoplasts expressing pporRFP divided by the total number of protoplasts using a hemocytometer and multiplied by 100 as shown in the formula below: …”

“…PEG-mediated DNA transformation was performed as previously described [21,35] , transferred to a 12-well plate (Corning Incorporated, Corning, NY, USA) and incubated at 28°C in the dark for 20 h. As a positive control, switchgrass mesophyll protoplasts were transformed using PEG as previously described [31]. The transformation efficiency (%) was calculated by counting the number of protoplasts expressing pporRFP divided by the total number of protoplasts using a hemocytometer and multiplied by 100 as shown in the formula below:…”

A versatile high throughput screening platform for plant metabolic engineering highlights the major role of ABI3 in lipid metabolism regulation

“…CRISPR) targets (Cao et al, 2014;Maćkowska et al, 2014;Schapire and Lois, 2016;You et al, 2014;Zhai et al, 2009). In addition, protoplast-based systems are now available for almost any plant of commercial interest, including lettuce (Lactuca sativa) (Sasamoto and Ashihara, 2014), grape (Vitis vinifera) (Wang et al, 2015) and bean (Phaseolus vulgaris) (Nanjareddy et al, 2016) and even the less expected, such as oil palm (Elaeis guineensis) (Masani et al, 2014), poplar (Populus euphratica) (Guo et al, 2015) and switchgrass (Panicum virgatum) (Burris et al, 2016).…”