title: A Robotic Platform for High-throughput Protoplast Isolation and Transformation.
abstract: Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a 'Bright Yellow' 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.
authors: Department of Plant Sciences, University of Tennessee, Knoxville.
authors: Elizabeth M
authors: EM
authors: Dlugosz
authors: Center for Renewable Carbon, University of Tennessee, Knoxville; Department of Mechanical, Aerospace, and Biomedical Engineering, University of Tennessee, Knoxville; slenagha@utk.edu.
authors: Scott C
authors: SC
authors: Lenaghan
authors: Department of Plant Sciences, University of Tennessee, Knoxville.
authors: C Neal
authors: CN
authors: Stewart
mesh_terms: D015870:Gene Expression; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D011523:Protoplasts / Q000166:cytology* / Q000378:metabolism; D012371:Robotics / Q000379:methods; D014026:Nicotiana / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic / Q000502:physiology*
chemical_list: D008164:Luminescent Proteins