Published: September 27th, 2016
A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.
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.
In recent years there has been significant impetus placed on the design of transgenic crops to overcome various diseases1, endow herbicide resistance2, confer drought3,4 and salt tolerance5, prevent herbivory6, increase biomass yield7, and decrease cell wall recalcitrance8. This trend has been aided by the development of new molecular tools for generating transgenic plants, including genome-editing using CRISPR and TALENs9, and gene silencing through dsRNA10, miRNA11, and siRNA12. While these technologies have simplified the generation of transgenic....
1. Establishment of Suspension Cell Cultures
In the current study, the doubling rate of BY-2 varied from 14-18 hr dependent on the temperature at which the cultures were incubated, consistent with previous reports of a mean cell cycle length of 15 hr. With this doubling rate, a 1:100 starting inoculum was used to initiate cultures, leading to cultures with a packed cell volume (PCV) of 50% in 5-7 days. In the current protocol, in which cultures were grown in 200 ml of media, a PCV of 100 ml was generated in 7 days, which provided en.......
The protocol described above has been successfully validated for protoplast isolation, enumeration, and transformation using the BY-2 tobacco suspension cell culture; however, the protocol could easily be extended to any plant suspension culture. At present, protoplast isolation and transformation has been achieved in numerous plants, including maize (Zea mays)10, carrot (Daucus carota)32, poplar (Populus euphratica)33, grape (Vitis vinifera)34
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|Bravo Liquid Handler
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|MultiFlo FX Multi-mode Dispenser
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