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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Genetics

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Published: September 27th, 2016

DOI:

10.3791/54300

1Department of Plant Sciences, University of Tennessee, Knoxville, 2Center for Renewable Carbon, University of Tennessee, Knoxville, 3Department of Mechanical, Aerospace, and Biomedical Engineering, University of Tennessee, Knoxville

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....

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1. Establishment of Suspension Cell Cultures

  1. Prepare liquid BY-2 media by adding 4.43 g Linsmaier & Skoog Basal media, 30 g of sucrose, 200 mg KH2PO4, and 200 µg of 2,4-dichlorophenoxyacetic acid (2,4-D) to 900 ml of distilled water and pH to 5.8 with 0.1 M KOH. After adjusting pH, adjust final volume to 1,000 ml with distilled water and autoclave. Media can be stored up to 2 weeks at 4 °C.
  2. Inoculate a 250 ml Erlenmeyer flask with 100 ml of liquid BY-2 media and a.......

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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.......

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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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This research was supported by Advanced Research Projects Agency - Energy (ARPA-E) Award No. DE-AR0000313.

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Name Company Catalog Number Comments
Orbitor RS Microplate mover Thermo Scientific
Bravo Liquid Handler Agilent
Synergy H1 Multi-mode Reader BioTek
MultiFlo FX Multi-mode Dispenser BioTek
Teleshake Inheco 3800048
CPAC Ultraflat Heater/cooler Inheco 7000190
Vworks Automation Software Agilent Software used to control and write protocols for Agilent Bravo
Momentum Software Thermo Scientific Task scheduling software for controlling Orbiter RS
Liquid Handling Control 2.17 Software Biotek Software used to control and write protocols for MultiFlo FX
IX81 Inverted Microscope Olympus
Zyla 3-Tap microscope camera Andor
ET-CY3/TRITC Filter Set Chroma Technology Corp 49004
Rohament CL AB Enzymes sample bottle low-cost cellulase
Rohapect UF AB Enzymes sample bottle low-cost pectinase
Rohapect 10L AB Enzymes sample bottle low-cost pectinase/arabinase
Linsmaier & Skoog Basal Medium Phytotechnology Laboratories L689
2,4 dichlorophenoxyacetic acid Phytotechnology Laboratories D295
propidium iodide Sigma Aldrich P4170
Poly (ethylene glycol) 4000 Sigma Aldrich 95904-250G-F Formerly Fluka PEG
Propidium Iodide Fisher Scientific 25535-16-4 Acros Organics
CaCl2 Sigma Aldrich C7902-1KG
Sodium Acetate Fisher Scientific BP333-500
Mannitol Sigma Aldrich M1902-1KG
Sucrose Fisher Scientific S5-3
KH2PO4 Fisher Scientific AC424205000
KOH Sigma Aldrich P1767
Gelzan CM Sigma Aldrich G1910-250G
6-well plate Thermo Scientific 103184
96-well 1.2 ml deep well plate Thermo Scientific AB-0564
96 well optical bottom plate Thermo Scientific 165305
Finntip 1000 Wide bore Pipet tips Thermo Scientific 9405 163
NaCl Fisher Scientific BP358-10
KCl Sigma Aldrich P4504-1KG
MES Fisher Scientific AC17259-5000
MgCl2 Fisher Scientific M33-500

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