A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Summary

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.

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.

Introduction

In recent years there has been significant impetus placed on the design of transgenic crops to overcome various diseases¹, endow herbicide resistance², confer drought^3,4 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....

Protocol

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 single piece of BY-2 callus (>1 cm in diameter) grown on solid BY-2 media (liquid BY-2 media with the addition of 1% agar) and seal with aluminum....

Results

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.......

Discussion

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

Materials

List of materials used in this article

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