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 plants, they have also created a bottleneck, where the sheer number of transgenic plants generated cannot be screened using traditional systems that rely on plant regeneration. Related to this bottleneck, while silencing and genome-editing constructs can be rapidly inserted into plants, many of the targeted traits fail to produce the desired effect, which is often not discovered until plants are analyzed in the greenhouse. In this work, we have developed a method for rapid, automated, high-throughput screening of plant protoplasts, specifically to address the current bottleneck in early screening of large numbers of genome-editing and gene silencing targets.

The use of protoplasts, as opposed to intact plant cells, has several advantages for the development of an automated platform. First, protoplasts are isolated after digestion of the plant cell wall, and with this barrier no longer present, transformation efficiency is increased¹³. In intact plant cells there are only two well established methods for transformation, biolistics¹⁴ and Agrobacterium-mediated transformation¹⁵. Neither of these methods can be easily translated to liquid handling platforms, as biolistics requires specialized equipment for transformation, whereas Agrobacterium-mediated transformation requires co-culture and subsequent removal of the bacteria. Neither are amenable for high throughput methods. In the case of protoplasts, transformation is routinely conducted using polyethylene glycol (PEG)-mediated transfection¹⁶, which only requires several solution exchanges, and is ideally suited for liquid handling platforms. Second, protoplasts, by definition, are single-cell cultures, and thus the problems associated with clumping and chain formation in plant cell cultures, are not observed in protoplasts. In terms of rapid screening using a plate-based spectrophotometer, clumping of cells, or cells in multiple planes will lead to difficulty in acquiring consistent measurements. Since protoplasts are also denser than their culture media, they sediment to the bottom of wells, forming a monolayer, which is conducive for plate-based spectrophotometry. Finally, while plant cell suspension cultures are primarily derived from callus¹⁷, protoplasts can be harvested from a number of plant tissues, leading to the ability to identify tissue-specific expression. For example, the ability to analyze root- or leaf-specific expression of a gene can be very important to phenotype prediction. For these reasons, the protocols developed in this work were validated using protoplasts isolated from the widely-used tobacco (Nicotiana tabacum L.) 'Bright Yellow' 2 (BY-2) suspension culture.

The BY-2 suspension culture has been described as the "HeLa" cell of higher plants, owing to its ubiquitous use in molecular analysis of plant cells¹⁸. Recently, BY-2 cells have been used to study the effects of plant stressors^19-22, intracellular protein localization^23,24, and basic cell biology^25-27 demonstrating the broad utility of these cultures in plant biology. An additional advantage of BY-2 cultures is the ability to synchronize the cultures with aphidicolin, which can lead to enhanced reproducibility for gene expression studies²⁸. Furthermore, methods have been developed for the extraction of BY-2 protoplasts using low-cost enzymes^29,30, as enzymes traditionally used for generating protoplasts are cost prohibitive for high-throughput systems. As such, the protocol described below has been validated using the BY-2 suspension culture, but it should be amendable to any plant cell suspension culture. Proof-of-concept experiments are performed using an orange fluorescence protein (OFP) reporter gene (pporRFP) from the hard coral Porites porites³¹ under the control of the CAMV 35S promoter.

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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 foil. Incubate culture at 28-30 °C with shaking for 5 days.
   NOTE: BY-2 callus is maintained on solid media for long term storage, as liquid cultures will rapidly overgrow. Culture volume can be adjusted as necessary, typically a 100 ml culture will be capable of loading thirty-three 6-well plates.
3. Transfer 2 ml of log phase BY-2 culture to 98 ml of fresh BY-2 media and incubate for 5-7 days at 28-30 °C with shaking.
   NOTE: Sub-culturing of established liquid cultures can be carried out regularly using 1:100 dilutions of 5-7 day old cultures, rather than inoculation with callus.
4. Mix the culture thoroughly, as cells will rapidly settle, and transfer 6 ml of the cell culture to a 15 ml conical bottom centrifuge tube and let the cells settle for at least 10 min. Adjust the packed cell volume to 50% of the total volume by removing supernatant.
5. Shake tube by inverting and pipet 500 µl into each well of a 6-well plate for digestion. Use wide-bore pipets to transfer the cells at this stage, as they are dense and will clog standard pipet tips.

2. Protoplast Isolation

1. Turn on all components of the robotic system (Figure 1A) and open the plate mover task scheduling software. The task scheduling program integrates the microplate mover with the other equipment to enable transfer of plates between each piece of equipment.
2. Prior to experimentation, define the technical specifications for labware (e.g., plates, lids) used in the protocol, as well as starting and ending positions for each piece of labware, in the plate mover software, as follows:
   1. Click Setup from the main tool bar in the plate mover software and select the Manage Container Types command.
   2. If the container type is listed in the existing container type library, then select the appropriate container.
   3. If the container type is not listed in the existing container type library then click Add to specify a new container type.
   4. After adding a new container type, insert the dimensions of the new container (height, width, stacking dimensions, and gripper offset) into the appropriate tabs and click Save.
      1. If the correct dimensions are not available from the manufacturer, then accurately determine all dimensions to the nearest millimeter using calipers. Errors in measuring the container dimensions will result in failure of the plate mover.
   5. Repeat steps 2.2.2 through 2.2.4.1 for all container types that the plate mover will encounter. After completion of this step for all labware, it is necessary to define the start and end location for each container (step 2.2.6).
      NOTE: For this protocol the labware includes the 6-well plate, the deep-well plate, and the 96-well fluorescent screening plate.
   6. To define the start and end position of each container, click the Start/End tab in a specific protocol. First, select the start position of each container. Next, check the box for Lidded/Unlidded at both the start and end position. Repeat for all containers.
      NOTE: If the container will be left on another piece of equipment (instead of the plate mover) the end location can be left blank.
3. At this stage manually load all plates into the starting position for the entire workflow. Load the 96-well fluorescent screening plates into Hotel 2, 6-well plates with BY-2 cells into Hotel 3, a 96 deep-well plate that will be used for transformation onto plate heater/chiller nest 2, and a 50 ml conical tube containing the enzyme solution (see Supplemental File, Section 1.2.2) onto the multi-mode dispenser.
   1. If transformation will be carried out in the protocol, pre-load the 96 deep-well plate with 10 µl of plasmid DNA per well (1 µg/µl, A_260/280 > 1.8) containing the OFP reporter construct and incubate on the plate heater/chiller at 4 °C. Each well will be used for a single transformation, thus the number of wells filled will depend on the experimental setup.
4. Load all supplies and plates on the automated liquid handling platform in the designated locations and define the position of each item in the automation control software (Figure 1B).
   1. Load a 96-well plate preloaded with 200 µl of 40% PEG in MMg (0.4 M mannitol, 100 mM MgCl₂, 4 mM MES, pH 5.7) in column 1, 200 µl of propidium iodide (PI, 1 µg/ml) in column 2, and 200 µl of ethanol in column 3 (nest 2), and a box of pipette tips in nest 8.
   2. To define the location of the labware in the automated liquid handling platform's software, select Tools from the menu and click Labware Editor.
   3. Select the type of labware from the pulldown menu or define a new labware type using the New Labware button.
   4. Define the position of each piece of labware selected by selecting the Main Protocol tab and clicking Configure Labware. Ensure that each piece of labware placed on the automated liquid handling platform is defined prior to running the protocol.
   5. To trigger the automated protocols, click the Profile Explorer window and select the name of the protocol to be run (Protoplast Isolation, Cell Count, and PEG-mediated Transformation).
   6. Click Run to initialize all devices that will be used in the protocol.
   7. Finally, in the Work Explorer window, click Add Work Unit to verify all containers/labware in the system and begin the automated protocol.
      NOTE: Full descriptions of the automated protocols are contained in the Supplemental File.

3. Establish Standard Curve for Cell Counts

1. Manually concentrate protoplasts at a concentration of 1 x 10⁶ protoplasts/ml in a volume of 1 ml. Centrifuge protoplasts at 1,000 x g for 10 min, and remove supernatant.
2. Manually add 900 µl of 70% ethanol (maintained at 4 °C) to the protoplast pellet and re-suspend the pellet. Incubate for 10 min on ice to fix cells.
3. Add 100 µl of PI (1 µg/ml) to the protoplasts to label the nuclei and allow detection by the plate reader.
4. Load 80 µl of liquid BY-2 media into each column and row of the 96-well fluorescent screening plate starting at column 2.
5. In column 1 of a 96-well fluorescent screening plate, add 70 µl of protoplasts and 50 µl BY-2 media to each well in the column.
6. Place the plate in nest 6 of the automated liquid handling platform, and run a 2-fold serial dilution.
   1. To conduct a serial dilution on the automated liquid handling platform, click Launch Serial Dilution Wizard and adjust the transfer volume (60 µl), number of mixes and volume (2, 70 µl), initial wells (Column 1, Rows A-F), dilution wells (Columns 2-11, Rows A-F), and aspirate and dispense properties (0.5 mm from well bottom).
   2. After setting the parameters, save and run the protocol.
      NOTE: Since all protoplasts are labeled with PI (due to fixation of cells), the fluorescence is proportionate to the protoplast concentration.
   3. After the serial dilution is completed, remove the plate from nest 9 and insert into the plate reader and run the standard curve protocol in the plate reader software.
      NOTE: A standard curve will then be generated by comparing the fluorescence from PI (excitation 536 nm/emission 620 nm) in each well with the known protoplast concentration.
7. Upon completion of the plate reader protocol, remove the plate and collect any transgenic cells for autoclaving or other de-vitalization procedures as defined in biosafety protocols'.

4. Microscopic Analysis of Transformation

1. Remove plate with transformed protoplasts (the product of Automated Protocol 3, Supplemental File) from Hotel 1 of the robotic system.
2. Turn on inverted microscope, camera, and fluorescent lamp.
3. Select the 10X objective for initial focusing on the protoplasts, turn on the halogen lamp, and close the shutter for the fluorescent lamp.
4. Load plate onto microscopic system and focus on the protoplasts using brightfield.
5. After focusing on protoplasts, turn off the halogen bulb and open the shutter for the fluorescent lamp.
6. Select the CY3/TRITC filter set for visualization of the pporRFP protein expressed by the transgenic protoplasts.
7. Scan each well to determine the number of protoplasts expressing the pporRFP fluorescent marker.
8. Calculate the transformation efficiency as the total number of protoplasts expressing the fluorescent marker the total number of protoplasts.
9. Collect any plates containing transgenic cells in approved biosafety bags for autoclaving or other de-vitalization procedures as defined in biosafety protocols'.

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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...

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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

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Materials

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