Enabling High-throughput Transgene Expression Studies Using Automated Liquid Handling for Etiolated Maize Leaf Protoplasts

Summary

This protocol describes the creation of a randomized transfection layout using an automated liquid handler, a protoplast isolation protocol for etiolated maize leaf, and a 96-well transfection procedure using a liquid handler.

Abstract

The field of plant biotechnology has witnessed remarkable advancements in recent years, revolutionizing the ability to manipulate and engineer plants for various purposes. However, as research in this field increases in diversity and becomes increasingly sophisticated, the need for early, efficient, dependable, and high-throughput transient screening solutions to narrow down strategies proceeding to stable transformation is more apparent. One method that has re-emerged in recent years is the utilization of plant protoplast, for which methods of isolation and transfection are available in numerous species, tissues, and developmental stages. This work describes a simple automated protocol for the randomized preparation of plasmid within a 96-well plate, a method for the isolation of etiolated maize leaf protoplast, and an automated transfection procedure. The adoption of automated solutions in plant biotechnology, exemplified by these novel liquid handling protocols for plant protoplast transfection, represents a significant advancement over manual methods. By leveraging automation, researchers can easily overcome the limitations of traditional methods, enhance efficiency, and accelerate scientific progress.

Introduction

Plant protoplast transfection, the introduction of foreign genetic material into plant cells devoid of cell walls, is a pivotal technique and, in the last half a century, encompasses numerous species in support of plant biotechnology research. However, the utilization of these methods can be painful and limited in scope, even with millions of protoplasts produced per isolation. Traditional methods of plant protoplast transfection are often laborious, time-consuming, prone to variability, and technically demanding, leading to niche systems with low reproducibility¹. However, the potential introduced by automated solutions in recent years illuminates the possibility of breathing new life into this 60-year young technique^2,3. With the potential of automating crucial but repetitive steps such as material preparation, poly-ethylene glycol (PEG) incubation, and subsequent transfection reagent dispensing, researchers can significantly reduce the physical handling requirements and other potential sources of human error⁴. Furthermore, the precise control and uniformity offered by automated liquid handling systems ensure consistent and reproducible transfection results.

While protoplast isolation is a meticulous process that involves chopping, digestion, incubation, filtration, and centrifugation, the transfection portion of these protocols is tailor-made for automated liquid handlers. The procedure for most protoplast transfection protocols is PEG-mediated, and mixing the isolated protoplast in the presence of PEG and purified plasmid DNA for a specified duration at a precise concentration (dependent on the species and tissue) allows these cells to take in the plasmid DNA⁵. This transfection is followed by a series of wash steps, culminating in an overnight incubation⁶. After the incubation period, if everything was designed and delivered properly, the experiment results in the expression of the component of interest and/or the potential of evaluating different regulatory components⁷. All the aspiration, dispensing, and agitation/mixing steps associated with this procedure would normally be handled by a manual pipette. Executing such a protocol by hand, one individual reaction at a time, is laborious and introduces unnecessary variation between samples while also limiting the capacity that can be evaluated at any given time. Automated protocols for the manipulation of mammalian or insect cells and chemical synthesis in the pharmaceutical industry have been in practice for several years^4,8,9. Protoplast utilization and protocols involving the automated liquid handling of plant materials are on the rise^10,11,12,13.

The adoption of automated liquid handling protocols for plant protoplast transfection holds great promise for research applications. Researchers can explore larger genetic libraries, screen for specific gene functions at an accelerated pace, and investigate complex genetic interactions related to plant stress more comprehensively¹⁴. The scalability of automated approaches utilizing 96-well pods combined with fluorescence screening enables high-throughput experimentation and allows scientists to rapidly generate data and insights that can fuel advancements in plant biotechnology¹¹. However, with this increase in throughput, leading to the generation of hundreds, if not thousands of data points, there must be additional quality control that accounts for any sources of error that may confound results¹⁵. One element that has been identified as a contributing factor across numerous scientific disciplines is the edge effect. Some mitigating strategies will suggest the best plate to use or fill inter-well space or the outermost wells with water to combat this phenomenon^16,17. However, these strategies add time, and if a specific disposable is unavailable, the only option is to settle for less or postpone. Alternatively, looking at strategies that account for this effect via a blocking scheme makes no sacrifice of throughput or delay to execution.

This etiolated maize leaf protoplast protocol and its two automated methods illustrated in Figure 1 seek to address the variability inherent to protoplast experiments by automating multiple portions of the canonical protoplast method, the allocation of plasmid material to the vessel used for transfection, and the transfection itself. These methods are demonstrated for the etiolated maize leaf protoplast platform as it is a well-characterized, simple, and efficient protoplast platform. All the steps detailed within are immediately accessible to protoplast transfection protocols, which utilize similar or the same buffer solutions. However, special attention to the unique characteristics of protoplast source tissue and species should be considered before the adoption of these techniques. These improvements through automation simplify the preparation of material for individual experiments and significantly improve the throughput, from one-by-one sequential transfection to 96 transfections handled simultaneously. This work will also show justification for the utilization of randomized incomplete blocks to account for plate positional bias.

Protocol

1. Transfection plate creation

1. Deck layout creation: To create a DNA plate randomization method, open the liquid handler software. Click the New Method button from the menu bar to make a new method. Add Instrument Setup line between the Start and Finish lines by pressing the Instrument Setup button on the left panel.
   NOTE: Relevant screenshots that follow along with this automated protocol can be found in Supplementary Figure 1, Supplementary Figure 2, and Supplementary Figure 3.
2. Click Instrument Setup line, find the correct labware from Labware category menu, and drag them onto the deck layout as shown in Supplementary Figure 1.
   NOTE: If the labware is not previously defined, then it will need to be programmed into the liquid handler according to the manufacturer's specification, but such instructions are outside the scope of this protocol.
3. Transfer from file setup: Press Transfer From File button at the step palettes and place the Transfer From File line below the Instrument setup line. Make sure that the tip selection and replacement are as shown in Supplementary Figure 3.
4. Check File has a header row and make sure the column information is correct.
5. Press Source area to activate it and click the Source Plate from the deck layout, define the target plate, and input the amount of DNA to be pipetted.
6. Press Customize button to define the pipetting technique in detail, such as tip touches on the wall.
7. Specify the amount of plasmid DNA to be transferred. This protocol uses 20 µL of 1 µg/µL plasmid DNA per transfection (well).
   NOTE: the amount of plasmid DNA that is transferred to the transfection plate prior to the protoplast transfection will depend on the protoplast platform being used.
8. Create a Transfer From File .csv document.
   1. This document is composed of two columns. Prepare the first column, i.e., the From column, which indicates the location of the stock plasmid DNA within the tube rack, i.e., for sample 1; this would be well A01. As this transfer happens three times (three reps), three rows should denote A01 in the From column.
   2. Prepare the second column, i.e., To column, used to specify the plasmid construct destination well of the 96-well plate. For example, sample 1 (A01) could be B02, D04 and H07. Save this as a .csv file to be compatible with the liquid handler software.
9. Before running the protocol, check that the deck positions are loaded, labware is correctly defined, the randomization document includes well locations for all the sample DNA in the tube rack, and that there is enough plasmid sample in the tubes to execute the protocol.
10. Expand File Properties menu of the Transfer From File step, select the .csv file created and run the protocol.
11. Cover transfection plates with an aluminum foil seal when the protocol has been completed successfully. Store transfection plates at -20 °C until ready to use. The isolated etiolated maize leaf protoplast will be added to this transfection plate in step 3.3.

2. Etiolated maize leaf protoplast isolation

1. To begin the isolation of etiolated maize leaf protoplast, obtain a protoplast-compatible genetic background such as NP222 which was used here¹⁸. Prepare only 3 buffers, namely MMg, W5, and WI buffers, listed in Table 1 before the start of the experiment and keep at 4 °C. Prepare digestion solution and PEG on the day of the experiment for best results.
2. Surface sterilize 25 seeds by first submerging them in a 25% commercial bleach solution and shaking them on a rotary platform shaker for approximately 15 to 20 min at room temperature.
3. After agitation, rinse the seeds thoroughly using sterile water (DI). Repeat the rinse step 5x. Imbibe the seeds in sterile water overnight at room temperature.
4. Sow the seed on moist, autoclaved soil the following day. Add dry clay pellets to wick away excess moisture from the soil to prevent fungal contamination.
5. Grow the maize seedlings in a 16 h light growth chamber at 28 °C (Relative Humidity (RH) of 30%) for approximately 3 days until the coleoptile is 1-2 cm above the soil and then transfer to a dark chamber with the same temperature and RH for additional 5 - 7 days.
6. Harvest the first true leaf tissue by cutting it just above the first collar.
7. Surface sterilize the leaf tissue briefly in 25% commercial bleach and 250 µL of 20% Tween 20 solution for 1 min. Rinse off the leaf material thoroughly 5x with DI water.
8. Pat dry (gently) the maize leaf tissue with lint-free tissues, and using a sharp blade, remove ~1.5 cm from both the tip and base of each leaf blade.
9. Cut the remaining leaf tissue into narrow (0.5 mm - 1 mm) transverse strips and place in a 100 x 25 mm Petri dish.
10. Filter-sterilize 25 mL of the digestion solution through a 0.22 µm syringe filter directly into the Petri dish containing the sliced tissue.
11. Vacuum infiltrate the leaf tissue with digestion solution by applying vacuum for 30 min at room temperature in a vacuum desiccator at approximately -75 mbar pressure.
    NOTE: House vacuum pressure for numerous academic and private labs should be sufficient for this step.
12. Place onto a rotary platform shaker and shake at 25 °C in the dark at 60 RPM for 2.5 to 3 h. When 10 min are left to the end of the digestion period, increase the speed to 90 RPM.
13. Pour the digestion solution and any undigested plant material through a funnel on top of a 40 µm sieve filter into a 50 mL tube.
14. Centrifuge the solution inside of the 50 mL tube at 150 x g for 4 min to pellet the protoplast. Remove the supernatant and resuspend the pellet in 10 - 15 mL of MMg Buffer solution.
15. Repeat the centrifugation and remove the supernatant. Resuspend in 5 - 10 mL fresh MMg buffer.
16. Using a hemocytometer, carefully measure the density of the isolated protoplast. Resuspend to an approximate density of 5 x 10⁵ protoplasts per mL.
17. Allow the isolate to rest on ice for ~30 min prior to transfection. During this time, prepare the PEG solution. Completely dissolve PEG into a solution using a 37 °C water bath and leave it there until transfection.

3. Automated protoplast transfection

NOTE: Steps 3.6 through 3.15 are managed completely by the automated liquid handler, except for the two user pauses at steps 3.10 and 3.13, which require manual centrifugation. Relevant screenshots that follow along with this automated protocol can be found in the supplement, Supplementary Figure 1, Supplementary Figure 3, Supplementary Figure 4, and Supplementary Figure 5.

1. Prepare the deck layout of the liquid handler for transfection according to the protoplast transfection method described in the following steps. Assemble the following materials: Destination plate (in this case, would be a 96-well black with a clear bottom microplate for fluorescence measurement), one box of 25 - 250 µL wide-bore automation tips for the PEG aspiration step, five boxes of 25 - 250 µL pipette automation tips for the remaining liquid handling steps, three plastic troughs as reagent reservoirs, one empty 96-well plate for waste collection, remaining transfection buffer wash solutions, W5 and WI.
2. Immediately before the transfection procedure starts, filter sterilize the PEG solution through a 0.22 µm sterile disposable vacuum filter unit into a fresh 50 mL tube.
3. From the protoplast resuspended in MMg buffer, add 100 µL (approximately 50,000 protoplast) to each well of the pre-prepared, defrosted transfection plate. To do this, pour the protoplast-containing buffer solution into a sterile 10 mL trough and use a multichannel pipette. Continue gentle agitation of the trough to prevent the protoplast from settling out of the solution during this manual transfer step.
4. Pour the PEG solution into the appropriate trough and set down the transfection plate, now containing protoplast and plasmid DNA containing transfection plate, prepared using step 1, in the specified location according to the deck layout.
5. To create a protoplast transfection method, open the liquid handler software and perform the steps described below.
   1. Moving labware among decks: Replace the empty tip box on the tip loading deck with new one by using the Move Labware command. Select the From and To decks from the Configuration view.
   2. For volumes greater than 200 µL: Do not replace tips between the transfers. Load tips for the first transfer step and unload after the last transfer step, which requires the Loading/Unloading options be correctly specified for each transfer step as in Supplementary Figure 3.
6. Deck layout creation: Like the transfection plate creation method, to create a DNA automated transfection method, open the liquid handler software. Click New Method button from the menu bar to make a new method. Add Instrument Setup line between Start and Finish lines by pressing the Instrument Setup button on the left panel. Create a deck layout according to the deck layout shown in Supplementary Figure 1.
7. Cell/DNA mixing: Add a step to mix protoplast and DNA prior to PEG addition using the Device Action button and setting up the device (shaking automated labware positioner or ALP), shaking speed (1500 rpm), and time to shake (10 s).
8. PEG addition and mixing: To initiate the transfection, add a Transfer step to add 110 µL of PEG from PEG reservoir using a 96-pod pipet. Ensure that the correct source and destination positions are programmed according to the specified deck layout. Program the transfer step to aspirate PEG 1 mm from the bottom of the reservoir and deposit the PEG 3 mm from the base of the 96-well plate containing the protoplast/DNA mixture. Add another shaking step after PEG addition by copying and pasting the previously programmed steps.
9. PEG incubation: Add a pause step by selecting the Pause button, select the shaker, and input the pre-determined incubation time, which starts from PEG addition.
   NOTE: The timer for the pause will continue unless there are any light curtain interruptions or disruptions that would result in an error message. Make sure that if manipulation of the deck is necessary, these messages are cleared before walking away.
10. W5 buffer addition/mixing: Add three lines of Transfer of 200 µL to transfer a total of 600 µL of W5 buffer to the transfection plate to quench the PEG incubation reaction. Follow W5 buffer addition by shaking and a user pause to allow centrifugation of the transfection plate.
11. User pause 1: Centrifuge the protoplast transfection plate at 100 x g for 4 min. Return the transfection plate, now with pelleted protoplast, back to the appropriate deck position. Add user pause by clicking the Pause button, except now with the Pause the whole system and display the message option selected.
    NOTE: The pause message pop-up needs to be cleared before the liquid handler will continue with the rest of the protocol.
12. Removal of supernatant: Add two lines of Transfer to remove 400 µL of supernatant and transfer to the waste plate. To avoid cell aspiration during supernatant removal, set the distance from the bottom as 6 mm.
13. WI addition/mixing Add 3 lines of Transfer to transfer 580 µL of WI buffer to the transfection plate. Add another shaking step after WI addition by copying and pasting the previously programmed steps. Proceed to the next user pause.
14. User pause 2: Centrifuge the protoplast transfection plate at 100 x g for 4 min. Return the transfection plate, now with pelleted protoplast, back to the appropriate deck position.
15. Removal of supernatant: Add four lines of Transfer to remove 680 µL of supernatant and transfer to the waste plate. To avoid cell aspiration during supernatant removal, set the distance from the bottom as 6 mm.
16. Transferring protoplasts to microplate: The final transfer of this protocol is composed of two 150 µL transfer steps. Prior to each individual step, repeatedly aspirate protoplasts at 50 µL/s at 2 mm from the bottom of the transfection plate and dispense at 3 mm. Then, utilizing the same pipette tips, transfer to the destination microplate.
    NOTE: Aspirating and dispensing buffer volume above the pellet prior to transfer to the microplate will disturb any remaining protoplasts pelleted in the bottom of the transfection plate to ensure there is no sample loss. Since randomization was done during the transfection plate creation, this concludes the automated protocol.
17. Incubate the microplate at room temperature in the dark for 24 to 48 h before the first fluorescence measurement.

Results

To obtain observational data supporting that edge effects may be affecting the response measurements; a pilot study was conducted to confirm those suspicions. For this study, the above methods were applied to three replicate 96-well plates with only a single treatment level; all protoplasts were transfected using pSYN11250¹⁹, a plasmid that constitutively expresses ZsGreen, with the goal of showing there exist systematic differences in response level for units on the edge of the plate as compared ...

Discussion

This manuscript describes a protocol for automating transfection plate creation and etiolated maize leaf protoplast isolation with an automated transfection. For the successful completion of the transfection plate creation portion of the protocol, it requires an automated liquid handling robot that is fitted with an 8-channel pod. For the transfection protocol, a 96-well pod is recommended for full and uniform 96-well plate transfection. The transfection method can be completed using an 8-channel pod, but special co...

Materials

Name	Company	Catalog Number	Comments
(2)β-mercaptoethanol	Sigma	M6250
2-(N-Morpholino)ethanesulfonic acid (MES) monohydrate	Sigma	69892
50mL centrifuge tubes with flat cap sterile	Fisher	22-010-064
96 Well Optical Btm Plt PolymerBase Black w/Lid Cell Culture Sterile PS .4mL Well	Fisher	12-566-70
Axygen Biomek FX/NX Robotic Tips, non-sterile, Wide Bore	Fisher	14-222-096
Axygen Robotic Tips 30uL filter, sterile, racked	Fisher	14-222-103
Bel-Art SP Scienceware Lab Companion Round Style Vacuum Desiccators	Fisher	08-648-10
Bemis 2 IN. X 250 Ft. Roll Laboratory Parafilm	Fisher	13-374-16
Biomek FXP	Beckman Coulter	902508
Calcium chloride dihydrate	Sigma	C5080
Chemglass Life Sciences Disposable Hemocytometer	Fisher	50-131-1352
Clorox Germicidal Bleach, Concentrated	Fisher	NC1871274
Corning Microplate Aluminum Sealing Tape	Fisher	07-200-684
Corning 96-Well assay Blocks, 2mL, 96 well standard	Fisher	07-200-701
DL-Dithiothreitol (DTT)	Sigma	10197777001
D-Mannitol	Sigma	M9546
Fisherbrand 60mL Plastic Syringe	Fisher	14-955-461
Fisherbrand Sterile Cell Strainer 40um	Fisher	22-363-547
Fisherbrand Petri Dishes with Clear Lid, Stackable, 100 mm x 25 mm, Case of 325	Fisher	FB0875711
Magnesium chloride hexahydrate	Sigma	M2670
Millex Syringe-driven Filter Unit Sterile 33mm PES .22um	Fisher	SLGPR33RS
Millex Syringe-driven Filter Unit Sterile 33mm PVDF.45um	Fisher	SLHAR33SS
MillliporeSigma Steriflip Sterile Disposable Vacuum Filter Units 50mL PES	Fisher	SCGP00525
Poly(ethylene glycol) 4000	Sigma	81240
Redi-Earth Plug & Seedling Mix	Wyatt Quarles	GP92747
Regular Duty Single Edge Razor Blades steel back .009RD	Fisher	12-640
Research Products International Corp Cellulase RS	Fisher	50-213-232
Research Products International Corp Macerozyme R-10	Fisher	50-213-444
Sodium chloride	Sigma	S7653
Tray Insert - 36 Cell - 6x6 Nested	Hummert	11635000
Tween-20	Sigma	P1379
VACUUBRAND ME1 Vacuum Pump, 100-120V, 50/60 Hz, US plug	VWR	97058-164