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

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.

Representative 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 to the second row, and central regions of the plate. The 36 wells on the outer edge of the plate are defined as block 1, the 28 wells in the second row from the edge as block 2, and the central 32 wells as block 3 - see Figure 2A bottom right panel. This arrangement can accommodate 28 treatment levels as a randomized complete block design (RCBD) or 32 treatment levels as a randomized incomplete block design (RIBD). In Figure 2A, for each replicate (rep), the raw data points for each well were normalized by the mean from that respective plate. In all three replicates of the experiment, the responses on the edge of the plate are, on average, 1-1.5x larger than the minimum. Figure 2B depicts block means as a percentage of the overall plate mean for each replicate. Table 2 shows one-way ANOVA tables from fitting a linear model with block as a fixed effect. For reasons related to the restricted randomization involved in block designs, inference based on hypothesis testing for the blocking factor is regarded as approximate at best and invalid at worst. However, fitting a model with a fixed blocking factor is useful to assess whether a blocking scheme is beneficial. Mn_Sq (block) represents the average squared deviation of the block means about the overall mean. Mn_Sq (Error) represents the average squared deviation of the individual observations about their respective group means, in this case, the overall mean since there is no treatment factor in the model. When Mn Sq (block) is larger than Mn Sq (Error), i.e., the F-ratio is greater than one, the size of the mean square error has been effectively reduced relative to a completely randomized design (CRD), thereby increasing statistical power to compare the treatments of interest and decreasing the width of confidence intervals estimating treatment contrasts. Figure 2B shows F-ratios that far exceed one, and the measured response tends to decrease as it moves toward the center on all three replicate plates. In all three replicates, 95% confidence intervals are observed at level 0.05 for at least one block that does not cover the observed plate mean. Therefore, it can be concluded that the blocking scheme substantially increases the precision of these estimates. Beyond having less precision, failure to account for spatial nuisance variability may lead to spurious results due to the possibility of confounding treatment effects with the edge effect.

Though there is a long history of etiolated maize protoplast isolation and transfection protocols dating back to the early 1990s, this protocol introduces some additional modifications to the traditional procedure to better facilitate a reproducible, bulk protoplast isolation for the purposes of high-throughput protoplast transfection²⁰. The seed and leaf tissue surface sterilization steps before digestion reduce fungal contamination without requiring aseptic media. Utilizing soil will also help to keep costs down relative to using specialty growth media or buying sterile single-use disposable containers. The protocol at numerous stages can be scaled to accommodate various levels of throughput. Approximately 2 g of first leaf tissue results in between 5 x 10⁶ - 10 x 10⁶ protoplasts. Since 5 x 10⁶ protoplasts are required per 96-well plate transfection, the isolation protocol, as it is written, can accommodate 2 runs of the transfection protocol. This protocol also utilizes MMg as the wash solution instead of the canonical W5 solution. This was derived originally from publications for Arabidopsis root protoplast as a means of reducing the number of buffer solutions used for any given step of the protoplast transfection procedure²¹. However, it was also employed for etiolated maize leaf protoplast within 2022²². Further improvement to the isolation and transfection of any protoplast protocol would be the simplification and reduction of buffer solutions. As it currently stands, this protocol switches between the canonical digestion buffer, W5 buffer, MMg buffer, and WI buffer. Simplifying the solutions would be very beneficial to improving the reproducibility of protoplast experiments and reducing the person-to-person or batch-to-batch variability between experiments. Noticeably, the last novelty of the protocol is the lack of complete removal for buffer solutions following PEG transfection. The reason that the automation is possible is because the protoplast, etiolated maize shown here and others we have tested, is that they seem to tolerate the dilution as a means of quenching the reaction rather than complete removal of the different buffer solutions¹¹. The reporter production, as indicated by our GFP transfection control used here, indicates that protoplast can tolerate up to 5% PEG with no negative impact on fluorescence intensity (Supplementary Figure 6). That value is more than double what the anticipated PEG concentration would be upon completion of the protocol described here.

Figure 1: Illustrative schematic of the protoplast isolation and transfection procedure. Steps, where automation has been introduced are indicated by the dashed red lines and the accompanying blue boxes. Numbers surrounded by a red circle correspond to the three methods described. Created with BioRender.com. Please click here to view a larger version of this figure.

Figure 2: Edge effect and the impact of mitigating replicate layouts. (A) Topographical maps showing fold change of fluorescence intensity for 96-well plates transfected with pSYN11250 relative to the plate mean for 3 independent replicate (Rep) experiments. The average of those experiments is also shown with the blocking scheme, indicated by the different color dotted lines laid over top. (B) Strip plots showing the block means as a percentage of the overall plate mean for replicate plates 1, 2, and 3 from left to right. The semi-transparent circles represent the raw data points after dividing by the plate mean. The error bars are 95% confidence intervals at level 0.05, with the central dash denoting the mean. The brown, golden, and gray colors indicate the edge, second row, and inner portion of the plate, respectively. The dashed red line at 100% represents the overall plate mean. Please click here to view a larger version of this figure.

Buffer	Reagent
Digestion	1.5% Cellulose Rs
	0.3% Macroenzyme
	0.6 M Mannitol
	10 mM MES (ph 5.7)
	1 mM CaCl2
	0.1% (w/v) BSA
	0.5 nM β-Mercaptoethanol or 0.5 mM DTT
Mmg	0.6 M Mannitol
	15 mM MgCl2
	4 mM MES, pH 5.7
W5	154 mM NaCl
	125 mM  CaCl2
	5 mM KCl
	2 mM MES, pH 5.7
WI	0.6 M Mannitol
	4 mM MES, pH 5.7
	4 mM KCl
PEG	30% PEG (w/v)
	0.6 M Mannitol
	100 mM CaCl2

Table 1: Buffer solutions for the isolation and transfection of etiolated maize protoplast.

Rep	Source	Df	Sum Sq	Mn Sq	F Value	Pr (>F)
Replicate 1	Block	2	0.26	0.13	10.64	<0.001
	Residual	93	1.135	0.012
Replicate 2	Block	2	0.507	0.25	21.41	<0.001
	Residual	93	1.102	0.012
Replicate 3	Block	2	0.179	0.09	4.48	0.014
	Residual	93	1.861	0.02

Table 2: One way ANOVA table for the three replicates of the 96-well pSYN11250 transfection experiment. For each replicate plate: the Source column denotes the source of variation, Df denotes degrees of freedom, Sum Sq denotes sum of squares, Mn Sq denotes mean squares (Sum Sq/Df), F value denotes the ratio of Mn_Sq (block) to Mn_Sq (error), and Pr(>F) denotes the p-value of the global F-test.

Supplementary Figure 1: Screenshots of the software dashboard. Selection categories that pertain to making a new Method as well as the deck layouts for randomization and transfection protocols. Please click here to download this File.

Supplementary Figure 2: Key steps in the setup for the transfection plate creation protocol. Screenshots taken during the creation of the transfection plate creation protocol. The number and title for each panel correspond to steps in the protocol. Please click here to download this File.

Supplementary Figure 3: Screenshots for labware manipulation and tip loading for the creation of the transfection protocol. These represent steps which occur numerous times throughout the protocol so to avoid repeated mention representative steps are shown here. Please click here to download this File.

Supplementary Figure 4: Cell-DNA mixing through User Pause 1. Screenshots taken during the creation of the transfection protocol. The number and title for each panel correspond to steps within the body of the protocol. Please click here to download this File.

Supplementary Figure 5: Supernatant removal following User Pause 1 through transfer of samples to microplate. Screenshots taken during the creation of the transfection protocol. The number and title for each panel correspond to steps within the body of the protocol. Please click here to download this File.

Supplementary Figure 6: ZsGreen transfected etiolated maize leaf protoplast treated with different concentrations of PEG in WI buffer time course. Bar chart showing the relative fluorescence intensity of protoplast following PEG treatment at 24, 48, and 72 h with measurement by a microplate reader. Error bar = SD, n = 4. Please click here to download this File.

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 consideration needs to be given to the amount of time that aspiration and dispensing steps take, as well as the time that can be attributed to changing tips between columns. Differences in the duration of PEG incubation are well-documented to have a significant impact on transfection efficiency and expression, and therefore, special attention to these contributing factors must be considered to prevent disparity in sample handling¹³. Before starting the liquid handler protocol, it is important to consider the protocol's steps and whether certain activities can be completed simultaneously. This protocol utilizes a device that loads tips on the 96-well pod from 1 position on the deck. The liquid handler for this protocol includes a function where the robot swaps tip boxes during the incubation periods or during the user pause steps to avoid adding additional time to the protocol. When making the decision to buy a liquid handler, think about functionality and potential time-saving conveniences.

While this protocol was developed to utilize Beckman Coulter NXp and Biomek FX devices, this protocol can be adapted to any comparable liquid handling device. All liquid handlers have some means of denoting how to transfer volumes from one location to another and how much. For this protocol, these devices used a Transfer from File line command. If the labware is correctly defined, a user can transfer from or to any vessel. In exceptional circumstances, such as if tube racks or adapters are not commercially available, 3D printing such connectors can be used. For typical protoplast transfection protocols, 20 µg DNA at a concentration of 1 µg/µL transfection is a standard volume of material for numerous protoplast platforms¹³_. During the transfection plate creation, as an added precaution, use 0.2 - 20 µL filter tips to minimize any contamination risk due to aerosolized plasmid during transfer. Removing the human component of preparation reduces variation over time and improves the reproducibility of these high-throughput experiments. Also, plates can be prepared well ahead of time. This preparation will alleviate the stress of the transfection scientist on the day of the experiment. However, it is important to avoid storing these plates of plasmid DNA at 4 °C for extended periods of time as samples may evaporate. Samples should be stored in a -20 °C freezer and can then be defrosted in the 4 °C refrigerator prior to transfection. Once this transfection plate creation protocol is programmed, it should be easily repeatable by supplying a new Transfer From File .csv and occupying the same deck locations for the samples, labware, and reagents.

For the automated transfection procedure (step 3), a surplus of PEG solution is prepared to ensure equal aspiration of the viscous liquid from the trough, typically an excess of 40 mL to account for the dead volume. Using exactly the required amount for the transfection can result in air being pulled into the pipettes and uneven volumes of PEG being aspirated across the 96-channel head. While this solution can also be prepared ahead of time, it is recommended to be prepared on the day of transfection. Sterilization of the PEG solution can be done using a syringe filter, but owing to viscosity it can be difficult. Using a vacuum filter unit is faster and can alleviate any frustration or pain associated with this activity. Like the PEG solution, an excess of the other transfection buffer solutions (W5, WI) is also provided to ensure equal pipetting across the 96-channel head. Buffer solutions (W5 and WI) can be prepared ahead of time in bulk to use for future liquid handler runs. While the reagents are not costly, there is a good amount of sacrifice of buffer solution. With the increasing number of runs per day, it may be better to use alternatives to the reservoir that would reduce the excess discarded at the end of the run. During the run of the liquid handler protocol, W5 and WI can be added to their respective troughs prior to protocol start, during the 15 min incubation, or during the user pause steps in the protocol. Be cautious when adding buffers during the incubation period due to safety features like a light curtain. Be sure to clear any warning messages from the software to prevent any unintended disruption of the protocol.

This protocol reflects a reliable, repeatable, and widely applicable improvement on previously documented automated protocols for the handling of protoplast^12,13. This method is adaptable to the seemingly endless repertoire of protoplast protocols as many depend on the same series of buffer manipulation steps. Mitigating for the edge effect through the RICBD during the automated transfection plate creation simultaneously reduces the amount of effort and variability while applying a statistically significant correction of the edge effect. A 96-well pod transfection of protoplast eliminates the possibility of any variation associated with the time of PEG incubation, conducting the reaction within all 96 wells simultaneously. While the protocol was intended to accomplish complete automation of the transfection steps, the user pauses will require physical intervention by the transfection scientist. In recent years, there have been many advances in imbalance-tolerant, automation-compatible centrifuges. With this equipment, it would be possible to automate the entire method without user involvement. Alternatively, other protocols have avoided centrifugation by allowing the protoplast to settle to the bottom of the well following transfection^12,13. However, this will add additional time to the transfection procedure and an excess of time incubating in the W5 buffer before overnight incubation in WI.

At numerous places in the transfection method, it describes the handling of volumes exceeding 200 µL where the liquid handling must be done using multiple transfers. Depending on the age and model of the liquid handler, this step could and should be done as one single volume. For the older liquid handlers, such as the model described here, the most that can be aspirated using a 96-well head is 200 µL. An obvious improvement to the method in both efficiency and accuracy would be to reduce in the number of transfers to minimize any potential risk of spilling, dripping or contamination. Other considerations for the improvement of liquid handling protocols are outlined in reviews of these technologies and their utilization within the field of biotechnology²³.

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