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 illumin....

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 corre....

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

List of materials used in this article

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