The overall goal of this protocol is to develop 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. This method can help answer key questions in the plant biotechnology field. The main advantage of this technique is that it enables high-throughput automated protoplast production and transformation for rapid parallel screening of promoter function in gene expression.
This procedure will be demonstrated using the widely-used tobacco bright yellow two, or BY-2, suspension culture. Start by growing a BY-2 culture in an Erlenmeyer flask, as described in the protocol text. On the day of protoplast isolation, mix the culture thoroughly as cells will rapidly settle, and transfer six milliliters of the culture to a 15 milliliter conical-bottom centrifuge tube.
Let the cells settle for at least 10 minutes. Adjust the packed cell volume to 50%of the total volume by removing the appropriate volume of supernatant. Shake the tube by inverting it and then use wide bore pipettes to pipette 500 microliters of cell suspension into each well of a six-well plate for digestion.
The first step in this procedure is to turn on all the components of the robotics system, which are the microplate mover, the liquid handler, the multi-mode plate reader, the multi-mode dispenser, the plate heater or chiller, and the computers. Next, open the plate mover task scheduling software that integrates the microplate mover with the other equipment to enable the transfer of plates between each piece of equipment. In the plate mover software, define the technical specifications for the labware used in the protocol.
Click Setup from the main toolbar and select the Manage Container Types command. Select the appropriate Container Type from the list of existing Container Types. Define the technical specifications for all the labware that the plate mover will encounter.
The next step is to define the Start and End positions for each container. Click the Start/End tab in a specific protocol. Select the Start position of each container and check the box for Lidded, Unlidded at both the Start and End positions.
After defining the Start and End positions for each piece of labware, manually load all the plates into the starting position for the entire workflow. Load the 96-well fluorescent screening plates into hotel two, the six-well plates with BY-2 cells into hotel three, a 96-deep well plate that will be used for transformation onto nest two of the plate heater or chiller, and a 50 milliliter conical tube containing the enzyme solution onto the multi-mode dispenser. To carry out transformation in the protocol, preload each well of the 96-deep well plate with 10 microliters of plasma DNA containing the orange fluorescent protein reporter construct and incubate on the plate heater or chiller at four degrees Celsius.
Load all the supplies and plates on the automated liquid handling platform in the designated locations. Load a 96-well plate preloaded with 200 microliters of a 40%polyethylene glycol solution per well in column one. Load a box of pipette tips in nest eight.
To define the location of each item in the automated liquid handling platforms software, select Tools from the menu and click Labware Editor. Select the type of labware from the pull-down menu. Select the Main Protocol tab and click Configure Labware to define the position of each piece of labware.
Click Run to initialize all devices that will be used in the protocol. Finally, in the Work Explorer window, click Add Work Unit to verify all labware in the system and begin the automated protocol. The most sensitive part of the procedure is the protoplast transformation.
Since the transformation is performed by the robotics system, it is critical that the automated protocols are set up correctly. For the purposes of this video, only selected steps in the automated protocols will be shown. During the transformation protocol, the automated liquid handling platform aspirates 70 microliters of the protoplast solution from the six-well plate and dispenses it into the 96-deep well plate in nest six.
Then, new pipette tips are used to slowly aspirate 70 microliters of the highly viscous polyethylene glycol solution from the preloaded deep well plate and completely dispense the solution into the applicable wells of the deep well plate. Next, the automated liquid handling platform moves the deep well plate from next six to nest nine where the plate mover picks up the plate and moves it to the plate shaker station and shakes it at 1500 RPM for 30 seconds. After a 20 minute incubation without shaking to allow equilibration of protoplasts, the plate mover moves the 96-deep well plate to the multi-mode dispenser and triggers the wash protocol.
When the automated protoplast isolation and transformation protocol is complete, remove the plate with the transformed protoplasts from hotel one of the robotics system. Turn on the inverted microscope, camera, and fluorescent lamp. Select the 10X objective for initial focusing on the protoplasts.
Turn on the halogen lamp and close the shutter for the fluorescent lamp. Load the plate onto the microscope system and focus on the protoplasts using brightfield. Next, turn off the halogen bulb and open the shutter for the fluorescent lamp.
Select the Cy3/TRITC filter set for visualization of the orange fluorescent protein expressed by the transgenic protoplasts. Scan each well to determine the number of protoplasts expressing the fluorescent marker. Enzyme digestion of protoplasts at the conditions specified in this protocol resulted in 2, 820, 000 protoplasts per six-well plate.
The loss in protoplast concentration after each transfer step in the protocol was evaluated. After the digestion protocol, 11, 500 protoplasts were transferred to each well of the 96-deep well plates. Once the transformation protocol was completed, 1, 230 protoplasts were transferred to the 96-well fluorescent screening plate.
To ensure that multiple pipetting stages did not damage the protoplasts, their viability was assayed by propidium iodide staining. The results showed no significant difference in viability after digestion, after transfer to the 96-well fluorescent screening plate, and after the transformation protocol. The transformation protocol was successful in generating transgenic cells that express orange fluorescent protein although the transformation efficiency was low, only about 2%This is due to the large binary plasmic used and the protocol not having been optimized specifically for BY-2 protoplasts.
Metrics obtained for the time course of the protocol showed that the major investment of time is spent during the Digestion stage. The complete duration of protoplast isolation and transformation was three hours, 50 minutes, and 53 seconds with no external input required from the operator. Once set up properly, this technique can be performed by the robot in less than four hours.
After its development, this technique is paving the way for researchers in the field of plant biotechnology to perform high-throughput crop genomic procedures, such as genome editing and promoter screening.
