1Horticulture Department, University of Wisconsin-Madison, 2Department of Zoology, Oregon State University
Su, S., Clark, K. A., Gibbs, N. M., Bush, S. M., Krysan, P. J. Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping. J. Vis. Exp. (57), e3280, doi:10.3791/3280 (2011).
It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.
1. Prepare Ice-Cap Seedling Plates with Agar Growth Media
Molten growth media (0.5X Murashige and Skoog (MS) basal salt mixture, 2 mM Morpholinoethanesulfonic acid (MES), 0.6% agar (w/v), pH 5.7) is dispensed into autoclaved Seedling Plates using a MicroFill Microplate Dispenser. One liter of media is needed per 18 Seedling Plates.
2. Sow Seeds into Ice-Cap Seedling Plates and Germinate Seedlings
We have previously described a semi-automated method for dispensing Arabidopsis seeds into Seedling Plates using a seed loading device (2). Although this device can be useful for some batches of seed, we have found that the variability in seed size displayed by different batches of Arabidopsis seeds limits the general utility of this device. We are in the process of developing an alternative automated method for seed dispensing, but at present we have found that the most effective strategy for dispensing seeds into the Seedling Plates is to perform this step by hand as described below.
3. Transfer Seedling Plates to Ice-Cap Fountain
After the Seedling Plates have been under light for four days, transfer the Seedling Plates to the Ice-Cap fountain. For Arabidopsis, the seedlings are typically left in the Ice Cap fountain for 10 to 14 days.
4. Harvest Root Tissue
Root tissue is harvested by freezing the water in the Root Plate and then peeling the Seedling Plate away from the Root Plate.
5. Store Seedling Plate at Low Temperature in the Dark
Following tissue harvest, the seedling plates can be stored in the dark at low temperature which will maintain the viability of the seedlings while genotype analysis is performed. The seedlings can be stored at these temperatures for several weeks without affecting viability.
6. DNA Extraction
DNA suitable for PCR reactions can be easily extracted from the root tissue samples. The yield of total DNA recovered from Arabidopsis root tissue samples is ca. 400 ng on average 2.
7. Transfer Seedlings with Desired Genotype to Soil
Once genotype analysis is complete, seedlings with the desired genotype can be transferred from the Seedling Plates to soil.
8. Representative Results:
An example of Arabidopsis seedlings grown using the Ice-Cap system is shown in Figure 4A. These seedlings had been in the Ice-Cap fountain for two weeks when the picture was taken and were ready for tissue harvest. Root tissue from these same seedling is shown in Figure 4B. The total amount of DNA extracted from one sample of Arabidopsis root tissue is typically in the range of 100 ng to 700 ng 2. This yield calculation is based on the use of only one portion of the crude root extract. Because only ca. 10% of the crude root extract is typically used for the DNA extraction process, it is possible to obtain significantly more genomic DNA for downstream analysis by retaining more of the crude extract. The total amount of genomic DNA obtained using the Ice-Cap procedure is sufficient to perform hundreds of PCR reactions 2.
Figure 1: Flow chart of the Ice-Cap process. A) Seedling Plate containing agar growth media on which seedlings germinate. Roots penetrate the agar and grow down towards the bottom of the plate. Holes in the bottoms of the wells of the plate are sealed with adhesive film. B) The Seedling Plate is stacked on top of a Root Plate containing water and a metal ball. Roots exit the holes in the bottoms of the wells of the Seedling Plate and grow into the wells of the Root Plate. C) Wooden skewers are inserted between the Seedling Plate and the Root Plate the day before the stacked Ice Cap plates are placed in a thermoblock in a dry ice/ethanol bath. D) Once the water in the Root Plate has frozen solid, the Seedling Plate is peeled away from the Root Plate, yielding a Root Plate with 96 tissue samples and a Seedling Plate with 96 viable seedlings. The wooden skewers facilitate the separation of the two plates during this step.
Figure 2: Photographs of a Seedling Plate and a Root Plate. A) A Seedling Plate and a Root Plate seen separately. B) A Seedling Plate stacked on top of a Root Plate. The steel balls used for DNA extraction can be seen in the wells of the Root Plate. Elastic bands are used to secure the two plates together.
Figure 3: The Ice-Cap Fountain. The Ice-Cap Fountain is used to maintain a constant water level at the precise height of the tops of the wells of the Root Plates. This continuous watering system ensures that the water in the wells of the root plates does not become depleted due to evaporation or transpiration. A) A home-made rack that supports the cookie sheet on which the stacked Ice-Cap Plates sit. B) A close-up view of one of the 1" nuts that provides a means for precisely adjusting the level of the cookie sheet so that a uniform water depth is achieved across the surface of the fountain. C) This image displays all of the parts needed to construct the home-made rack for an Ice-Cap Fountain. D) The assembled Ice-Cap Fountain. A submersible fountain pump constantly moves water from the lower reservoir to the cookie sheet, which rests on top of the home-made rack. A spring loaded clamp is used to attach the hose to the edge of the cookie sheet.
Figure 4: Arabidopsis seedlings grown using the Ice-Cap process. A) Top view looking down into two wells of a Seedling Plate. Each well contains an Arabidopsis seedling. B) Side view of two wells of a Root Plate. Roots can be seen twisting around the inner surface of the wells. The seedlings in this picture had been in the Ice-Cap Fountain for ca. two weeks and are at the growth stage where tissue harvest would normally be performed.
The Ice-Cap method presented here enables one scientist to collect tissue samples from hundreds, or even thousands, of individual plants in a single day and efficiently extract genomic DNA from those samples for use in genotyping reactions. By giving an individual scientist the ability to genotype thousands of seedlings in a short period of time, the Ice-Cap method has the potential to facilitate a number of genetic experiments that would not otherwise be practical to perform. Several examples are provided below.
The idea behind iTILLING is to produce an ephemeral mutant population, which stands in contrast to the durable mutant populations created for traditional TILLING projects. For iTILLING, seeds from a bulked M2 population are sown into 50 to 100 Ice Cap blocks, and DNA is extracted using the Ice-Cap method. Arabidopsis seedlings are then placed at 4°C while mutation screening is performed using the Ice-Cap DNA preps. Tomato seedlings are stored at 12°C.. Once seedlings carrying desired mutations have been identified, they are recovered from the Ice-Cap plates and transferred to soil. iTILLING is not intended to provide a long-lasting mutant population for an entire research community to screen. Instead, iTILLING provides a strategy by which one scientist can screen a custom mutant population for mutations in a handful of genes quickly, efficiently, and at a relatively low cost.
We have found Ice-Cap to be an effective method for growing and genotyping Arabidopsis, tomato, and rice. We expect that other species of plants should also be amenable to Ice-Cap. One limitation on the variety of species that can be accommodated by Ice-Cap is the size of the seeds. Only plants with seeds small enough to fit into the wells of a 96-well plate will be compatible with Ice-Cap in its present configuration.
No conflicts of interest declared.
This work was funded by a grant from the National Science Foundation (grant number MCB-0447750).
|GenoGrinder||Spex/CertiPrep, Metuchen, NJ||2000 Geno/Grinder||Machine for shaking Root Plates for DNA extraction|
|ABgene Thermo-Sealer||ABgene Limited||AB-0384||Manual heat sealing machine used to Root Plates prior to tissue disruption|
|Centrifuge equipped with swinging microplate carriers||Beckman Coulter Inc.||Allegra 25R||Any centrifuge with swinging microplate carriers would be suitable|
|MicroFill Microplate Dispenser||BioTek||AF1000A||Automated liquid dispenser for filling microplates|
|Bialetti brand 17x11 inch Commercial Large Cookie Sheet||Target Department Store||www.bialetti.com||This specific cookie sheet has the ideal depth for Ice-Cap|
|Custom Made Metal Rack||Hardware Store||See text for full description|
|33 Liter plastic storage box||Target Department Store||Sterilite Brand||For Ice-Cap Fountain.
Plastic storage box large enough to hold the 17x11 inch cookie sheet
|30 gallon per hour Fountain Pump||Sunterra||104506||Small submersible fountain pump available at garden supply stores|
|Hose for fountain pump||Any Supplier||Hose compatible with fountain pump|
|Plastic Clamp||Hardware Store||Clamp to hold hose to cookie sheet|
|Clear plastic lids from Falcon Microtest flat bottom 96-well polystyrene plates||BD Biosciences||351172||Clear plastic lids for covering Ice-Cap Seedling Plates|
|3M Micropore surgical tape||Fisher Scientific||19-027-761|
|3/32-inch diameter stainless steel balls||Hartford Technologies, Rocky Hill, CT||034-006-1K||Used to disrupt root tissue after harvest|
|3 inch Elastic hair bands||Local Grocery Store||Used to secure Root Plate to Seedling Plate|
|4 mm diameter wooden skewers||Local Grocery Store||These skewers are typically used to prepare kabobs|
|Fisher Scientific Nunc brand, 1-mL filter plates without frit||Fisher Scientific||278012||96-well Seedling Plates with holes in bottom.|
|Fisherbrand 96-Well Tall-Chimney PCR Plate||Fisher Scientific||14-230-242||96-well Root Plates|
|Agar - cell culture tested||Sigma-Aldrich||A1296|
|4-Morpholinoethanesulfonic acid (MES)||Sigma-Aldrich||M2933|
|Murashige and Skoog (MS) basal salt mixture||Sigma-Aldrich||M-5524|
|Easy Peel heat sealing foil||ABgene Limited||AB-0745||Heat sealing film for sealing Root Plates prior to tissue disruption|
|Duck Brand Extra Wide Packaging Tape, 3.00" x 54.6 yd||Office Max||Item no. 21242139||Adhesive tape for sealing microplates containing DNA extracts for storage|
|Sealing Roller||Bio-Rad||MSR-0001||Hand held rolling tool for pressing adhesive sealing tape onto microplates|
|96-well metal thermal block||Cole-Parmer||36400-66||Used to freeze the Root Plates for tissue harvest|
|Whatman Filter paper||Whatman, GE Healthcare||1002 090||Used for surface sterilizing seeds|
|Disposable Pasteur Pipette||Fisher Scientific||13-678-6A||5 ¾"disposable Pasteur pipette used for transferring seeds to Seedling Plate|
|Pyrex baking dish (20 cm x 30 cm)||Target Department Store||Used to construct a dry ice/ethanol bath.|