High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

This protocol improve seed surface sterilization, a fundamental step in functional genomics of the model species Arabidopsisthaliana. The main advantages of this technique are ease and high throughput, allowing the processing of hundreds of samples per day. With some simple modifications, this method can be applied to many other model and non-model plant species.

For first-time users, please pay attention to the temperature of the medium and the centrifugation speed as they are both critical for seed survival. To begin, prepare 70%ethanol by adding 95%technical ethanol to distilled water and mixing thoroughly. Next, prepare 5%bleach solution by adding five milliliters of household bleach to 95 milliliters of sterile distilled water.

Then, add a few drops of nonionic detergent to the bleach solution, and mix thoroughly. Next, prepare half-strength Murashige and Skoog medium by adding 2.2 grams of MS medium powder including vitamins and 10 grams of sucrose in 800 milliliters of distilled water. Adjust the pH of the medium to 5.7 using one molar potassium hydroxide, then bring the volume up to one liter using distilled water.

Aliquot 500 milliliters of the medium into a one-liter bottle, and add four grams of agar to prepare a solid medium. After autoclaving, allow the medium to cool down to 50 to 53 degrees Celsius in a water bath. Then pour it into Petri dishes under the laminar flow hood.

To prepare selective medium, add kanamycin to the medium, and pour it into Petri dishes as demonstrated earlier. To set up the aspirator, connect the vacuum pump inlet to one end of a polyethylene tube of a suitable size. Then connect the other end of the tube to the outlet of the two-way lid of the decantation bottle.

Wrap the tubing's junction tightly with a sealing film to ensure airtight connection. Next, connect a second polyethylene tube to the inlet of the screw cap on the decantation bottle. Then connect the other side of the tube to the outlet of an aquarium valve fitted with a thin polyethylene tube.

If necessary, wrap the junction with the sealing film to eliminate air leakage. After labeling two batches of 48 1.5-milliliter microcentrifuge tubes with progressive numbers, add 100 to 200 Arabidopsis seeds to each of the 96 sterile microcentrifuge tubes, roughly one to two millimeters above the bottom of the conical end of the tube. Next, using a 10-milliliter sterile serological pipette, add around one milliliter of 70%ethanol into each tube, and carefully close the lids.

Shake the tubes at an oscillation frequency of eight hertz for three minutes in a shaker. Then remove the adapters from the shaker and transfer them into the basket of a benchtop microcentrifuge for spinning down the seeds using the pulse function. After transferring the tubes to a rack, open all tubes under the laminar flow hood.

Do not touch the part of the lids fitting into the tubes to avoid contamination. Then, under the laminar flow hood, fit a sterile 200-microliter yellow tip onto the aquarium valve inlet of the homemade aspirator, and switch on the pump. Insert the tip just above the level of the seeds to avoid touching the seeds when sucking the liquid.

Next, using a 10-milliliter sterile serological pipette, aliquot one milliliter of 5%bleach solution into each tube. Close the lids before placing the tubes back into the shaker adapters, then shake the tubes as demonstrated previously. After spinning down the seeds using the pulse function of a benchtop centrifuge, fit a new sterile 200-microliter yellow tip onto the aquarium valve, and switch on the pump.

Insert the tip above the level of the seeds to avoid touching the seeds when sucking the bleach solution. Next, using a 10-milliliter sterile serological pipette, aliquot one milliliter of sterilized water into each tube. After fitting a new sterile 200-microliter yellow tip onto the aquarium valve, switch on the pump.

Insert the tip just above the level of the seeds to avoid touching the seeds when sucking the water. Finally, aliquot 500 microliters of sterilized water into each tube, and close all the lids in the laminar flow hood. The seeds are now ready to be sown.

If required, keep the tubes at room temperature for up to a few hours or at four degrees Celsius overnight. Using a one-milliliter pipette, transfer the seeds with 300 to 400 microliters of sterile water into a Petri dish. After transferring 10 tubes, pour 1.5 to two milliliters of melted half-strength Murashige and Skoog medium without antibiotics into each plate.

Quickly swirl the plate to distribute the seeds, and tape the plates on opposite sides. Wrap the plates in plastic or aluminum foil, and place them in a refrigerator for three days in the dark to obtain uniform germination. Germination analyses performed at days two, three, four, and seven indicated no significant differences among 10 to 40 minutes of sterilization time with 70%ethanol.

However, when the sterilization time exceeded 40 minutes, the germination rates declined. Correspondingly, green cotyledon emergence rates also decreased. Based on the study, three minutes for 70%ethanol and three minutes for 5%bleach were selected as the minimum time to sterilize the seeds.

To demonstrate that the seeds used originally were contaminated with microorganisms, non-sterile seeds were sown directly on the plates. Compared to sterile seeds, non-sterile seeds showed a fungal appearance after two days, which spread all over the plates after a seven-day germination. A cross-contamination assay performed using a single sterile pipette tip to process different seed samples showed that no green cotyledons were observed in the Columbia-0 genotype seeds sown in plates with kanamycin.

In parallel, in Columbia-0 seeds sown in plates without kanamycin, all cotyledons appeared green after germination. These results indicate no carryover of contamination between samples despite using a single pipette tip to remove the sterile solution. This procedure is the basis for many other functional genomics methods like gene overexpression, genome editing, subcellular localization, promoter activity, and protein-protein and protein-DNA interactions.