The overall goal of this procedure is to provide standardized conditions for successful seed sterilization of a large number of Arabidopsis genotypes while preventing microbial growth. This method allows you to sterilize hundreds of seed samples at a time, thus simplifying a critical plant material preparation step in the fields of genomics, proteomics, metabolomics and other high-throughput studies. The main advantage of this technique is that is provides conditions in which efficient sterilization is combined with a high germination rate for a large number of seed samples.
To begin, prepare a 50%bleach solution with distilled water. To every 200 milliliters of bleach solution, add 50 microliters of TWEEN 20 detergent. The bleach solution can be stored for up to a month as long as it is only opened in sterile conditions.
Next, aliquot 100 seeds into a 1.5-milliliter microcentrifuge tube. This can be done manually, or by using a custom-made seed robot. Now, in a laminar flow hood, add 500 microliters of the 50%bleach solution to the seeds.
Then, transfer the tubes to a rotator and incubate the seeds in the bleach with mild agitation for 10 minutes. After 10 minutes, aspirate the bleach solution from the tubes removing as much as possible. Then, add back 500 microliters of sterile distilled water and invert the tubes to mix.
Allow the seeds to settle, then aspirate the water and replace it with another aliquot to wash the seeds again. Perform a total of six such washes with sterilized distilled water. Once the seeds have been thoroughly washed with water, suspend them in one milliliter of water before plating them.
Before starting the sterilization process, calculate the volumes of bleach and hydrochloric acid required to produce chlorine gas. Use the provided equation to obtain the recommended 6%chlorine gas. Make sure to precisely calculate the volume of HCl required to ensure successful sterilization and to prevent splashing.
Be sure to wear gloves and a lab coat during this protocol and change the gloves whenever they are wetted with acid or bleach. Under a fume hood, transfer the seed vials into a rack with their lids open. Then, place the rack into a plastic container in which the sterilization can take place and prepare paraffin film strips to seal the lid to the container.
Now, load the beaker with 100 milliliters of bleach and then place a 250-milliliter beaker inside. Using an excess of bleach is a safety feature and the beaker should be at least twice the total volume of the solution it contains. Also, always handle the bleach and acids separately.
Next, add three milliliters of hydrochloric acid to the beaker, and then immediately close the container and seal it with parafilm. Monitor the container for one hour to ensure the gas is accumulating during the sterilization. Chlorine gas should either be visible as a faint yellow haze inside the container or the solution can be checked for turning yellow.
Periodically check the seal on the container. If the lid gets unseated or the paraffin seal begins to loosen, tightly reseal the lid with an additional layer of paraffin film. After the one-hour sterilization, open a corner of the container to vent the gas over a period of three hours.
After three hours of venting, cap all the tubes and store the sterilized seeds in dry conditions until they are plated. Now, neutralize the reaction by slowly adding 1.5 grams of sodium bicarbonate powder while stirring with a glass rod to dissolve the solids. Keep adding sodium bicarbonate until the carbon dioxide gas bubbles have stopped forming.
Then, measure the pH of the solution, and if needed, add additional sodium bicarbonate until the pH is neutral. Provided the solution is not smelly, it can be disposed of. Otherwise, wait until all the smells dissipate.
Add 500 microliters of sterilized distilled water to each tube of gas-sterilized seeds. Then, pour the seeds onto an MS plate and spread them out evenly using a sterile single-use inoculating loop or a sterile pipette tip. After three days of seed stratification at four degrees Celsius, transfer the plates to 23 degrees Celsius with 16 hours of daily light exposure.
Keep the lids on top so that the roots grow into the medium. After eight days of growth, score the fraction of seeds that have germinated. Germination is counted when the radical has projected outside the seed coat, and the two cotyledons are visible.
Also, count the number of seeds affected by mold to determine the effectiveness of the sterilization conditions. Four different concentrations of bleach at five different exposure times were tested on Columbia wild-type seeds. High bleach concentrations adversely affected germination with too much exposure.
However, high germination rates were observed with 40 and 50%bleach at all exposure times. Seeds sterilized with high bleach concentrations had high mortality and often showed growth defects but were usually completely mold-free. Sterilization by 50%bleach for 10 minutes was determined to have the optimal balance of mold inhibition and healthy germination.
To optimize chlorine gas sterilization conditions, three different concentrations of chlorine gas were used to sterilize Columbia wild-type seeds over two different lengths of time. There was a significant interaction between time and gas concentration. At one hour, there was no effect on germination, whereas the highest gas concentration was detrimental at three hours.
Thus, a one-hour sterilization period was chosen. Mold growth was effectively inhibited by three-hour gas exposures or for one-hour exposures using six or 17%gas. Thus, a 6%gas concentration for one hour was determined to be optimal for germination and mold inhibition.
Once mastered, this technique can be used to successfully sterilize hundreds of seed lines per day if it is performed properly. Gas-sterilized seeds can also be stored for an extended period of time in dry conditions. While attempting this procedure, it's important to remember to use provided equations to calculate required volumes of HCl and bleach.
Following this procedure, other methods like genome-wide association studies can be performed in order to link a variety of different genotypes and the phenotypes of interest. Good luck with your experiments.
