Plant callus culture provides a solution for the accurate, efficient, and fast evaluation of metabolic degradation of xenobiotics in plants. Plant callus culture can exclude microbiome or fungal interference and photochemical degradation, simplify the matrix effect of intact plants, standardize the cultivation conditions, shorten the treatment duration, and require less experimental effort. The method proposed here could provide insight into the uptake behavior and metabolic mechanisms of different organic pollutants on crops and are useful towards efforts on environmental risk assessments.
To begin, autoclave all the equipment and perform all operations in a UV sterilized ultra clean work bench. Sterilize the vernalized seed surface with 75%ethanol for 20 minutes. Rinse them with sterile deionized water three times and again sterilize them with 20%hydrogen peroxide for 20 minutes.
After washing the seeds with sterilized deionized water six times, aseptically germinate them by sowing on autoclaved, hormone-free Murashige and Skoog medium of pH 5.8 containing 1%agar gel and incubate at 26 degrees Celsius with 16 hours photo period for 15 days. After 15 days of seed incubation, cut the hypocotyl and cotyledon of the seedling into small pieces of 0.5 centimeters to obtain the explants. Transform the explants in a Petri dish containing 15 to 20 milliliters of autoclaved Murashige and Skoog medium supplemented with Oxymon and Phycocyanin and incubate in the dark at 26 degrees Celsius for three to four weeks to induce the callus.
Using a sterile scalpel and forceps, separate the callus of approximately one centimeter diameter formed from the initial explants. For treatment, dissolve 2, 4-dibromophenol in 10 milliliters of aseptic liquid Murashige and Skoog medium. Then add three grams of separated carrot callus to the prepared 2, 4-dibromophenol solution.
After preparing the medium in blank control as described in the manuscript, incubate all the flasks in the dark. To prepare the sample, separate the callus carefully from the 2, 3-dibromophenol treatment and control flasks by filtration with 0.45 micron glass fiber filters. Wash the callus three times with ultrapure water before collecting it.
Freeze dry all the collected callus and homogenize 0.2 grams of dried callus with a high throughput tissue grinder at 70 hertz for three minutes. Use a glass microsyringe to add 50 microliters of surrogate deuterated 4-n-nanophenol into the homogenized callus and vortex for one minute. Add five milliliters of the solution containing an equal ratio of methanol and water to the spiked callus and sonicate it for 30 minutes to extract the 2, 4-dibromophenol and metabolites.
After extraction, centrifuge the suspension at 8, 000 G at four degree Celsius for 10 minutes and collect the supernatant by pipetting. Pass the extract through hydrophilic lipophilic balanced solid phase extraction or HLB-SPE cartridge with a flow rate of one milliliter per minute. Dilute the analytes by passing six milliliters of methanol through the HLB-SPE cartridge.
Then concentrate the obtained eluent to one milliliter under a gentle stream of nitrogen gas for instrumental analysis. For analysis, open the column heater door. Then install the ultra performance liquid chromatography column by connecting the column inlet to the injection valve and the outlet to the mass spectrometer's inlet.
Insert the end of the solvent tubes A and B into the corresponding solvent bottles. Place the sample vials by serial number in the corresponding locations of the sample trays and reinsert the sample trays into the sample chamber. In the software window, click on Instrument, then Inlet Method to edit the conditions for the liquid chromatogram.
Select MS Method and set up the parameters of mass spectrum analysis. Click on File and then New to create and name the database. Load the sample program created above by selecting MS File, followed by Inlet File and Inject Volume.
Save the database in the sample folder of the project by clicking File and Save. Next, select Run and Start in the main software window and click on Acquire Sample Data, followed by the OK button in the start sample list run to collect data. To process the data, select the target data row and click on the chromatogram window to view the MS scan chromatogram.
In the chromatogram window, click on Display, followed by TIC. After clicking on the scan wave DS, add trace and OK to get the daughter mass spectra scan. The chromatogram of 2, 4-dibromophenol-treated carrot callus extract showed the presence of eight different metabolites compared to the control samples.
Additionally, the absence of parent 2, 4-dibromophenol peak from the chromatogram indicates the rapid metabolism of 2, 4-dibromophenol in the carrot callus under experimental conditions. Furthermore, 2, 4-dibromophenol incubated in carrot callus led to the formation of metabolites by direct conjugation with glucose and amino acids. All equipment as well as the Murashige and Skoog medium should be autoclaved to make sure that the differentiation and maintenance of the plant callus are performed under aseptic conditions.
Omics approaches following this procedure allow the creation of a clear phytotoxic mechanistic picture of environmental pollutants.
