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Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves
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Summary June 15th, 2019
This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.
Transcript
Tea is one of most popular drinks throughout world. Pesticides are sometimes used to protect tea plants from pests. Some systemic pesticides may be made for this by enzymes in tea tree.
And then integrated through oxidation, hydrolysis, or reduction reactions. Plant cell suspension cultures can be used as an, easily to study plant metabolism behavior of pesticides. This method has several advantages.
Firstly, it can be operated on conditions of free microorganisms, thus, avoiding the interference of the pesticide degradation caused by microbes. Secondly, it can provide constant material for us at any time. Finally, it can also be used easily to compare theories of pesticide behavior at a single experiment.
In this study, we establish tea cell suspension cultures from variable colors of tea leaves. The optimal cultures status of cell suspensions was then used to compare the dissipation behavior of six pesticides. This experiment will be conducted by Jiao Weiting and Ge Guoqin.
This is our tea sterile plantlets which works as the source of explant. Cut along the middle wing of a sterile leaf using scissors. And then, subdivide each half into small pieces of about 0.3 times 0.3 centimeters in a Petri dish.
Place the sterile explants onto MS visual medium containing 2, 4-D and KT.Six explants can be placed in a 300 milliliter flask. Culture the leaf explants at a constant temperature of 25 degree Celsius in the dark. After 28 days, select the first generation of induced callus and then transfer to fresh flask.
Acquire the loose and variable callus after three to four subcultures. Cut up the big ones, variable and loose calluses from the solid medium into small pieces using a sterile surgical blade under sterile conditions. We're about three grams of the small pieces of a callus.
Place the callus into B5 containing 2, 4-D and the KT.Culture the liquid cell suspension at a constant temperature in the shaking incubator in the dark. After seven to ten days of culturing, remove the culture flask, and leave them to stand for a few minutes. Take the supernatant as sediment here will force the culture to fresh medium.
Remove the precipitated large calluses. Obtain the final well-groomed cell suspension culture after four to three subculture cycles of 28 days each. Keep a sample of living cells at one hundred degrees celsius for ten minutes as the control cell before viability staining.
Centrifuge all cell suspension culture for eight minutes at 6, 000 G.Remove the supernatant before suspending the cells into 5 milliliter of PBS buffer and shake it for one minute by hand. Add 2.5 milliliters of the TTC solution and shake by hand again. Incubate the mixture for one hour in a standing incubator at 13 degrees Celsius.
Add an aliquot of four hundred microliters on view to sterilize the stock solution of all neonicotinoids Thiamethoxam, Acetimoprid, Imidacloprid, and Imideproxase. All to organic first phase. Dimethoate and omethoate into the cell suspension cultures respectively.
Culture samples of cell suspensions with insecticides at constant temperature, and shaking incubator speed. Take the samples on different days. To test the sample containing a neonicotinoid, remove a 1 milliliter aliquot of the homogeneous cell culture.
Place it into a 1.5 milliliter plastic centrifuge tube, and centrifuge it at 4, 000 G for two minutes. Pass the supernatant through a 0.22 micrometer filter membrane before analysis by HPLC-UV and UPLC QTOF. To test the sample, continue in the organic first phase.
Remove a 500 microliter aliquot of the cell culture and place into a 35 milliliter centrifuge tube or 1.5 milliliter plastic centrifuge tube. Add 0.1 grams of sodium chloride and five milliliters extract solvent into the 35 milliliter centrifuge tube of the 500 microliter samples. Vortex the mixture for one minute.
And then allow them to rest for 10 minutes. Take 2.5 milliliters of supernatant into a 10 milliliter glass tube and evaporate to near-dryness using a nitrogen evaporator at 14 degrees Celsius. Dissolve the residue with one milliliter acetone.
Vortex for one minute, pass it through a 0.22 micrometer filter membrane before analysis by GC-FPD. Put five plants in four liter plastic pots for fifteen days. Add zero microgram per milliliter or 100 microgram per milliliter of Thiamethoxam or dimethoaxe into plastic pots, respectively.
Prepare the intact plant sample according to the previous method, except for pre-soaking and then analyze by Orbitrap mass spectrometry. Use an HPLCUV to detect the content and metabolic products of the Thiamethoxam and Acetimoprid at wavelengths of 254 nanometers, and of Imidacloprid and Imideproxase at 270 nanometer. Detect the content of dimethoate and omethoate by GC-FPD using a tubular column.
Detect the metabolites of insecticides in cell culture using the UPRC QTOF with a Carbon-18 column. Detect the metabolites of insecticides in test plant extract using UPLC Orbitrap mass spectrometry. The callus of one sterile plantlet has lower contamination browning but higher inductivity that that of callus from picked tea leaves.
The callus derived from sterile leaves was always bright yellowish, while the callus derived from picked leaves was white with brown spots. When KT concentration was 0.1 milligram per liter, the callus was yellowish in color and loose in texture. The growth rate of callus was the highest, up to 61.5%when the concentration of 2, 4-D was one milligram per liter with the concentration of KT was 0.5 milligrams per liter.
The growth data of callus was the best and the growth rate of callus was the highest reached 46.9%Therefore, the best combination of plant homent was one milligram per liter of 2, 4-D and 0.1 milligram per liter of KT for the tea callus growth. The callus completely covered the leaves by the fourth subculture, and when the subculture cycle was 28 days. The callus had grown vigorously with yellowish color and loose texture and no browning.
After comparing the gross data of callus and the color of cell suspension, the B5 liquid medium was more suitable for cell suspension culture. The OD value of the cell suspension culture was used to represent the amount of cell growth. During the 75 days of cultivation, the ratio of 15 grams per 40 milliliter had an OD value significantly higher than that of the other two ratios.
Cell viability within the tea cell suspension culture was tested by TTC staining. The colorless TTC compound can be converted to red formadin by dehydrogenase in mitochondria of living cells, but the color cannot be changed in dead cells. And this is the whole process of establishing a tea cell suspension culture.
This is the first comparative study of the metabolism of different pesticides in tea using a cell suspension culture technique. Several pesticide metabolites were identified, and some of them were further investigated in kept tea plants. This new method could serve as the model for pesticide metabolic studies in tea or other plants.
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