Name: 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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This work was supported by the National Key Research &#38; Development Program (2016YFD0200900) of China, the National Natural Scientific Foundation of China (No. 31772076 and No. 31270728), China Postdoctoral Science Foundation (2018M630700), and Open Fund of State Key Laboratory of Tea Plant Biology and Utilization (SKLTOF20180111).

1. Tea callus culture
NOTE: Sterile leaves were derived from in vitro-grown plantlet lines first developed in the research group17. All procedures up to section 5 were carried out in a sterile laminar flow hood, except for the culture time in an incubator.
<ol>
	<li>Adjust the pH of the two media (Murashige and Skoog [MS] basal medium and Gamborg&#39;s B5 liquid medium) to 5.8 prior to autoclaving (121 &#176;C, 20 min).</li>
	<li>Cut along the middle vein of a sterile...

<ol>
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This article presents the detailed process of establishing a model of pesticide metabolism in tea plant tissue, including the selection of explants, the determination of cell viability, and the establishment of a tea cell suspension culture with high metabolic activity. Any parts of a plant tissue could be used to initiate callus in a sterilized environment25. Tea leaves were chosen for callus initiation in this study, not only because leaves to tend to be less contaminated than the parts below gr...

Tea is one of the most widely consumed non-alcoholic beverages in the world1,2. Tea is produced from the leaves and buds of the woody perennial Camellia sinensis L. Tea plants are grown in vast plantations and are susceptible to numerous insect pests3,4. Organophosphorus and neonicotinoid insecticides are often used as systemic insecticides5 to protect tea plants from pests such as whiteflies, leaf hoppers, and some lepidopteran species6,7. After application, these insecticides are absorbed or translocated into the plant. Within the plant, these systemic insecticides may be transformed through hydrolysis, oxidation or reduction reactions by plant enzymes. These transformation products can be more polar and less toxic than the parent compounds. However, for some organophosphates, the bioactivities of some products are higher. For example, acephate is metabolized into the more toxic methamidophos8,9, and dimethoate into omethoate10,11. Plant metabolic studies are thus important for determining the fate of a pesticide within a plant12.
Plant tissue cultures have been proven to be a useful platform for investigating the pesticide metabolism, with the identified metabolites similar to those found in intact plants13,14,15. The use of tissue cultures, particularly cell suspension cultures, has several advantages. Firstly, experiments can be carried out free of microorganisms, thus avoiding the interference of pesticide transformation or degradation by microbes. Secondly, tissue culture provides consistent materials for use at any time. Thirdly, the metabolites are easier to extract from tissue cultures than from intact plants, and tissue cultures often have fewer interring compounds and lower complexity of compounds. Finally, tissue cultures can more easily be used to compare a series of pesticides metabolism in a single experiment16.
In this study, a cell suspension derived from the leaves of sterile-grown tea plantlet was successfully established. The tea cell suspension culture was then used to compare the dissipation behaviors of six systemic insecticides.
This detailed protocol is intended to provide some guidance so that researchers can establish a plant tissue culture platform useful for studying the metabolic fate of xenobiotics in tea.

<table border="0" cellpadding="0" cellspacing="0" style="width:674px;" width="673">
	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Acetamiprid (99.8%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">46717</td>
			<td style="width:123px;">CAS No: 135410-20-7</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:188px;">Acetonitrile (CAN, 99.9%)</td>
			<td style="width:188px;">Tedia</td>
			<td style="width:175px;">AS1122-801</td>
			<td style="width:123px;">CAS No: 75-05-8</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Agar</td>
			<td style="width:188px;">Solarbio Science &#38; Technology</td>
			<td style="width:175px;">A8190</td>
			<td style="width:123px;">CAS No: 9002-18-0</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Clothianidin (99.8%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">525</td>
			<td style="width:123px;">CAS No: 210880-92-5</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:188px;">Dimethoate (98.5%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">109217</td>
			<td style="width:123px;">CAS No: 60-51-5</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Imidacloprid (99.8%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">91029</td>
			<td style="width:123px;">CAS No: 138261-41-3</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Imidaclothiz (99.5%)</td>
			<td style="width:188px;">Toronto Research Chemical</td>
			<td style="width:175px;">I275000</td>
			<td style="width:123px;">CAS No: 105843-36-5</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Kinetin (KT, &#62;98.0%)</td>
			<td style="width:188px;">Solarbio Science &#38; Technology</td>
			<td style="width:175px;">K8010</td>
			<td style="width:123px;">CAS No: 525-79-1</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:188px;">Omethoate (98.5%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">105491</td>
			<td style="width:123px;">CAS No: 1113-02-6</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Polyvinylpolypyrrolidone (PVPP)</td>
			<td style="width:188px;">Solarbio Science &#38; Technology</td>
			<td style="width:175px;">P8070</td>
			<td style="width:123px;">CAS No: 25249-54-1</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:188px;">Sucrose</td>
			<td style="width:188px;">Tocris Bioscience</td>
			<td style="width:175px;">5511</td>
			<td style="width:123px;">CAS No: 57-50-1</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Thiamethoxam (99.8%)</td>
			<td style="width:188px;">Dr. Ehrenstorfer</td>
			<td style="width:175px;">20625</td>
			<td style="width:123px;">CAS No: 153719-23-4</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:188px;">Triphenyltetrazolium Chloride (TTC, 98.0%)</td>
			<td style="width:188px;">Solarbio Science &#38; Technology</td>
			<td style="width:175px;">T8170</td>
			<td style="width:123px;">CAS No: 298-96-4</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:188px;">2,4-Dichlorophenoxyacetic Acid (2,4-D, &#62;98.0%)</td>
			<td style="width:188px;">Guangzhou Saiguo Biotech</td>
			<td style="width:175px;">D8100</td>
			<td style="width:123px;">CAS No: 94-75-7</td>
		</tr>
		<tr height="77">
			<td height="77" style="height:77px;width:188px;">chiral column</td>
			<td style="width:188px;">Agilent CYCLOSIL-B</td>
			<td>112-6632</td>
			<td style="width:123px;">Chromatography column (30 m &#215; 0.25 mm &#215; 0.25 &#956;m)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:188px;">Gas chromatography (GC)</td>
			<td style="width:188px;">Shimadu</td>
			<td>2010-Plus</td>
			<td style="width:123px;">Paired with Flame Photometric Detector (FPD)&#160;&#160;</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:188px;">High-performance liquid chromatography (HPLC)</td>
			<td>Agilent</td>
			<td>1260</td>
			<td style="width:123px;">Paired with Ultraviolet detector (UV)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:188px;">HSS T3 C18 column</td>
			<td style="width:188px;">Waters</td>
			<td>186003539</td>
			<td style="width:123px;">Chromatography column (100 mm &#215; 2.1 mm &#215; 1.8 &#956;m)</td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:188px;">Ultra-high-performance liquid chromatography (UPLC)</td>
			<td style="width:188px;">Agilent</td>
			<td>1290-6545</td>
			<td style="width:123px;">Tandem quadrupole time-of-flight mass spectrometer (QTOF)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:188px;">Ultra-high-performance liquid chromatography (UPLC)</td>
			<td style="width:188px;">Thermo Scientific</td>
			<td>Ultimate 3000-Q Exactive Focus</td>
			<td style="width:123px;">Connected to a Orbitrap mass spectrometer</td>
		</tr>
	</tbody>
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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

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

The induction of callus from leaves harvested from field-grown tea trees and from leaves excised from tea plantlets grown in vitro in a sterile environment was compared by measuring contamination, browning, and induction after 28 days of cultivation on MS media (Figure 1A). Callus growth was recorded at 20, 37, 62 and 90 days of culture (Figure 1B). The callus derived from the in vitro-grown leaves showed more vigorous growth tha...

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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

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.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

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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