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

Summary

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.

Abstract

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 ± 1 °C. After 3−4 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 µg 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.

Introduction

Tea is one of the most widely consumed non-alcoholic beverages in the world^1,2. 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 pests^3,4. Organophosphorus and neonicotinoid insecticides are often used as systemic insecticides⁵ to protect tea plants from pests such as whiteflies, leaf hoppers, and some lepidopteran species^6,7. 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 methamidophos^8,9, and dimethoate into omethoate^10,11. 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 plants^13,14,15. 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.

Protocol

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.

1. Adjust the pH of the two media (Murashige and Skoog [MS] basal medium and Gamborg's B5 liquid medium) to 5.8 prior to autoclaving (121 °C, 20 min).
2. Cut along the middle vein of a sterile leaf using scissors, and then subdivide each half into small pieces of about 0.3 cm x 0.3 cm in a petri dish.
3. Place the sterile explants (the small leaf pieces) onto MS basal media containing the plant hormones 2,4-D (1.0 mg L^-1) and KT (0.1 mg L^-1). Six explants can be placed in a 300 mL flask containing 100 mL of MS basal media.
4.  Culture the above leaf explants at a constant temperature of 25 °C in the dark. After 28 days, select the first generation of induced callus and transfer to fresh flask (a subculture). Acquire the loose and friable callus after 3−4 subcultures.

2. Tea cell suspension culture

1. Cut the vigorous, friable and loose calluses from the solid medium into small pieces (range here 0.5−2 mm) using a sterile surgical blade under sterile conditions.
2. Weigh about 3 g of the small pieces of callus. Place the callus into a 150 mL flask containing 20 mL of B5 liquid media supplemented with 2,4-D (1.0 mg L^-1) and KT (0.1 mg L^-1).
3. Culture the liquid cell suspension at a constant temperature (25 ± 1 °C) in a shaking incubator at 120 rpm in the dark.
4. After 7 to 10 days of culturing, remove the culture flasks and let them stand for a few minutes.
5. Take all the supernatant as seed material for subculture to fresh medium (subculture ratio of suspension mother liquid to fresh medium ranged between 1:1 and 1:2). Remove the precipitated, large calluses.
6. Obtain the final well-grown cell suspension culture after 3−4 subculture cycles of 28 days each.

3. Triphenyl tetrazolium chloride assay of cell viability

1. Kill a sample of living cells at 100 °C for 10 min as a control cell before viability staining.
2. Centrifuge all cell suspension culture for 8 min at 6000 x g. Remove the supernatant before suspending the cells in 2.5 mL of phosphate-buffered saline (PBS) buffer (pH 7.3), and shake it for 1 min by hand.
3. Add 2.5 mL of the 0.4%triphenyl tetrazolium chloride (TTC) solution and shake by hand again.
4. Incubate the mixture for 1 h in a standing incubator (30 °C).

4. Treatment and sampling of tea cell suspension cultures with insecticides

1. Add an aliquot of 400 µL of filter-sterilized stock solution (500 µg mL^-1) of four neonicotinoids (thiamethoxam, acetamiprid, imidacloprid, and imidaclothiz) or two organophosphates (dimethoate and omethoate) into the cell suspension cultures, respectively.
   NOTE: If the aim is to compare xenobiotic behaviors, use the same mother batch of cell suspensions to test the different compounds.
2. Culture the samples of cell suspensions with insecticides at constant temperature (25 ± 1 °C) and shaking incubator speed (120 rpm). Take the samples (see step 4.3 or 4.4) on 0, 3, 5 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 and 75 days.
3. To test a sample containing a neonicotinoid, remove a 1 mL aliquot of the homogeneous cell culture, place it into a 1.5 mL plastic centrifuge tube, and centrifuge at 4000 x g for 2 min.
   1. Pass the supernatants through a 0.22-µm pore-size filter membrane before analysis by high-performance liquid chromatography-ultraviolet (HPLC-UV) and ultra-high performance liquid chromatography-quadrupole time-of-flight (UPLC-QTOF) mass spectrometry (Table of Materials).
4. To test a sample containing an organophosphate, remove a 500 µL aliquot of the cell culture and place into a 35 mL centrifuge tube or a 1.5 mL plastic centrifuge tube (prepare the latter sample like that of neonicotinoid).
   1. Add 0.1 g of sodium chloride and 5 mL of acetone/ethyl acetate (3:7, v/v) into the 35 mL centrifuge tube of the 500 µL samples.
   2. Vortex the mixtures for 1 min, and then allow them to rest for 10 min.
   3. Take 2.5 mL of the supernatant into a 10 mL glass tube and evaporate to near-dryness using a nitrogen evaporator at 40 °C.
   4. Dissolve the residue with 1 mL acetone, vortex for 1 min, pass it through a 0.22-µm filter membrane before analysis by gas chromatography-flame photometric detector (GC-FPD).

5. Sample preparation of intact tea plant with insecticides

NOTE: The intact tea plant trial was conducted in a hydroponic system using tea seedlings grown in 50 mL of a nutrient solution (30 NH₄+, 10 NO₃-, 3.1 PO₄-, 40 K⁺, 20 Ca²⁺, 25 Mg²⁺, 0.35 Fe²⁺, 0.1 B³⁺, 1.0 Mn²⁺, 0.1 Zn²⁺, 0.025 Cu²⁺, 0.05 Mo⁺, and 10 Al³⁺, in mg L^-1)¹⁸. An experimental greenhouse was under a light-dark cycle (12 h of light and 12 h of darkness) at 20 °C at Anhui Agricultural University.

1. Put five plants in a 4 L plastic pot for 15 days.
2. Add 0 ppm (control) or 100 ppm of thiamethoxam or dimethoate into plastic pots, respectively.
3. Prepare the intact plant sample according to the previous method, except for presoaking¹⁹, and then analyze with mass spectrometry for an accurate mass spectrum.

6. Instrument analysis

1. HPLC analysis of the metabolic behavior of neonicotinoids
   1. Use an HPLC-UV (Table of Materials) to detect the content and metabolic products of thiamethoxam and acetamiprid at a wavelength of 254 nm, and of imidacloprid and imidaclothiz at 270 nm in samples from section 4.3.
      NOTE: The HPLC-UV condition was the same as the previous study¹⁹.
2. GC analysis of the metabolic behavior of organophosphates
   1. Detect the content of dimethoate and omethoate in samples from section 4.4 by a GC-FPD using a chiral column (Table of Materials).
   2. Use nitrogen as the carrier gas and set the flow rate at 1.0 mL min^-1.
   3. Set the initial temperature to 120 °C, and hold it for 5 min. Increase the temperature to 150 °C at 30 °C min^-1 and hold for 3 min. Increase to 170 °C at 10 °C min^-1 and hold for 7 min. Finally increase to 210 °C at 30 °C min^-1 and then hold for 5 min.
   4. Set the injection temperature to 200 °C in splitless mode; Set the detector temperature to 250 °C.
   5. Set the injection volume to 1 µL.
3. UPLC-QTOF analysis of the insecticide metabolites in cell culture
   1. Detect the metabolites of the insecticides in cell culture (samples from section 4.3) using UPLC-QTOF with a C18 column (Table of Materials).
   2. Set the flow rate to 0.2 mL min^-1. Set the injection volume to 10 µL.
   3. For the neonicotinoid-treated samples, set the initial mobile phase to 85% A (5 mM ammonium formate water) and 15% B (acetonitrile). Over 10 min, increase mobile phase B to 38% and return to 15% over 1 min, hold for 9 min.
   4. For the organophosphate-treated samples, set the initial mobile phase to 55% A (0.1% formic acid water) and 45% B (acetonitrile). Over 5 min, increase mobile phase B to 70%, then return to 45% of B over 0.5 min, hold for 2.5 min.
   5. Set the QTOF operation parameters as follows: gas temperature, 325 °C; drying gas (nitrogen), 10 L min^-1; sheath gas temperature, 350 °C; sheath gas flow, 11 L min^-1; capillary voltage, 4000 V; nozzle voltage, 1000 V; fragmentor voltage, 100 V for neonicotinoid insecticides or 110 V for organophosphorus insecticides; skimmer voltage, 65 V; operating in positive ion mode.
   6. Set the instrument to the full scan spectrum and target MS/MS mode.
   7. Process the data using accurate mass tools; Infer the metabolites with no standard products from the MS/MS annotation as well as the literature^{12,15,20,21,22}.
4. UPLC-Orbitrap analysis of the insecticide metabolites in intact plant extract
   1. Detect the metabolites of insecticides in intact plant extract using UPLC-Orbitrap mass spectrometry (Table of Materials).
   2. Set the mass spectrometry (Table of Materials) operation parameters as follows: sheath gas pressure, 35 arb; gas temperature, 300 °C; nozzle voltage, 3.5 KV; capillary temperature, 350 °C.
   3. Set the elution programs as the above (steps 6.3.3 and 6.3.4) for UPLC-QTOF analysis of cell culture.

Results

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...

Discussion

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...

Materials

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