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, th....

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.......

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