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* These authors contributed equally
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.
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.
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, th....
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.
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.......
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.......
This work was supported by the National Key Research & 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).
....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 |
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