Chinese Academy of Agricultural Sciences 10 articles published in JoVE Environment Source and Route of Pyrrolizidine Alkaloid Contamination in Tea Samples Weiting Jiao1,2, Tingting Shen2, Luyao Wang1, Lei Zhu1,3, Qing X. Li4, Chen Wang3, Hongping Chen3, Rimao Hua1, Xiangwei Wu1 1Key Laboratory of Agri-food Safety of Anhui Province, School of Resource & Environment, Anhui Agricultural University, 2State Key Laboratory of Tea Plant Biology and Utilization, School of Tea and Food Science & Technology, Anhui Agricultural University, 3Key Laboratory of Tea Quality and Safety & Risk Assessment, Tea Research Institute, Chinese Academy of Agricultural Sciences, Ministry of Agriculture, 4Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa The present protocol describes the contamination of pyrrolizidine alkaloids (PAs) in tea samples from PA-producing weeds in tea gardens. Biology Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis Jinxi Yuan1, Jie Zhang1, Yan Zhang1, WuYun QiQiGe1, Wei Liu2, Shanchun Yan1, Guirong Wang2 1Key Laboratory of Sustainable Forest Ecosystem Management - Ministry of Education, Northeast Forestry University, 2Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis. Biology Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance Yutong Liu1, Mingming Sun1, Zipeng Jiang1, Xinfang Wang2, Bingguang Xiao3, Aiguo Yang1, He Meng1, Lirui Cheng1 1Key Laboratory of Tobacco Genetic Improvement and Biotechnology, Tobacco Research Institute, Chinese Academy of Agricultural Sciences, 2Institute of Rural Development, Zhejiang Academy of Agricultural Sciences, 3Yunnan Academy of Tobacco Agricultural Sciences Here, a protocol is presented for the efficient and accurate screening of tobacco genotypes for Phytophthora nicotianae resistance in seedlings. This is a practical approach for precision breeding, as well as molecular mechanism research. Behavior Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner) Dong Ai1,2, Bing Wang2, Zhennan Fan1, Yuyao Fu1, Caihong Yu1, Guirong Wang2,3 1School of Chemical and Environmental Engineering, China University of Mining and Technology, 2State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 3Guangdong Laboratory of Lingnan Modern Agriculture, Shenzhen; Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences Presented here is a protocol of Helicoverpa armigera (Hübner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo. Biology Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts Lu Zhang1, Wenwen Liu1, Xifeng Wang1 1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission. Environment Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions Zhongyi Wang*1, Jiaming Li*2, Lu’an Qian*3, Linna Liu1, Jun Qian1, Bing Lu1, Zhendong Guo1 1Academy of Military Medical Sciences, 2Chinese Academy of Agricultural Sciences, 3The Experimental High School Attached to Beijing Normal University Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions. Environment Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens Cai Wang1, Huifang Wang1, Tao Ma1,2, Qiang Xiao3, Panrong Cao4, Xuan Chen5, Hongpeng Xiong1, Wenquan Qin1, Zhaohui Sun1, Xiujun Wen1 1Guangdong Key Laboratory for Innovation Development and Utilization of Forest Plant Germplasm, College of Forestry and Landscape Architecture, South China Agricultural University, 2College of Agriculture, South China Agricultural University, 3Tea Research Institute, Chinese Academy of Agricultural Sciences, 4College of Horticulture, South China Agricultural University, 5College of Coast and Environment, Louisiana State University Here, we present a protocol to investigate the pupation preference of mature larvae of Ectropis grisescens in response to soil factors (e.g., substrate type and moisture content) using choice bioassays. We also present a protocol of no-choice bioassays to determine the factors that affect the pupation behaviors and survivorship of E. grisescens. Chemistry Extraction of Ramie Fiber in Alkali Hydrogen Peroxide System Supported by Controlled-release Alkali Source Chaoran Meng1, Zhaoling Li1, Chaoyun Wang2, Chongwen Yu1,2, Xuerong Bi1, Siyi Wang1 1College of Textiles, Donghua University, 2Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences Presented here is a protocol for extraction of ramie fiber in alkali hydrogen peroxide system supported by controlled-release alkali source. Immunology and Infection Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm Xianming Yang1, Pengjun Xu2, Robert I. Graham3, He Yuan1, Kongming Wu1 1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 2Tobacco Research Institute, Chinese Academy of Agricultural Sciences, 3Crop and Environment Sciences, Harper Adams University Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts. Biology A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response Ritesh Kumar*1, Shu Wei Wu*1, Arya Bagus Boedi Iswanto1, Dhinesh Kumar1, Xiao Han2, Jae-Yean Kim1 1Division of Applied Life Science (BK21 plus program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, 2Biotechnology Research Institute, Chinese Academy of Agricultural Sciences This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.
