title: A simple improved-throughput xylem protoplast system for studying wood formation.
abstract: Isolated protoplasts serve as a transient expression system that is highly representative of stable transgenics in terms of transcriptome responses. They can also be used as a cellular system to study gene transactivation and nucleocytoplasmic protein trafficking. They are particularly useful for systems studies in which stable transgenics and mutants are unavailable. We present a protocol for the isolation and transfection of protoplasts from wood-forming tissue, the stem-differentiating xylem (SDX), in the model woody plant Populus trichocarpa. The method involves tissue preparation, digestion of SDX cell walls, protoplast isolation and DNA transfection. Our approach is markedly faster and provides better yields than previous protocols; small (milligrams)- to large (20 g)-scale SDX preparations can be achieved in ~60 s, with isolation of protoplasts and their subsequent transfection taking ~50 min. Up to ten different samples can be processed simultaneously in this time scale. Our protocol gives a high yield (~2.5 × 10(7) protoplasts per g of SDX) of protoplasts sharing 96% transcriptome identity with intact SDX.
authors.affiliation: 1] State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China. [2] Department of Forestry and Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Ying-Chung
authors.initials: YC
authors.lastname: Lin
authors.affiliation: 1] State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China. [2] Department of Forestry and Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Forestry and Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.affiliation: 1] State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China. [2] Department of Forestry and Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, North Carolina, USA. [3] College of Forestry, Shandong Agricultural University, Taian, Shandong, China.
authors.forename: Quanzi
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Forestry, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Ying-Hsuan
authors.initials: YH
authors.lastname: Sun
authors.affiliation: Department of Forestry and Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Shi
authors.affiliation: Department of Forestry and Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Chien-Yuan
authors.initials: CY
authors.lastname: Lin
authors.affiliation: 1] State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China. [2] Department of Forestry and Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Jack P
authors.initials: JP
authors.lastname: Wang
authors.affiliation: Department of Forestry and Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Hsi-Chuan
authors.initials: HC
authors.lastname: Chen
authors.affiliation: Department of Forestry and Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Chuang
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China.
authors.forename: Guan-Zheng
authors.initials: GZ
authors.lastname: Qu
authors.affiliation: Department of Forestry and Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Sederoff
authors.affiliation: 1] State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China. [2] Department of Forestry and Environmental Resources, Forest Biotechnology Group, North Carolina State University, Raleigh, North Carolina, USA. [3] Department of Forest Biomaterials, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Vincent L
authors.initials: VL
authors.lastname: Chiang
mesh_terms: D002473:Cell Wall / Q000378:metabolism; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D053263:Gene Regulatory Networks / Q000235:genetics*; D049452:Green Fluorescent Proteins; D032107:Populus / Q000166:cytology / Q000254:growth & development*; D011523:Protoplasts / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014934:Wood / Q000166:cytology / Q000254:growth & development*; D052584:Xylem / Q000737:chemistry*
chemical_list: D049452:Green Fluorescent Proteins