An efficient protocol for perennial ryegrass mesophyll protoplast isolation and transformation, and its application on i
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Yu et al. Plant Methods (2017) 13:46 DOI 10.1186/s13007-017-0196-0
Open Access
METHODOLOGY
An efficient protocol for perennial ryegrass mesophyll protoplast isolation and transformation, and its application on interaction study between LpNOL and LpNYC1 Guohui Yu1, Qiang Cheng2, Zheni Xie1, Bin Xu1* , Bingru Huang3* and Bingyu Zhao4
Abstract Background: Perennial ryegrass (Lolium perenne L.) is an important temperate grass used for turf and forage pur‑ poses. With the increasing accumulation of genomic and transcriptomic data of perennial ryegrass, an efficient proto‑ plast and transient gene expression protocol is highly desirable for in vivo gene functional studies in its homologous system. Results: In this report, a highly efficient protoplast isolation (5.6 × 107 protoplasts per gram of leaf material) and transient expression (plasmid transformation efficiency at 55.2%) was developed and the detailed protocol presented. Using this protocol, the subcellular locations of two ryegrass proteins were visualized in chloroplasts and nuclei, respectively, and protein–protein interaction between two chlorophyll catabolic enzymes (LpNOL and LpNYC1) was recorded in its homologous system for the first time. Conclusion: This efficient protoplast isolation and transformation protocol is sufficient for studies on protein subcel‑ lular localization and protein–protein interaction, and shall be suitable for many other molecular biology applications where the mesophyll protoplast system is desirable in perennial ryegrass. Keywords: Ryegrass, Lolium, Protoplast, Transient gene expression Background Perennial ryegrass (Lolium perenne L.) is the most widely distributed and cultivated turf and forage grass in temperate zones. World-wide consorted programs are working on the molecular genetics of this grass species. The draft genome of this perennial ryegrass was recently published [1] and the assembly of the genome dataset is undergoing. The accumulation of genomic and transcriptomic datasets provided unprecedented opportunity *Correspondence: [email protected]; [email protected] 1 College of Agro‑grassland Science, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China 3 Department of Plant Biology and Pathology, Rutgers, the State University of New Jersey, New Brunswick, NJ 08901, USA Full list of author information is available at the end of the article
to conduct functional genomic studies in perennial ryegrass. Stable genetic transformation systems have been established in perennial ryegrass, yet the whole transformation takes months to obtain rooted transgenic plants [2, 3]. The plant protoplast system provides a complementary or, sometimes, an alternative way to the stable genetic transformation system for gene functional analysis in many cases, such as protein subcellular localization, in vivo protein–protein and protein-DNA interactions, protein trafficking and signal transduction, etc. Currently, mesophyll protoplast-based transient expression assays are routinely used in biological st
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