Genetic Transformation of Protoplasts Isolated from Leave of Lolium temulentum and Lolium perenne

Type Chapter
Original languageEnglish
Title of host publicationCereal Genomics
EditorsLuis M. Vaschetto
PublisherSpringer Nature
ISBN (Electronic)978-1-4939-9865-4
ISBN (Print)978-1-4939-9864-7
DOI
Publication statusE-pub ahead of print - 21 Sep 2019

Publication series

NameMethods in Molecular Biology
Volume2072
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029
View graph of relations
Citation formats

Abstract

Transient expression of inserted recombinant DNA in plant protoplasts is a widely used tool for functional genomics research. Recently it has been utilized to screen potential sgRNA guides for CRISPR-mediated genome editing. However, little research has been conducted into the use of transient expression of protoplasts in Lolium perenne (a globally important pasture, hay, and amenity grass), and no studies have been conducted into Lolium temulentum (a weed in cereal crops but a potentially useful model species for Lolium research). In this chapter, we describe a methodology of protoplast extraction and transformation from 14-day-old leaf mesophyll cells from L. perenne and L. temulentum. We believe this is the first report of a procedure for obtaining high density, viable protoplasts from L. temulentum. The method of polyethylene glycol (PEG)-mediated transformation is also described to achieve genetic transformation of protoplasts

Keywords

  • protoplasts, lolium temulentum, lolium perenne, transient expression, PEG-mediated transformation