Protoplast Isolation, Transfection, and Gene Editing for Soybean (Glycine max )

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Protoplast is a versatile system for conducting cell-based assays, analyzing diverse signaling pathways, studying functions of cellular machineries, and functional genomics screening. Protoplast engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. This system allows the direct delivery of DNA, RNA, or proteins into plant cells and provides a high-throughput system to validate gene-editing reagents. It also facilitates the delivery of homology-directed repair templates (donor molecules) into plant cells, enabling precise DNA edits in the genome. There is a great deal of interest in the plant community to develop these precise edits, as they may expand the potential for developing value-added traits which may be difficult to achieve by other gene-editing applications and/or traditional breeding alone. This chapter provides improved working protocols for isolating and transforming protoplast from immature soybean seeds with 44% of transfection efficiency validated by the green fluorescent protein reporter. We also describe a method for gene editing in soybean protoplasts using single guide RNA molecules.

Original languageEnglish (US)
Pages (from-to)173-186
Number of pages14
JournalMethods in molecular biology (Clifton, N.J.)
Volume2464
DOIs
StatePublished - 2022

Bibliographical note

Funding Information:
This work was partially supported by a grant from United Soybean Board (1920-152-0131-B) to F.Z. and R.S.

Publisher Copyright:
© 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

  • CRISPR/Cas9
  • Green fluorescent protein (GFP)
  • Immature seeds
  • Polyethylene glycol (PEG)
  • Transformation

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't

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