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Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)

Sandgrind, Sjur and Li, Xue-Yuan and Ivarson, Emelie and Ahlman, Annelie and Zhu, Li-Hua (2021). Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre). Frontiers in Genome Editing. 3 , 757540
[Research article]

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Abstract

Field cress (Lepidium campestre) is a potential oilseed crop that has been under domestication in recent decades. CRISPR/Cas9 is a powerful tool for rapid trait improvement and gene characterization and for generating transgene-free mutants using protoplast transfection system. However, protoplast regeneration remains challenging for many plant species. Here we report an efficient protoplast regeneration and transfection protocol for field cress. Important factors such as type of basal media, type/combination of plant growth regulators, and culture duration on different media were optimized. Among the basal media tested, Nitsch was the best for protoplast growth in MI and MII media. For cell wall formation during the early stage of protoplast growth, relatively high auxin concentrations (0.5 mg L−1 NAA and 2,4-D), without addition of cytokinin was preferred for maintaining protoplast viability. After cell wall formation, 1.1 mg L−1 TDZ combined with either 0.05 mg L−1 NAA or 2,4-D was found to efficiently promote protoplast growth. On solid shoot induction medium, 1.1 mg L−1 TDZ without any auxin resulted in over 80% shoot generation frequency. A longer culture duration in MI medium would inhibit protoplast growth, while a longer culture duration in MII medium significantly delayed shoot formation. Using this optimized protoplast regeneration protocol, we have established an efficient PEG-mediated transfection protocol using a vector harboring the GFP gene, with transfection efficiencies of 50–80%. This efficient protoplast protocol would facilitate further genetic improvement of field cress via genome editing, and be beneficial to development of protoplast regeneration protocols for related plant species.

Authors/Creators:Sandgrind, Sjur and Li, Xue-Yuan and Ivarson, Emelie and Ahlman, Annelie and Zhu, Li-Hua
Title:Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)
Series Name/Journal:Frontiers in Genome Editing
Year of publishing :2021
Volume:3
Article number:757540
Number of Pages:9
Language:English
Publication Type:Research article
Article category:Scientific peer reviewed
Version:Published version
Copyright:Creative Commons: Attribution 4.0
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 1 Natural sciences > 106 Biological Sciences (Medical to be 3 and Agricultural to be 4) > Genetics (medical genetics to be 30107 and agricultural genetics to be 40402)
Keywords:Braccicaceae, CRISPR/Cas9, oilseed crop, domestication, protoplast regeneration, transfection
URN:NBN:urn:nbn:se:slu:epsilon-p-114730
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-114730
Additional ID:
Type of IDID
DOI10.3389/fgeed.2021.757540
ID Code:26825
Faculty:LTV - Fakulteten för landskapsarkitektur, trädgårds- och växtproduktionsvetenskap
Department:(LTJ, LTV) > Department of Plant Breeding (from 130101)
Deposited By: SLUpub Connector
Deposited On:26 Jan 2022 10:11
Metadata Last Modified:26 Jan 2022 10:21

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