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

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 r...

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Autores principales:	Sjur Sandgrind, Xueyuan Li, Emelie Ivarson, Annelie Ahlman, Li-Hua Zhu
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	Braccicaceae CRISPR/Cas9 oilseed crop domestication protoplast regeneration transfection Biotechnology TP248.13-248.65 Genetics QH426-470
Acceso en línea:	https://doaj.org/article/1a0048b3836246f5be87b31bb656dcb4
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spelling	oai:doaj.org-article:1a0048b3836246f5be87b31bb656dcb42021-11-16T05:47:02ZEstablishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)2673-343910.3389/fgeed.2021.757540https://doaj.org/article/1a0048b3836246f5be87b31bb656dcb42021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fgeed.2021.757540/fullhttps://doaj.org/toc/2673-3439Field 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.Sjur SandgrindXueyuan LiEmelie IvarsonAnnelie AhlmanLi-Hua ZhuFrontiers Media S.A.articleBraccicaceaeCRISPR/Cas9oilseed cropdomesticationprotoplast regenerationtransfectionBiotechnologyTP248.13-248.65GeneticsQH426-470ENFrontiers in Genome Editing, Vol 3 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Braccicaceae CRISPR/Cas9 oilseed crop domestication protoplast regeneration transfection Biotechnology TP248.13-248.65 Genetics QH426-470
spellingShingle	Braccicaceae CRISPR/Cas9 oilseed crop domestication protoplast regeneration transfection Biotechnology TP248.13-248.65 Genetics QH426-470 Sjur Sandgrind Xueyuan Li Emelie Ivarson Annelie Ahlman Li-Hua Zhu Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)
description	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.
format	article
author	Sjur Sandgrind Xueyuan Li Emelie Ivarson Annelie Ahlman Li-Hua Zhu
author_facet	Sjur Sandgrind Xueyuan Li Emelie Ivarson Annelie Ahlman Li-Hua Zhu
author_sort	Sjur Sandgrind
title	Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)
title_short	Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)
title_full	Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)
title_fullStr	Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)
title_full_unstemmed	Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)
title_sort	establishment of an efficient protoplast regeneration and transfection protocol for field cress (lepidium campestre)
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/1a0048b3836246f5be87b31bb656dcb4
work_keys_str_mv	AT sjursandgrind establishmentofanefficientprotoplastregenerationandtransfectionprotocolforfieldcresslepidiumcampestre AT xueyuanli establishmentofanefficientprotoplastregenerationandtransfectionprotocolforfieldcresslepidiumcampestre AT emelieivarson establishmentofanefficientprotoplastregenerationandtransfectionprotocolforfieldcresslepidiumcampestre AT annelieahlman establishmentofanefficientprotoplastregenerationandtransfectionprotocolforfieldcresslepidiumcampestre AT lihuazhu establishmentofanefficientprotoplastregenerationandtransfectionprotocolforfieldcresslepidiumcampestre
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Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)

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