Home About Browse Search
Svenska


Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)

Li, Xue-Yuan and Sandgrind, Sjur and Moss, Oliver and Guan, Rui and Ivarson, Emelie and Wang, Eu Sheng and Kanagarajan, Selvaraju and Zhu, Li-Hua (2021). Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.). Frontiers in Plant Science. 12 , 680859
[Research article]

[img] PDF
2MB

Abstract

Difficulty in protoplast regeneration is a major obstacle to apply the CRISPR/Cas9 gene editing technique effectively in research and breeding of rapeseed (Brassica napus L.). The present study describes for the first time a rapid and efficient protocol for the isolation, regeneration and transfection of protoplasts of rapeseed cv. Kumily, and its application in gene editing. Protoplasts isolated from leaves of 3-4 weeks old were cultured in MI and MII liquid media for cell wall formation and cell division, followed by subculture on shoot induction medium and shoot regeneration medium for shoot production. Different basal media, types and combinations of plant growth regulators, and protoplast culture duration on each type of media were investigated in relation to protoplast regeneration. The results showed that relatively high concentrations of NAA (0.5 mg l(-1)) and 2,4-D (0.5 mg l(-1)) in the MI medium were essential for protoplasts to form cell walls and maintain cell divisions, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin were required, and among all the combinations tested, 2.2 mg l(-1) TDZ in combination with auxin 0.5 mg l(-1) NAA gave the best result with up to 45% shoot regeneration. Our results also showed the duration of protoplast culture on different media was critical, as longer culture durations would significantly reduce the shoot regeneration frequency. In addition, we have optimized the transfection protocol for rapeseed. Using this optimized protocol, we have successfully edited the BnGTR genes controlling glucosinolate transport in rapeseed with a high mutation frequency.

Authors/Creators:Li, Xue-Yuan and Sandgrind, Sjur and Moss, Oliver and Guan, Rui and Ivarson, Emelie and Wang, Eu Sheng and Kanagarajan, Selvaraju and Zhu, Li-Hua
Title:Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)
Series Name/Journal:Frontiers in Plant Science
Year of publishing :2021
Volume:12
Article number:680859
Number of Pages:11
Publisher:FRONTIERS MEDIA SA
ISSN:1664-462X
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:Brassica napus, CRISPR, Cas9, gene editing, glucosinolate transporter, GTR gene, protoplast regeneration
URN:NBN:urn:nbn:se:slu:epsilon-p-113177
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-113177
Additional ID:
Type of IDID
DOI10.3389/fpls.2021.680859
Web of Science (WoS)000674999100001
ID Code:25458
Faculty:LTV - Fakulteten för landskapsarkitektur, trädgårds- och växtproduktionsvetenskap
Department:(LTJ, LTV) > Department of Plant Breeding (from 130101)
Deposited By: SLUpub Connector
Deposited On:20 Sep 2021 12:25
Metadata Last Modified:20 Sep 2021 12:31

Repository Staff Only: item control page

Downloads

Downloads per year (since September 2012)

View more statistics

Downloads
Hits