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

Xueyuan Li,Oliver Moss,Emelie Ivarson,Sjur Sandgrind,Li-Hua Zhu,Eu Sheng Wang,Rui Guan,Selvaraju Kanagarajan

doi:10.3389/fpls.2021.680859

Xueyuan Li, Oliver Moss + Show 6 more

Open Access

https://doi.org/10.3389/fpls.2021.680859

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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.

Highlights

The CRISPR/Cas9 technology has become a prevailing tool for plant genome editing owing to its high precision, efficiency and simplicity in use (Arora and Narula, 2017)
We tested several plant growth regulators (PGRs) combinations in MI medium, and found that the combination of 0.5 mg l−1 2,4-D and 0.5 mg l−1 NAA gave the best result in terms of protoplast viability among all PGR combinations tested
Our results showed that addition of cytokinin, like TDZ, BAP or zeatin, in combination with auxin in MI medium did not improve protoplast viability or growth compared with auxin alone

Summary

Introduction

The CRISPR/Cas technology has become a prevailing tool for plant genome editing owing to its high precision, efficiency and simplicity in use (Arora and Narula, 2017). As stable transformation of plants normally results in regeneration of mutation lines with integration of foreign DNA into the plant genome, this gene editing system raise regulatory concerns related to genetically modified plants in some countries (Woo et al, 2015). Application of the protoplast approach for gene editing in crop species reported so far were mainly for research purpose (Nicolia et al, 2015; Woo et al, 2015; Malnoy et al, 2016; Kim et al, 2017; Liang et al, 2017; Lin et al, 2018), while in most cases no protoplast regeneration results were reported. Development of an efficient and reliable protoplast regeneration method is essential for the application of all currently available CRISPR gene editing systems for directly producing transgene-free mutants for many plant species

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