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

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.

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

“…Protoplasts from different species, genotypes, and different tissues, may require different culture conditions for successful regeneration. Some of the critical parameters include protoplast isolation method, medium composition, culture duration, and callus development phase suitable for shoot induction, which has been shown to be critical for successful protoplast regeneration in our earlier report on rapeseed ( Li et al, 2021 ). To obtain a high regeneration frequency it is often necessary to optimize the abovementioned important parameters and other culture conditions, which is very time- and labor-intensive.…”

“…Polyethylene glycol (PEG)-mediated transfection is an alternative and effective approach to deliver CRISPR/Cas9 vectors or ribonucleoprotein complexes into protoplasts, which enables generation of transgene-free mutated lines ( Woo et al, 2015 ). The CRISPR/Cas9 protoplast transfection system has been used successfully to edit genes in several plant species ( Kim et al, 2017 ; Liang et al, 2017 ; Lin et al, 2018 ; González et al, 2020 ; Li et al, 2021 ), and the protoplast transfection system has also been successfully used for gene editing in plants by base editors ( Molla et al, 2020 ) or prime editing ( Lin et al, 2020 ). However, as protoplast regeneration remains a major obstacle for obtaining mutated lines for most plant species, the method has mainly been used to evaluate mutation efficiencies of sgRNAs of target genes, not for trait improvement in general.…”

Targeted Gene Knockouts by Protoplast Transformation in the Moss Physcomitrella patens

“…Instead, the protoplast transformation of plasmids that transiently express in plant cells will generate transgene-free knockout mutants. Although protoplast transformation and regeneration are a bottleneck and are currently developing in many plant species (Hsu et al, 2021;Li et al, 2021), PEG-mediated protoplast transformation has been widely used in P. patens study for gene targeting, including steps of cell wall degradation, protoplast resuspension, plasmid transformation, and protoplast regeneration and selection of knockouts on growth medium (Schaefer et al, 1991;Schaefer, 2001;Cove et al, 2009a). The same protocol can be utilized in the CRISPR-Cas9 system, except for different vectors for transformation (Lopez-Obando et al, 2016;Collonnier et al, 2017;Radin et al, 2021).…”

Invited Mini-Review Research Topic: Utilization of Protoplasts to Facilitate Gene Editing in Plants: Schemes for In Vitro Shoot Regeneration From Tissues and Protoplasts of Potato and Rapeseed: Implications of Bioengineering Such as Gene Editing of Broad-Leaved Plants

“…CRISPR/Cas delivery via CRISPR vectors or DNA-free ribonucleoprotein (RNP) complexes into plant cells is based on tissue culture for most plant species. The ability to induce shoot formation in vitro is highly variable among the species, genotypes, and accessions which are largely genetically based and constitutes a key limiting step for successful gene editing ( Zhu and Welander, 2000 ; Zhu et al, 2001 ; Li et al, 2009 ; Burbulis et al, 2010 ; Ivarson et al, 2013 ; Farooq et al, 2019 ; Bidabadi and Jain, 2020 ; Li et al, 2021 ). Broad-leaved plants are generally easier to regenerate and, thus, relatively more amendable for bioengineering than for example cereals.…”

“…Callus formation and shoot generation are for many plant species two crucial and limiting steps in shoot generation. Many factors influence callus formation and shoot induction and formation, including genotype, explant age and type, medium composition (nutrients, type and concentration of sugars, gelling agent, type and combination of PGRs, and additional additives, e.g., silver nitrate ( Pawlicki and Welander, 1994 ; Akasaka-Kennedy et al, 2005 ; Ben Ghnaya et al, 2008 ; Li et al, 2011 ; Roh et al, 2012 ; Li et al, 2013 ; Li et al, 2021 ). The cultural conditions such as temperature, duration, illumination quantity, quality could also have a significant effect on shoot regeneration ( Akasaka-Kennedy et al, 2005 ; Afshari et al, 2011 ; Bidabadi and Jain, 2020 ).…”