Enhancing Erucic Acid and Wax Ester Production in Brassica carinata through Metabolic Engineering for Industrial Applications.

Metabolic engineering enables oilseed crops to be more competitive by having more attractive properties for oleochemical industrial applications. The aim of this study was to increase the erucic acid level and to produce wax ester (WE) in seed oil by genetic transformation to enhance the industrial applications of B. carinata. Six transgenic lines for high erucic acid and fifteen transgenic lines for wax esters were obtained. The integration of the target genes for high erucic acid (BnFAE1 and LdPLAAT) and for WEs (ScWS and ScFAR) in the genome of B. carinata cv. 'Derash' was confirmed by PCR analysis. The qRT-PCR results showed overexpression of BnFAE1 and LdPLAAT and downregulation of RNAi-BcFAD2 in the seeds of the transgenic lines. The fatty acid profile and WE content and profile in the seed oil of the transgenic lines and wild type grown in biotron were analyzed using gas chromatography and nanoelectrospray coupled with tandem mass spectrometry. A significant increase in erucic acid was observed in some transgenic lines ranging from 19% to 29% in relation to the wild type, with a level of erucic acid reaching up to 52.7%. Likewise, the transgenic lines harboring ScFAR and ScWS genes produced up to 25% WE content, and the most abundant WE species were 22:1/20:1 and 22:1/22:1. This study demonstrated that metabolic engineering is an effective biotechnological approach for developing B. carinata into an industrial crop.

Improved fatty acid composition of field cress (Lepidium campestre) by CRISPR/Cas9-mediated genome editing.

The wild species field cress (Lepidium campestre) has the potential to become a novel cover and oilseed crop for the Nordic climate. Its seed oil is however currently unsuitable for most food, feed, and industrial applications, due to the high contents of polyunsaturated fatty acids (PUFAs) and erucic acid (C22:1). As the biosynthesis of these undesirable fatty acids is controlled by a few well-known major dominant genes, knockout of these genes using CRISPR/Cas9 would thus be more effective in improving the seed oil quality. In order to increase the level of the desirable oleic acid (C18:1), and reduce the contents of PUFAs and C22:1, we targeted three important genes FATTY ACID ELONGASE1 (FAE1), FATTY ACID DESATURASE2 (FAD2), and REDUCED OLEATE DESATURASE1 (ROD1) using a protoplast-based CRISPR/Cas9 gene knockout system. By knocking out FAE1, we obtained a mutated line with almost no C22:1, but an increase in C18:1 to 30% compared with 13% in the wild type. Knocking out ROD1 resulted in an increase of C18:1 to 23%, and a moderate, but significant, reduction of PUFAs. Knockout of FAD2, in combination with heterozygous FAE1fae1 genotype, resulted in mutated lines with up to 66% C18:1, very low contents of PUFAs, and a significant reduction of C22:1. Our results clearly show the potential of CRISPR/Cas9 for rapid trait improvement of field cress which would speed up its domestication process. The mutated lines produced in this study can be used for further breeding to develop field cress into a viable crop.

Corrigendum: Improved fatty acid composition of field cress (Lepidium campestre) by CRISPR/Cas9-mediated genome editing.

[This corrects the article DOI: 10.3389/fpls.2023.1076704.].

Corrigendum: Efficient protoplast regeneration protocol and CRISPR/Cas9-mediated editing of glucosinolate transporter (GTR) genes in rapeseed (Brassica napus L.).

[This corrects the article DOI: 10.3389/fpls.2021.680859.].

Mutations introduced in susceptibility genes through CRISPR/Cas9 genome editing confer increased late blight resistance in potatoes.

The use of pathogen-resistant cultivars is expected to increase yield and decrease fungicide use in agriculture. However, in potato breeding, increased resistance obtained via resistance genes (R-genes) is hampered because R-gene(s) are often specific for a pathogen race and can be quickly overcome by the evolution of the pathogen. In parallel, susceptibility genes (S-genes) are important for pathogenesis, and loss of S-gene function confers increased resistance in several plants, such as rice, wheat, citrus and tomatoes. In this article, we present the mutation and screening of seven putative S-genes in potatoes, including two DMR6 potato homologues. Using a CRISPR/Cas9 system, which conferred co-expression of two guide RNAs, tetra-allelic deletion mutants were generated and resistance against late blight was assayed in the plants. Functional knockouts of StDND1, StCHL1, and DMG400000582 (StDMR6-1) generated potatoes with increased resistance against late blight. Plants mutated in StDND1 showed pleiotropic effects, whereas StDMR6-1 and StCHL1 mutated plants did not exhibit any growth phenotype, making them good candidates for further agricultural studies. Additionally, we showed that DMG401026923 (here denoted StDMR6-2) knockout mutants did not demonstrate any increased late blight resistance, but exhibited a growth phenotype, indicating that StDMR6-1 and StDMR6-2 have different functions. To the best of our knowledge, this is the first report on the mutation and screening of putative S-genes in potatoes, including two DMR6 potato homologues.

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

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.

Tissue Culture and Refreshment Techniques for Improvement of Transformation in Local Tetraploid and Diploid Potato with Late Blight Resistance as an Example.

Potato (Solanum tuberosum) is among the best producers of edible biomass in terms of yield per hectare and a variety of different regional cultivars are used as a staple commodity in many countries. However, this crop is attacked by several diseases, with the worst being the late blight disease caused by Phytophthora infestans. Stacking of resistance (R) genes from wild Solanum relatives are interesting prospects for the sustainable control of late blight. Therefore, we optimized methods for the efficient generation and screening of R-gene-containing transformants in tetraploid and diploid hybrid potato genotypes. Using these methods, a high transformation efficiency was achieved for the transformation of tetraploid and diploid potato lines with a triple resistance (3R) gene construct. Transformation efficiencies were improved by optimizing several factors affecting regeneration, including the quality of the starting plant material, and the composition of the plant growth regulators used during selective regeneration. A refreshment protocol was designed to alleviate in vitro related stress in stock plants, which significantly improved the growth vigor and resulted in a 4- to 10-fold increase in transformation efficiency. Furthermore, long-term exposure to exogenous Indole-3-butyric acid that is usually used for the initiation of roots in vitro, was found to cause aberrant morphological phenotypes in potato.