Sjur Sandgrind
Research Scientist
Authors
Oda Eline Sandmo Ånesland Eline Seim Magne Nordang Skårn Arti Rai Sjur Sandgrind May Bente Brurberg Tage ThorstensenAbstract
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Abstract
Brassica carinata is an important oil crop with significant potential for food and industrial production. The application of the CRISPR/Cas9 genome editing tool in B. c arinata could accelerate its breeding cycle. However, no efficient DNA-free gene editing method currently exists for this species. Protoplast-based CRISPR editing presents a promising solution, though it is often challenging for many crop species. In this study, we investigated several critical factors influencing in vitro shoot regeneration, including genotype, sugar type, selection and combination of plant growth regulators (PGRs), and culture duration on different media throughout various stages of protoplast development. As a result, we developed a highly efficient, five-stage protoplast regeneration protocol for B. carinata based on specific stages of protoplast development. Key findings of this study include the requirement for high concentrations of NAA and 2,4-D in the initial medium (MI) for cell wall formation, while a lower auxin concentration relative to cytokinin was necessary for active cell division (MII). For callus growth and shoot induction, a high cytokinin-to-auxin ratio was essential (MIII), and an even higher cytokinin-to-auxin ratio was optimal for shoot regeneration (MIV). For shoot elongation, low levels of BAP and GA 3 were sufficient (MV). Our results also demonstrated that the duration of culture on different media and maintaining appropriate osmotic pressure at the early stages were crucial for successful protoplast regeneration. With this optimized protocol, we achieved an average regeneration frequency of up to 64% and a transfection efficiency of 40% using the GFP marker gene. This efficient protoplast regeneration protocol is now being employed for genome editing in our lab and is expected to significantly enhance the application of the CRISPR system in both basic research and the genetic improvement of B. carinata over the long term.
Authors
Sjur Sandgrind Xueyuan Li Emelie Ivarson Eu Sheng Wang Rui Guan Selvaraju Kanagarajan Li-Hua ZhuAbstract
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.