Moss, Oliver
- Department of Plant Breeding, Swedish University of Agricultural Sciences
Doctoral thesis2025Open access
Enhancing rapeseed seedcake quality for feed and food using CRISPR-Cas RNP gene editing
Moss, Oliver
Abstract
Rapeseed is primarily grown for oil production, but its seed meal (RSM) is a valuable by-product rich in high-quality protein with potential for high-quality feed and food applications. However, its use in animal feed is limited by antinutritional factors, including glucosinolates (GSLs), sinapine, and phytic acid (PA), which impair nutrient uptake, reduce palatability, and negatively affect animal health. Due to the lack of natural genetic variation in these traits, traditional breeding has reached a plateau, while conventional genetic modification faces regulatory restrictions. To overcome these challenges and accelerate crop improvement, protoplast-based CRISPR RNP gene editing offers a promising transgene-free approach. However, a limitation in implementing this type of CRISPR gene editing in rapeseed is the regeneration of protoplasts into plants.
In this thesis, we developed an efficient regeneration protocol for rapeseed protoplasts, enabling the application of transgene-free CRISPR gene editing to reduce the levels of antinutritional factors. By targeting key genes involved in their biosynthesis and transport, we achieved high mutation efficiency and successfully edited multiple gene copies simultaneously. Knockout of GSL transporter genes (BnGTRs) reduced GSL content by up to 64%. Mutants of sinapine biosynthesis genes (BnSGT, BnSCT, and BnREF1) showed reductions of 45%, 31%, and 73%, respectively, with BnREF1 emerging as the most effective target gene. Additionally, disruption of BnITPK1 and BnITPK4 in the PA biosynthesis pathway led to PA reductions of up to 32% and 62%, respectively. These findings establish a strong foundation for transgene-free gene editing in rapeseed and highlight its potential for enhancing RSM’s nutritional value and agricultural viability.
Keywords
Rapeseed; protoplasts; CRISPR; RNP; transgene-free; antinutritional factors; glucosinolates; sinapine; phytic acid; raps; protoplaster; transgenfri; antinutritionella faktorer; glukosinolater; sinapin; fytinsyra
Published in
Acta Universitatis Agriculturae Sueciae
2025, number: 2025:24
Publisher: Swedish University of Agricultural Sciences
SLU Authors
UKÄ Subject classification
Genetics and Breeding in Agricultural Sciences
Publication identifier
- DOI: https://doi.org/10.54612/a.1dk92laqgh
- ISBN: 978-91-8046-459-8
- eISBN: 978-91-8046-509-0
Permanent link to this page (URI)
https://res.slu.se/id/publ/132964