Industrial Scale Gene Editing in <i>Brassica napus</i>

Abstract

In plants, an increasing number of traits and new characteristics are being developed using gene editing. Simple traits represented by a single gene can be managed through backcross breeding, but this is typically not the case for more complex traits which may result from the function of a large number of genes. Here, we demonstrate two case studies of improving oleic oil content and developing pod shatter reduction in Brassica napus by using gene editing tools on an industrial scale. There are four BnaFAD2 genes involved in oleic oil content and eight BnaSHP genes involved in pod shatter tolerance. In order to develop these two traits, we delivered nuclease ribonucleoproteins with Gene Repair OligoNucleotides (GRONs) into protoplasts, with subsequent regeneration into plants on an industrial scale, which encompassed robust tissue culture protocols, efficient gene editing, robotics sampling, and molecular screening, vigorous plant regeneration, growth, and phenotyping. We can produce precise loss-of-function-edited plants with two improved agronomically important complex traits, high oleic oil or pod shatter reduction, in elite canola varieties within 1–3 years, depending on the trait complexity. In the edited plants carrying loss of function of four BnaFAD2 genes, the seed fatty acid oleic acid content reached 89% compared to 61% in the non-edited wildtype control. The plants carrying eight edited BnaSHP genes achieved 51% pod shatter reduction in multiple year field testing in the target environment compared to the wildtype control.

Keywords

• canola
• <i>Brassica napus</i>
• gene editing
• multiplex gene editing
• non-transgenic gene editing
• pod shatter