Production of oilseed rape with increased seed shattering resistance

Rapeseed (Brassica napus) production is limited by the crop’s natural propagation mechanism which involves growing siliques that dry out upon maturity and break easily. The resulting pre-harvest yield loss makes shatter resistance an important breeding goal. Studies on Arabidopsis thaliana revealed a set of transcription factors controlling dehiscence zone establishment. INDEHISCENT (IND) and ALCATRAZ (ALC) are major regulators of tissue differentiation in the critical parts of the silique. While ALC is required for the development of a partially degraded separation layer, IND also regulates the essential lignification of neighboring cells, probably through induction of NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1 and 3 (NST1/3) expression. This study aimed at producing rapeseed lines with robust siliques through the use of Bnalc, Bnind, and Bnnst1 mutations. CRISPR/Cas9-mediated gene editing of the two BnALC homoeologs of cultivar ‘Haydn’ efficiently yielded four mutant alleles in a single transgenic T1 plant which were stably inherited. A tensile force test suggested the increased shatter resistance of T2 double mutants. However, the effect was masked by the innate silique robustness of ‘Haydn’. The Bnalc phenotype was therefore confirmed with EMS-induced mutant alleles in the shatter-prone cultivar ‘Express’. Bnind mutations derived from the same ‘Express’ mutant population were utilized for detailed analyses of shatter mechanics. Three phenotyping tests consistently identified a double mutant with especially robust siliques. No lignification defects were observed. Instead, shatter resistance was accounted to a broader replum-valve joint area in combination with smaller cells therein. CRISPR/Cas9-induced mutagenesis of four BnNST1 homoeologs yielded a chimeric T1 plant with multiple mutant alleles per gene copy which now have to be fixed in the progeny. Altogether, the developed mutant material provides novel variation for shatter resistance breeding.


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