van Belle Jarst, Schaart Jan G, Dechesne Annemarie C, Fei Danli, Cisneros Abraham Ontiveros, Serafini Michele, Visser Richard G F, van Loo Eibertus N
Wageningen University and Research, Plant Breeding, Wageningen, The Netherlands.
Linnaeus Plant Sciences B.V., Wageningen, The Netherlands.
Plant Biotechnol J. 2025 May;23(5):1399-1412. doi: 10.1111/pbi.14593. Epub 2025 Jan 27.
Mutants with simultaneous germline mutations were obtained in all three F5H genes and all three FAD2 genes (one to eleven mutated alleles) in order to improve the feed value of the seed meal and the fatty acid composition of the seed oil. In mutants with multiple mutated F5H alleles, sinapine in seed meal was reduced by up to 100%, accompanied by a sharp reduction in the S-monolignol content of lignin without causing lodging or stem break. A lower S-lignin monomer content in stems can contribute to improved stem degradability allowing new uses of stems. Mutants in all six FAD2 alleles showed an expected increase in MUFA from 8.7% to 74% and a reduction in PUFA from 53% to 13% in the fatty acids in seed oil. Remarkably, some full FAD2 mutants showed normal growth and seed production and not the dwarfing phenotype reported in previous studies. The relation between germline mutation allele dosage and phenotype was influenced by the still ongoing activity of the CRISPR/Cas9 system, leading to new somatic mutations in the leaves of flowering plants. The correlations between the total mutation frequency (germline plus new somatic mutations) for F5H with sinapine content, and FAD2 with fatty acid composition were higher than the correlations between germline mutation count and phenotypes. This shows the importance of quantifying both the germline mutations and somatic mutations when studying CRISPR/Cas9 effects in situations where the CRISPR/Cas9 system is not yet segregated out.
为了提高种子粕的饲用价值和种子油的脂肪酸组成，在所有三个F5H基因和所有三个FAD2基因中都获得了具有同时种系突变的突变体（一到十一个突变等位基因）。在具有多个突变F5H等位基因的突变体中，种子粕中的芥子碱含量降低了高达100%，同时木质素的S-单木质醇含量急剧降低，而不会导致倒伏或茎杆折断。茎中较低的S-木质素单体含量有助于提高茎的降解性，从而使茎有新的用途。所有六个FAD2等位基因的突变体在种子油脂肪酸中显示出预期的单不饱和脂肪酸（MUFA）从8.7%增加到74%，多不饱和脂肪酸（PUFA）从53%减少到13%。值得注意的是，一些完全FAD2突变体表现出正常生长和种子生产，而不是先前研究中报道的矮化表型。种系突变等位基因剂量与表型之间的关系受到CRISPR/Cas9系统仍在进行的活性影响，导致开花植物叶片中出现新的体细胞突变。F5H的总突变频率（种系突变加上新的体细胞突变）与芥子碱含量之间以及FAD2与脂肪酸组成之间的相关性高于种系突变计数与表型之间的相关性。这表明在CRISPR/Cas9系统尚未分离的情况下研究CRISPR/Cas9效应时，量化种系突变和体细胞突变的重要性。
