Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (2): 80-87.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0823

Previous Articles     Next Articles

Targeted Editing of BoZDS in Broccoli by CRISPR/Cas9 Technology

HUANG Wen-li1(), LI Xiang-xiang1, ZHOU Wen-ting1, LUO Sha1, YAO Wei-jia1, MA Jie1,2, ZHANG Fen1, SHEN Yu-sen3, GU Hong-hui3, WANG Jian-sheng3, SUN Bo1()   

  1. 1. College of Horticulture, Sichuan Agricultural University, Chengdu 611130
    2. Bijie Institute of Agricultural Science, Bijie 551700
    3. Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021
  • Received:2022-07-04 Online:2023-02-26 Published:2023-03-07

Abstract:

ζ-Carotene desaturase(ZDS)is one of the key enzymes regulating carotenoids biosynthesis. Here having the high-generation inbred broccoli(Brassica oleracea var. italica)line ‘ZN09’ as testing materials and BoZDS as target gene, then two target sites on the first exon of BoZDS were selected to construct CRISPR/Cas9 vector for stable genetic transformation. For the target site 1, the transformation efficiency and the mutation rate were 0.80% and 15.79%, respectively, and three heterozygous mutants were generated. For the target site 2, the transformation efficiency was 0.84%, and the mutation rate was 36.84%, and seven mutants were generated, including two homozygous, two heterozygous, and three chimeric mutants. All of the mutant plants showed albino or mottled phenotype. The L* and a* values of mutant plants were significantly higher than those of wild-type plants, while the b* value decreased. To sum up, a stable genetic system for CRISPR/Cas9-mediated gene editing in broccoli was established, and BoZDS was effectively edited. Our results provide the technical support for gene function study and germplasm innovation of broccoli by using gene editing technology.

Key words: broccoli, CRISPR/Cas9, gene editing, stable genetic transformation, ζ-carotene desaturase(ZDS)