Agrobacterium-mediated transformation and gene editing of kohlrabi (Brassica oleracea var. gongylodes)
摘要
Brassica oleracea L. var. gongylodes (kohlrabi) is an economically important vegetable. Due to the complicated in vitro regeneration, this plant has never been subjected to genetic transformation of any kind. Therefore, developing appropriate methods to improve the genome of this variety remains a challenge. A CRISPR/Cas9 genetic construct with three gRNAs targeting the BoCAPRICE (CPC) gene, which encodes a transcription factor with an R3 MYB motif, has been introduced to Agrobacterium tumefaciens to produce knockout mutants of kohlrabi. Existing methods for transforming other B. oleracea varieties were not effective for any of the six kohlrabi cultivars tested; therefore, we developed a new protocol. Addition of 5 mg L−1 AgNO3 to the culture medium reduced browning after inoculation, and a limited period of herbicide application increased the efficiency of shoot elongation up to four times and allowed successful rooting. A total of five glufosinate-resistant shoots were obtained from 250 explants of Vienna White, two from 250 explants of Gulliver, one from 250 explants of Delicacy White, and none from the other three cultivars. Of all the shoots, only three of the cultivar Vienna White were PCR-positive for the Cas gene. In two of the three, the expression level of the BoCPC gene was significantly reduced. A 547-bp deletion was detected in one plant, but it was either heterozygous or chimeric. The described protocol opens prospects for generating new kohlrabi cultivars with improved agricultural traits. Our results support the use of Vienna White as a model cultivar for kohlrabi.