Establishment of regeneration, transformation, and genome editing procedures for a seed-propagated carnation (Dianthus caryophyllus L.) variety

Abstract Carnations ( Dianthus caryophyllus L.) are amongst the three most commercially valuable cut flowers worldwide. However, traditional breeding methods are often time-consuming and labor-intensive. Although genome editing is used as an alternative method for creating new varieties, the high heterozygosity of carnations inhibits the ability to maintain varietal characteristics in null segregants except for target-derived traits. The use of homozygous lines is a possible solution. Therefore, this study aimed to establish regeneration, transformation, and genome editing methods using seed-carnation varieties. The effects of four auxins (indole-3-butyric acid, IBA; a-naphthaleneacetic acid, NAA; 2,4-dichlorophenoxyacetic acid, 2,4-D; and 3-indoleacetic acid, IAA) and five cytokinins (6-benzyladenine, BA; thidiazuron, TDZ; kinetin, KT; zeatin, ZT; and N 6 -2-isopentenyl adenine, 2IP) on callus and shoot induction were evaluated. The combination of 0.05 mg/l 2,4-D and 4 mg/l TDZ had the highest shoot formation rate at 28%. In addition, shoot hyperhydricity was reduced by increasing the size of culture vessels. Sucrose, agar, and AgNO 3 concentrations, as well as pH, were optimized to facilitate regeneration. Hygromycin at 12.5 mg/l was subsequently used as the selection agent after Agrobacterium -mediated transformation. Finally, the phytoene desaturase gene was knocked out using the CRISPR/Cas9 system. The obtained albino shoot had a one-base deletion or two-base insertion in the genome sequence. To our knowledge, this is the first study to establish a system for genome editing of callus-derived shoots from a homozygous seed-propagated carnation, which may contribute to the rapid breeding of the new varieties.