Development of a Transformation System and Locus Identification Pipeline for T-DNA in Chrysanthemum seticuspe, A Model Species for Hexaploid Cultivated Chrysanthemum

Chrysanthemum is one of the most popular flowers worldwide and has high aesthetic and commercial value. However, the cultivated varieties of chrysanthemum are hexaploid and highly heterozygous, which makes gene editing and gene function research difficult. Gojo-0 is a diploid homozygous line bred from a self-compatible mutant of Chrysanthemum seticuspe and is expected to become a model plant of the genus Chrysanthemum. After assessment of different growth regulator combinations, the optimal concentrations of alpha-naphthaleneacetic acid (NAA) and 6-benzyladenine (6-BA) in the regeneration system were 1.0 mg center dot L-1 and 0.2 mg center dot L-1, respectively. In the genetic transformation system, the selected concentrations of kanamycin, hygromycin and glufosinate-ammonium were 10 mg center dot L-1, 2.5 mg center dot L--(1) and 0.6 mg center dot L-1 for bud generation and 12 mg L-1, 1.5 mg center dot L-1 and 0.5 mg center dot L-1 for rooting. The transgenic plants were verified by not only PCR detection and GUS staining, but also identification of the T-DNA insertion locus using high-throughput sequencing. Our results lay the foundation for gene functional research on chrysanthemum and will help with the identification of transgenic plants.