Generation of Gene-Edited Chrysanthemum morifolium Using Multi-Copy Transgenes as Targets and Markers

Abstract

The most widely used gene editing technology-the CRISPR/Cas9 system-employs a bacterial monomeric DNA endonuclease known as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) and single-guide RNA (sgRNA) that directs Cas9 to a complementary target DNA. However, introducing mutations into higher polyploid plant species, especially for species without genome information, has been difficult. Chrysanthemum morifolium (chrysanthemum) is one of the most important ornamental plants, but it is a hexaploid with a large genome; moreover, it lacks whole-genome information. These characteristics hinder genome editing in chrysanthemum. In the present study, we attempted to perform gene editing using the CRISPR/Cas9 system to introduce mutations into chrysanthemum. We constructed transgenic chrysanthemum plants expressing the yellowish-green fluorescent protein gene from Chiridius poppei (CpYGFP) and targeted CpYGFP for gene editing. We compared the activity of a Cauliflower mosaic virus (CaMV) 35S promoter and parsley ubiquitin promoter in chrysanthemum calli and chose the parsley ubiquitin promoter to drive Cas9. We selected two sgRNAs to target different positions in the CpYGFP gene and obtained transgenic calli containing mutated CpYGFP genes (CRISPR-CpYGFP-chrysanthemum). A DNA sequencing analysis and fluorescence observations indicated that cells containing the mutated CpYGFP gene grew independently of cells containing the original CpYGFP gene in one callus. We finally obtained the CRISPR-CpYGFP-chrysanthemum shoot containing a mutation in the CpYGFP sequence. This is the first report of gene editing using the CRISPR/Cas9 system in chrysanthemum and sheds light on chrysanthemum genome editing.

Application of CRISPR/Cas9 to Tragopogon (Asteraceae), an evolutionary model for the study of polyploidy

“…In contrast to gene knockdown triggered by RNAi and VIGS, gene knockout can be achieved by CRISPR/Cas9. Along with a few CRISPR/Cas9 applications in species of Asteraceae (Kishi-Kaboshi et al, 2017;Woo et al, 2015), our study demonstrates the potential of using CRISPR/Cas9 to uncover the genetic mechanisms underlying Asteraceae inflorescence development, as well as other morphological or process-oriented features of this large and economically and ecologically important family of flowering plants.…”

“…Albino calli and shoots were generated in both species. This appears to be the first demonstration of CRISPR/Cas9 in a naturally formed neopolyploid system, and only the third case in species of Asteraceae following Lactuca sativa (Bertier, Ron, Huo, Bradford, & Michelmore, ; Woo et al, ) and Chrysanthemum morifolium (Kishi‐Kaboshi, Aida, & Sasaki, ). This work illustrates the potential of applying CRISPR/Cas9 technology in studies of evolutionary and functional biology and provides a guide for others working on nongenetic model systems.…”

Parsley <i>ubiquitin</i> promoter displays higher activity than the CaMV 35S promoter and the chrysanthemum <i>actin 2</i> promoter for productive, constitutive, and durable expression of a transgene in <i>Chrysanthemum morifolium</i>

“…To our knowledge, with the exception of our analysis using chrysanthemum calli (Kishi-Kaboshi et al 2017), there has only been one other previously published report, a patent application, that describes PcUbi promoter activity in plants (Plesch and Ebneth 2011). In this patent application, the PcUbi promoter was shown to lead transgene expression in buds, roots, stems, flowers, and seeds of transgenic A. thaliana, based on histochemical analysis (Plesch and Ebneth 2011).…”

“…Furthermore, genome editing has become the latest approach to breeding technology for many plant species (Kumar and Jain 2015), and could be applied to improve several traits in chrysanthemums. To obtain information about gene function in chrysanthemums, and to we also analyzed the activity of the parsley ubiquitin (PcUbi) promoter in plant organs, which exhibited greater activity than the 35S promoter in calli (Kishi-Kaboshi et al 2017). The PcUbi promoter was shown to lead transgene expression in buds, roots, stems, flowers, and seeds of transgenic A. thaliana (Plesch and Ebneth 2011), but its activity in chrysanthemum plants has not been analyzed.…”

Sustainability Challenges and Innovations in the Value Chain of Flowering Potted Plants for the German Market

“…Up to now, GM technologies were considered too costly for the floricultural industry [61]. However, the emergence of new and cheaper GM technologies, which was already implemented in different ornamental crops offers new prospects [62,63]. Adopting GM technologies might shorten the breeding process and eventually reduce the overall costs of the development of novelties.…”