Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale

Abstract

Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses. Dendrobium officinale is a special orchid species that can grow without seed vernalization. Because the whole-genome sequence of D. officinale is publicly available, this species is poised to become a convenient research model for the evolutionary, developmental, and genetic studies of Orchidaceae. Despite these advantages, the methods of genetic manipulation are poorly developed in D. officinale. In this study, based on the previously developed Agrobacterium-mediated gene transformation system, we identified several highly efficient promoters for exogenous gene expression and successfully applied the CRISPR/Cas9 system for editing endogenous genes in the genome of D. officinale. These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and the β-glucuronidase (GUS) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by the Agrobacterium-mediated transformation system. Fluorescence emission from the transformed plants confirmed the successful transcription and translation of SG genes into functional proteins. We compared the GUS activity under different promoters including four commonly used promoters (MtHP, CVMV, MMV and PCISV) with CaMV 35S promoter and found that MMV, CVMV, and PCISV were as effective as the 35S promoter. Furthermore, we applied the CRISPR/Cas9-mediated genome editing system successfully in D. officinale. By selecting five target genes (C3H, C4H, 4CL, CCR, and IRX) in the lignocellulose biosynthesis pathway, we showed that, for a given target, this system can generate edits (insertions, deletions, or substitutions) at a rate of 10 to 100%. These results showed that our two genetic manipulation tools can efficiently express exogenous genes and edit endogenous genes in D. officinale. These efficient research tools will not only help create novel D. officinale varieties, but will also facilitate the molecular genetic investigation of orchid biology.

Application of CRISPR/Cas9 genome editing system for molecular breeding of orchids

“…In order to make the CRISPR/Cas9 constructs, two DNA oligonucleotides were chemically synthesized for each gRNA. The two oligonucleotides were complemen tary for the corresponding sequence to the spacer, and the sequences GGC and AAAC were added to the 50 end of the forward and reverse oligonucleotides, respectively, to allow the formation of cohesive ends of BsaI restriction sites following annealing (Kui et al 2017).…”

Utilization of transcription factors for controlling floral morphogenesis in horticultural plants

“…Genome editing is also expected to contribute to the generation of new floral traits and flower cultivars. In ornamental flowers, several studies on genome editing have been reported, such as in orchid ( Kui et al 2017 ), petunia ( Zhang et al 2016 ), chrysanthemum ( Kishi-Kaboshi et al 2017 ), and Japanese morning glory ( Watanabe et al 2017 ). Many more ornamental flowers could now also be subjected to genome editing.…”

Post genomics era for orchid research

“…For P. equestris , the transformation system has been established, but the regeneration time is long, about 2–3 years. With these advantages, also being a popular medicinal plant for multiple pharmaceutical effectives such as immunomodulation, anti-oxidation, anti-fatigue, genome editing has been recently reported in D. officinale to knockout the expression of several lignocellulose biosynthesis genes, including C3H, C4H, 4CL, CCR and IRX (Kui et al 2016 ). In addition, Kui et al, has adopted Agrobacterium to deliver the CRISPR/Cas9 construct and compare five promoters with Cauliflower mosaic virus 35S promoter, and identified their compatible promoter activities.…”