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.
Highlights
The orchid family (Orchidaceae) is the second largest family of flowering plants with blooms that are often colorful and fragrant
The polymerase chain reaction (PCR) result confirms that the gene Superfolder green fluorescent protein (SG) has been successfully inserted into the genome of D. officinale and the Agrobacterium-mediated gene transformation can work efficiently
The results showed that mirabilis mosaic virus (MMV), cassava vein mosaic virus (CVMV), and PCISV are as effective as the 35S promoter with strong expression in all protocorms examined, while Medicago truncatula hypothetical protein (MtHP) is less effective than the 35S promoter
Summary
The orchid family (Orchidaceae) is the second largest family of flowering plants with blooms that are often colorful and fragrant This family encompasses about 6–11% of all seed plants (Pillon and Chase, 2007). Gene silencing has been achieved by gene editing using the new technology called CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9). This system has two major advantages: firstly, complete knock out of target gene instead of partial knock down due to dosage difference in RNAi (Barrangou et al, 2015); secondly, stable genomic change that can be maintained in the offspring which is an important advantage for application in breeding programs (Liu et al, 2016). There are only published genome sequences of two orchid species: Dendrobium officinale (Yan et al, 2015; Zhang G.Q. et al, 2016) and Phalaenopsis equestris (Cai et al, 2015)
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