Establishment of a CRISPR/Cas9 genome editing system in the Indonesian orchid, Dendrobium lineale Rolfe
Lailia Zubaidah is a Master’s student in the Laboratory of Biotechnology, Faculty of Biology, Universitas Gadjah Mada, Indonesia. She is working on a thesis entitled “Genome editing in Dendrobium lineale Rolfe an Indonesian orchid using CRISPR/Cas9.” This study focuses on a plant DNA transformation method to transfer T-DNA into the plant genome, and characterize transformant plants. Orchid is one of the most popular ornamental plants in the world. It is appreciated for its beautiful flowers and the adaptability of the plants in containers. Dendrobium is endemic in Indonesia. It has high value for horticultural or medicinal uses. This orchid can be used as a parental plant in crosses to produce characteristics to meet the consumers’ needs. However, the juvenile stage is long until the flowering stage is reached. We wanted to evaluate new phenotypes of Dendrobium with appealing leaves. We considered the use of biotechnological applications to obtain the consumers’ expectations without requiring the flowering state. CRISPR/Cas9 is a new method for genome editing. It has been developed and used on plants. CRISPR/Cas9 can induce double strand breaks (DSBs) in DNA with the Cas9 nuclease enzyme, followed by repair using two mechanisms called non-homologous end joining (NHEJ) and homologous direct repair (HDR). The genome sequence information of Dendrobium facilitated the sgRNA design based on the similarity of the homologous gene of interest. This method was successfully used in Dendrobium officinale. The objective of our study was to determine whether CRISPR/Cas9 could be used as a tool for genome editing in Dendrobium lineale. The rapid confirmation of phenotype mutations could be seen when the chloroplast gene was used as an indicator. Variegation of a yellowish-white pattern on the leaves can be found in nature. This phenomenon could also be induced by mutating the chloroplast genes, such as VAR2 (VARIEGATED2). The interest gene encodes filamentous sensitive (FtsH) enzyme family, a metalloptrotease ATP dependent that exists in the thylakoid membrane. According to Yu et al. (2004) var2 mutation in Arabidopsis thaliana showed variegation in leaves. Agrobacterium-mediated transformation is a tool widely used to transfer T-DNA into the plant genome. Eight-week-old protocorms were used as plant materials. The construct of T-DNA Ubi :: Cas9 :: U3 :: VAR2/pRGEB32 in the Agrobacterium tumefaciens strain EHA 105 was transferred into the orchid’s protocorm. In this study, we optimized hygromicin sensitivity with various concentrations of hygromicin antibiotics and inoculation times (15, 30, and 45 min) to introduce A. tumefaciens into the plant genome. Furthermore, genotypic and phenotypic analysis was checked from putative transformants of D. lineale. The results showed that 18.9 µM hygromicin on New Phalaenopsis (NP) medium was the concentration closest to the lethal dose (LD50). The highest efficiency of transformation was obtained with a 45-min infection period (1.16%), compared to 15 min (0.17%) and 30 min (0.25%). Confirmation of T-DNA integration into the plant genomes was obtained by PCR analysis using primers from several genes, i.e., Cas9, HPT, VAR2 and trnl-F cpDNA as internal control. The sequence analysis showed mutation in the form of substitution at the target site, and slightly different color changes on the leaves in the transformant plants, as compared to that in wild type (WT). The result of this study showed that the Cas9 and sgRNA complex disrupted the DNA of the D. lineale genome in genotype analysis but phenotypes showed less color differences between transformants and non-transformants. This study remains in progress to establish the extent of the effectiveness of using CRISPR/Cas9 on orchids. References: Yu, F., Park, S., and Rodermel, S.R. (2004). The Arabidopsis FtsH metalloprotease gene family: interchangeability of subunits in chloroplast oligomeric complexes. Plant J. 37, 864–876.
Lailia Zubaidah was the second winner of the ISHS Young Minds Award for the best oral presentation at the II International Symposium on Tropical and Subtropical Ornamentals, which was held virtually in Indonesia in July 2021.
Lailia Zubaidah, Laboratory of Biotechnology, Faculty of Biology, Universitas Gadjah Mada, Indonesia, e-mail: firstname.lastname@example.org
The article is available in Chronica Horticulturae