| Penulis/Author |
LAILIA ZUBAIDAH (1); YULI SETIAWATI (2); SRI NOPITASARI (3); Muhammad Dylan Lawrie (4); Aries Bagus Sasongko, S.Si., M.Biotech. (5); Prof. Dr. Ir. Aziz Purwantoro, M.Sc. (6); Ir. Jaka Widada, M.P., Ph.D. (7); Prof. Dr. Endang Semiarti, M.S., M.Sc. (12) |
| Abstrak/Abstract |
Orchid is an ornamental plant which has high economic value due to its appearance as a potted plant. Modern biotechnology can be used as a tool to improve the phenotype, not only the beauty of the flowers but also the leaves. One of the method is CRISPR/Cas9 which could knock out target gene such as chloroplast gene that cause variegated leaf in orchid. Variegation is a pattern in the leaf plants that have a yellow or white phenotype, this caused by mutation of VARIEGATED2 (VAR2) gene. Mutagenesis can be induced by CRISPR/Cas9 genome editing system through Agrobacterium-mediated transformation. The objective of this study was to determine whether CRISPR/Cas9 effective for editing VAR2 gene as a tool for genome editing in Dendrobium lineale orchid. The methods were conducted as follow: 1. The determination of protocorm resistency in Hygromycin, eight-weeks old orchid protocorms which were cultured on NP medium containing (0; 3.79; 7.58; 11.37; 15.17 and 18.96) µM Hygromycin antibiotic for transformant selection: 2. Agrobacterium-mediated transformation in various time co-cultivation (15, 30, and 45 min), the construct of T-DNA Ubi::Cas9::U3::VAR2/pRGEB32 in the A. tumefaciens strain EHA 105 was transferred into orchid’s protocorm: 3. Detection the integration of T-DNA in transformant genome by PCR: 4. Mutation detection by sequence analysis. The results showed that 18.96µM Hygromycin is the optimum concentration that closest to LD 50. The highest efficiency of transformation (1.16%) was obtained in the 45 min infection period, compared to 0.17% in 15 min and 0.25% in 30 min. PCR results showed that several genes, i.e Cas9 (402 bp), HPT (545 bp), VAR2 (723 bp) and cpDNA trnL-F (1200 bp, as internal control) were amplified. Sequence analysis showed that there was a mutation at the target site. This research is still in progress to know the effectiveness of using CRISPR/Cas9 in orchids. |
