Articles
Establishment of CRISPR/Cas9 genome editing system in Indonesian orchid Dendrobium lineale Rolfe
Article number
1334_25
Pages
205 – 214
Language
English
Abstract
Orchid is a decorative plant that has high aesthetic value because of its appearance as a potted plant.
Modern biotechnology can be practiced as a tool to extend the phenotype, not only the beauty of the flowers but also the leaves.
One of the methods is CRISPR/Cas9 which could knock out target genes such as the chloroplast gene that causes variegated leaves in orchids.
Variegation is a pattern in the leaf plants that have a yellow or white phenotype, this is caused by mutation of the VARIEGATED2 (VAR2) gene.
CRISPR/Cas9 can be facilitated mutagenesis using Agrobacterium-mediated transformation.
This study aimed to ensure that CRISPR/Cas9 is effective for editing VAR2 gene as a tool to edit the genome of Dendrobium lineale. The methods were conducted as follows: 1) the determination of protocorm resistance 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), pRGEB32 vector contained the T-DNA construct Ubi::Cas9::U3::VAR2 in Agrobacterium tumefaciens EHA 105, bacteria were transferred to orchidRSQUOs protocorm; 3) detection of the integration of T-DNA in the transformant genome by PCR; 4) mutation detection by sequence analysis.
The results indicated that 18.96 µM Hygromycin is the optimum concentration that is closest to LD50. The optimum transformation efficiency (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 the genes, i.e., Cas9 (402 bp), HPT (545 bp), VAR2 (723 bp) and cpDNA trnL-F (1200 bp, as internal controls) were amplified.
The analysis of the sequence displayed 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.
Modern biotechnology can be practiced as a tool to extend the phenotype, not only the beauty of the flowers but also the leaves.
One of the methods is CRISPR/Cas9 which could knock out target genes such as the chloroplast gene that causes variegated leaves in orchids.
Variegation is a pattern in the leaf plants that have a yellow or white phenotype, this is caused by mutation of the VARIEGATED2 (VAR2) gene.
CRISPR/Cas9 can be facilitated mutagenesis using Agrobacterium-mediated transformation.
This study aimed to ensure that CRISPR/Cas9 is effective for editing VAR2 gene as a tool to edit the genome of Dendrobium lineale. The methods were conducted as follows: 1) the determination of protocorm resistance 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), pRGEB32 vector contained the T-DNA construct Ubi::Cas9::U3::VAR2 in Agrobacterium tumefaciens EHA 105, bacteria were transferred to orchidRSQUOs protocorm; 3) detection of the integration of T-DNA in the transformant genome by PCR; 4) mutation detection by sequence analysis.
The results indicated that 18.96 µM Hygromycin is the optimum concentration that is closest to LD50. The optimum transformation efficiency (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 the genes, i.e., Cas9 (402 bp), HPT (545 bp), VAR2 (723 bp) and cpDNA trnL-F (1200 bp, as internal controls) were amplified.
The analysis of the sequence displayed 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.
Authors
L. Zubaidah, Y. Setiawati, S. Nopitasari, M.D. Lawrie, A.B. Sasongko, A. Purwantoro, J. Widada, K. Ninomiya, Y. Asano, S. Matsumoto, Y. Yoshioka, E. Semiarti
Keywords
CRISPR/Cas9-VAR2, variegation, Agrobacterium-mediated genetic transformation, Hygromycin, T-DNA
Groups involved
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