[This article belongs to Volume - 55, Issue - 07]
Gongcheng Kexue Yu Jishu/Advanced Engineering Science
Journal ID : AES-06-07-2023-590

Title : Study of Hygromycin Effect for Regeneration and Transformation of CRISPR/Cas9_gRNA-OsCKX2 Cassette into Rice Genome cv. Mentik Susu Through Agrobacterium-Mediated Transformation
Fitria Roviqowati, Samanhudi, Tri Joko Santoso, Atmitri Sisharmini, Aniversari Apriana, Ahmad Yunus,

Abstract : The in vitro regeneration system of rice plants is known to be very well established compared to other plants. However, several rice genotypes are still recalcitrant for regeneration and genetic transformation, including the local rice cultivar Mentik Susu. Therefore, improving and optimizing tissue culture system protocols for regenerating fertile plants is a prerequisite for the application of genome editing. The purpose of this study was to evaluate the concentration of hygromycin and its effect on regeneration response, and introduce the CRISPR/Cas9 cassette construct that carries gRNA from the OsCKX2 gene into the rice cv. Mentik susu through A. tumefaciens-mediated transformation. The immature embryo of rice cv. Mentik susu is used for regeneration and transformation study. Analysis of the sensitivity level of explants derived from immature embryos of rice cv. Mentik susu was conducted at different hygromycin concentrations added in the callus induction medium, i.e. 0, 10, 15, 20, 25, and 30 mg L-1 as treatments. The A. tumefaciens vector strain LBA4404 harboring a CRISPR/Cas9_gRNA/OsCKX2 plasmid was used in for genetic transformation of rice cv. Mentik susu. The results showed that the hygromycin concentration of 10 mg L-1 was the suitable concentration that could be used for callus selection during the transformation using A. tumefaciens. Regeneration study of callus of rice Mentik Susu revealed that callus regeneration without adding of hygromycin in the medium produced 93 plants (81%), whereas the regeneration with hygromycin addition produced 2 plants (8.7%). Molecular detection presence of the OsCKX2 gene target construct obtained 23 putative Mentik Susu rice lines positive for the hptII gene and the Cas9 gene produced 9 lines. Further analysis of the transgenic plants are required to identify the occurrence of mutagenesis which is induced by CRISPR/cas9 system.