Transformation of oil palm calli using CRISPR/Cas9 System: toward genome editing of oil palm

Abstract

Genome editing using CRISPR/Cas9 construct containing sgRNA is the newest technology used to assemble organisms with desired characters through knock out of the target gene. As part of the effort to develop oil palm planting material tolerant to Ganoderma, this research aimed to obtain a technique for introducing CRISPR/Cas9 modules containing sgRNA into oil palm tissue. Based on differential expression studies, two genes encoding isoflavone reductase (IFR) and methallothionine-like protein (MT) were selected as the target for edited genes. The CRISPR-IFR and CRISPR-MT modules each carrying a sequence of gene recognition (sgRNA) were constructed and transformed into oil palm calli using Agrobacterium tumefaciens. The transformed calli were cultured on a modified DF medium for callus propagation, and then sub-cultured into DF media for induction of somatic embryos. PCR analyses using specific primers flanking the DNA targeted region were carried out and the results showed that both constructs had been introduced into the cells. DNA sequence analysis indicated the presence of substitution of several bases in the target area of both genes. The transformed calli grew in a selection medium containing hygromycin. Development of somatic embryos was detected after 3-4 weeks culturing on modified DF media.