CRISPR/Cas9‐mediated knockout of an oil palm defense‐related gene to the pathogenic fungus Ganoderma boninense

Asmini Budiani(1), Imam Bagus Nugroho(2), Dini Astika Sari(3), Inez Palupi(4), Riza Arief Putranto(5*)

(1) Indonesian Research Institute for Biotechnology and Bioindustry, Jalan Taman Kencana No. 1, Bogor, Jawa Barat 16128
(2) Indonesian Research Institute for Biotechnology and Bioindustry, Jalan Taman Kencana No. 1, Bogor, Jawa Barat 16128
(3) Indonesian Research Institute for Biotechnology and Bioindustry, Jalan Taman Kencana No. 1, Bogor, Jawa Barat 16128
(4) Agrotechnology Department, Faculty of Agriculture, Universitas Jenderal Soedirman, Jalan dr. Suparno PO BOX 125 Purwokerto, Jawa Tengah 53123
(5) Indonesian Research Institute for Biotechnology and Bioindustry, Jalan Taman Kencana No. 1, Bogor, Jawa Barat 16128
(*) Corresponding Author


Oil palm plantation in Indonesia is significantly affected by basal stem rot disease caused by the pathogenic fungus Ganoderma boninense. Tolerant oil palm cultivars toward G. boninense have been developed through a breeding program accelerated by the implementation of the CRISPR/Cas9 technology. This study was conducted to perform a gene knockout (KO) of oil palm that confers a putative defense‐related trait toward G. boninense. A plasmid pCRISPR_EMLP containing modules, i.e., 35S‐CaMV‐promoter‐driven CRISPR/Cas9, U6‐promoter‐driven sgRNA to the target EgEMLP gene (EL695076), and hygromycin resistance gene as the selectable marker, was established for Agrobacterium‐mediated delivery into oil palm calli (OPC). The transformed OPCs were regenerated and screened in DF (de Fossard) media containing hygromycin. The working concentration of hygromycin was successfully optimized for selection at 20 ppm. Through PCR‐based selection using HYG primers, we succeeded in discerning positive transformed OPC clones. The sequenced PCR products of genomic DNA as the template amplified using EMLP1 primers showed a point mutation, causing a frameshift in the edited EgEMLP and premature stop codon. Furthermore, in silico modeling demonstrated that the mutation resulted in a change in the C‐terminal region, affecting the tertiary protein structure. Moreover, electrophoresis analysis of PCR products of cDNA as the template from transformed OPC clones showed several samples with faint or undetected bands. This indicated that the CRISPR/Cas9 module induced a mutation that could destabilize the transcribed mRNA, e.g., premature degradation. Altogether, this study has successfully implemented CRISPR/Cas9 gene editing in oil palm in a model gene that is responsible for putative defense‐related traits toward the pathogenic fungus G. boninense.


gene editing; plant‐microbe interaction; Ganoderma boninense; defense trait; SNP

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Budiani A, Nugroho IB, Minarsih H, Riyadi I. 2019. Regeneration of oil palm plantlets introduced by P5CS gene using Agrobacterium­mediated transfor­ mation. E­Journal Menara Perkebunan 87(2):123– 130. doi:10.22302/

Budiani A, Putranto RA, Riyadi I, Minarsih H, Faizah R. 2018. Transformation of oil palm calli using CRISPR/Cas9 System: toward genome editing of oil palm. In: IOP Conf Ser: Earth and Environ Sci., volume 183, page 012003. doi:10.1088/1755­ 1315/183/1/012003.

Chiruvella KK, Liang Z, Wilson TE. 2013. Re­ pair of double­strand breaks by end joining. Cold Spring Harbor Perspect Biol 5(5):a012757. doi:10.1101/cshperspect.a012757.

Hahn F, Eisenhut M, Mantegazza O, Weber APM. 2018. Homology­directed repair of a defective glabrous gene in Arabidopsis with Cas9­based gene targeting. Front Plant Sci 9. doi:10.3389/fpls.2018.00424.

Ho CL, Tan YC. 2015. Molecular defense response of oil palm to Ganoderma infection. Phytochemistry 114:168–177. doi:10.1016/j.phytochem.2014.10.016.

Horvath P, Barrangou R. 2010. CRISPR/Cas, the im­ mune system of bacteria and archaea. Science 327(5962):167–170. doi:10.1126/science.1179555.

Hou H, Atlihan N, Lu ZX. 2014. New biotechnology en­ hances the application of cisgenesis in plant breeding. Front Plant Sci 5:389. doi:10.3389/fpls.2014.00389. Hushiarian R, Yusof NA, Dutse SW. 2013. Detection and control of Ganoderma boninense: strategies and per­ spectives. SpringerPlus 2(1):555. doi:10.1186/2193­1801­2­555.

Jaganathan D, Ramasamy K, Sellamuthu G, Jayabalan S, Venkataraman G. 2018. CRISPR for crop im­ provement: an update review. Front Plant Sci 9. doi:10.3389/fpls.2018.00985.

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012. A programmable dual­

RNA–guided DNA endonuclease in adaptive bac­ terial immunity. Science 337(6096):816–821. doi:10.1126/science.1225829.

Mali P, Esvelt KM, Church GM. 2013. Cas9 as a ver­ satile tool for engineering biology. Nat Methods 10(10):957–963. doi:10.1038/nmeth.2649.

Ommelna BG, Jennifer AN, Chong KP. 2012. The poten­ tial of chitosan in suppressing Ganoderma boninense infection in oil­palm seedlings. J Sustain Sci Manag 7(2):186–192.

Shen H, Strunks GD, Klemann BJPM, Hooykaas PJJ, de Pater S. 2017. CRISPR/Cas9­induced double­ strand break repair in Arabidopsis nonhomologous end­joining mutants. G3: Genes, Genomes, Genet 7(1):193–202. doi:10.1534/g3.116.035204.

Tan YC, Yeoh KA, Wong MY, Ho CL. 2013. Ex­ pression profiles of putative defence­related proteins in oil palm (Elaeis guineensis) colonized by Gan­ oderma boninense. J Plant Physiol 170(16):1455– 1460. doi:10.1016/j.jplph.2013.05.009.

Telem RS, Wani HS, Singh NB, Nandini R, Sad­ hukhan R, Bhattacharya S, Mandal N. 2013. Cisgenics­a sustainable approach for crop im­ provement. Curr Genomics 14(7):468–476. doi:10.2174/13892029113146660013.

Yang TH. 2007. Social Factors, Transaction Costs and Industrial Organization. Int Sociol 22(4):435–461. doi:10.1177/0268580907078008.


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