Molecular and Phenotypic Analyses of Inpari HDB/K15 F2 Lines Containing sd1 Mutant Gene Resulted from Genome Editing Method

Clara S. A. Fitriyanti, Suharsono Suharsono Suharsono, Tri Joko Santoso


Inpari HDB variety is resistant to bacterial leaf blight (BLB) disease, but due to its tall stature, the variety is susceptible to lodging. Inpari HDB with semidwarf stature, therefore, is of high interest for lodging resistant performance. sd1 gene encoding GA20ox-2 enzyme is one of the genes responsible for imparting semidwarf stature of rice. In previous study, sd1 mutant rice cv. Kitaake (K15) was developed by using CRISPR/Cas9 technology. The objective of this study was to analyze molecularly and phenotypically F2 lines containing sd1 mutant gene resulted from a cross between Inpari HDB and K15 to develop semidwarf
Inpari HDB rice variety. Thirty F2 Inpari HDB/K15 lines were analyzed at molecular level using DNA sequencing method together with phenotypic assessment of the lines to verify the integration of sd1 mutant gene. DNA sequencing analysis showed that 9 out of the 30 F2 Inpari HDB/K15 lines were sd1 mutants. The remaining F2 lines contained 17 heterozygotes and 4 nonmutants. All the nine mutant lines demonstrated shorter plant stature and showed more tiller number per plant compared to the nonmutant lines. The sd1 mutant gene in the F2 lines showed pleiotropic effects on panicle number and
showed no effects on other traits, such as flowering time, panicle length, filled and unfilled grain percentages. This study showed the introduction of sd1 mutant gene generated semidwarf Inpari HDB lines. The semidwarf Inpari HDB lines obtained from this research should be further evaluated to confirm their lodging resistant performances.


Oryza sativa; semidwarf; GA20ox-2; CRISPR/Cas9; mutant.

Full Text:



Ashikari, M., Sakakibara, H., Lin, S., Yamamoto, T., Takashi, T., Nishimura, A., Angeles, E.R., Qian, Q., Kitano, H. & Matsuoka, M. (2005) Plant science: Cytokinin oxidase regulates rice grain production. Science. [Online] 309 (5735), 741–745. Available from: [Accessed 19 April 2017].

Bibikova, M., Golic, M., Golic, K.G. & Carroll, D. (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics, 161, 1169–1175 [Accessed 29 August 2019].

Butler, N.M., Atkins, P.A., Voytas, D.F. & Douches, D.S. (2015) Generation and inheritance of targeted mutations in potato (Solanum tuberosum L.) using the CRISPR/Cas system. PLOS One. [Online] 10 (12), 1–12. Available from: [Accessed 29 September 2019].

Char, S.N., Neelakandan, A.K., Nahampun, H., Frame, B., Main, M., Spalding, M.H., Becraft, P.W., Meyers, B.C., Walbot, V., Wang, K. & Yang, B. (2017) An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize. Plant Biotechnology Journal. [Online] 15 (2), 257–268. Available from: [Accessed 21 March 2018].

Chen, M., Zhao, Z., Chen, L., Zhou, F., Zhong, Z., Jiang, L. & Wan, J. (2013) Genetic analysis and fine mapping of a semi-dwarf gene in a centromeric region in rice (Oryza sativa L.). Breeding Science. [Online] 63 (2), 164–168. Available from: [Accessed 29 September 2019].

Dello, R., Linhares, F.S., Scacchi, E., Casamitjana-Martinez, E., Heidstra, R., Costantino, P. & Sabatini, S. (2007) Cytokinins determine Arabidopsis root-meristem size by controlling cell differentiation. Current Biology. [Online] 17 (8), 678–682. Available from: [Accessed 28 September 2019].

Deltcheva, E., Chylinski, K., Sharma, C.M., Gonzales, K., Chao, Y., Pirzada, Z.A., Eckert, M., Vogel, J. & Charpentier, E. (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. [Online] 471 (7340), 602–607. Available from: [Accessed 21 March 2018].

Deveau, H., Barrangou, R., Garneau, J.E., Labonté, J., Fremaux, C., Boyaval, P., Romero, D.A., Horvath, P. & Moineau, S. (2008) Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of Bacteriology. [Online] 190 (4), 1390–1400. Available from: [Accessed 28 March 2018].

Doyle, J.J. & Doyle, J.L. (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin. [Online] 19 (1), 11–15. Available from: [Accessed 29 September 2018].

Hirano, K., Ordonio, R.L. & Matsuoka, M. (2017) Engineering the lodging resistance mechanism of post-green revolution rice to meet future demands. Proceedings of the Japan Academy Series B: Physical and Biological Sciences. [Online] 93 (4), 220–233. Available from: [Accessed 29 September 2019].

Hosamani, V., Pradeep, S., Sridhara, S. & Kalleshwaraswamy, C.M. (2009) Biological studies on paddy ear head bug, Leptocorisa oratorius Fabricius (Hemiptera: Alydidae). Academic Journal of Entomology, 2 (2), 52–55.

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A. & Charpentier, E. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. [Online] 337 (6096), 816–821. Available from: [Accessed 21 March 2018].

Mojica, F.J.M., Díez-Villaseñor, C., García-Martínez, J. & Almendros, C. (2009) Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology. [Online] 155 (3), 733–740. Available from: [Accessed 12 June 2015].

Murai, M., Komazaki, T. & Sato, S. (2004) Effects of sd1 and Ur1 (Undulate rachis-1) on lodging resistance and related traits in rice. Breeding Science. [Online] 54 (4), 333–340. Available from: [Accessed 6 July 2020].

Ogi, Y., Kato, H., Maruyama, K. & Kikuchi, F. (1993) The effect on the culm length and other agronomic characters caused by semidwarfing genes at the sd-1 locus in rice. Japan Journal of Breeding. [Online] 5743, 267–275. Available from: [Accessed 6 July 2020].

Okuno, A., Hirano, K., Asano, K., Takase, W., Masuda, R., Morinaka, Y., Ueguchi-Tanaka, M., Kitano, H. & Matsuoka, M. (2014) New approach to increasing rice lodging resistance and biomass yield through the use of high gibberellin producing varieties. PLOS One. [Online] 9 (2), 1–13. Available from: [Accessed 19 February 2020].

Pan, C., Ye, L., Qin, L., Liu, X., He, Y., Wang, J., Chen, L. & Lu, G. (2016) CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Scientific Reports. [Online] 6 (January), 1–9. Available from: [Accessed 11 October 2018].

Santoso, T.J., Enggarini, W., Trijatmiko, K.R. & Sitepu, M.B. (2016) Introduksi konstruk CRISPR-Cas9/Gen Ga20ox-2 ke padi dan identifikasi mutan-mutan padi melalui analisis molekuler dan sekuensing. Laporan Tahunan Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumber Daya Genetik Pertanian. Badan Penelitian dan Pengembangan Pertanian, Kementerian Pertanian. Bogor, BB Biogen.

Sasaki, A., Ashikari, M., Ueguchi-Tanaka, M., Itoh, H., Nishimura, A., Swapan, D., Ishiyama, K., Saito, T., Kobayashi, M., Khush, G.S., Kitano, H. & Matsuoka, M. (2002) A mutant gibberellin-synthesis gene in rice: New insight into the rice variant that helped to avert famine over thirty years ago. Nature. [Online] 416 (6882), 701–702. Available from: [Accessed 21 October 2015].

Sasmita, P., Satoto, Guswara, A., Suharna, Rahmini & Handoko, D.D. (2019) Deskripsi varietas unggul baru padi (Inbrida Padi Sawah Irigasi [INPARI] Hibrida Padi [HIPA] Inbrida Padi Gogo [INPAGO] Inbrida Padi Rawa [INPARA]). [Online] Tersedia pada: [Diakses 7 Juli 2020].

Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., Zhang, K., Liu, J., Xi, J.J., Qiu, J.L. & Gao, C. (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nature Biotechnology. [Online] 31 (8), 686–688. Available from: [Accessed 21 October 2015].

Spielmeyer, W., Ellis, M.H. & Chandler, P.M. (2002) Semidwarf (sd-1), “green revolution” rice, contains a defective gibberellin 20-oxidase gene. Proceedings of the National Academy of Sciences of the United States of America. [Online] 99 (13), 9043–9048. Available from: [Accessed 22 October 2015].

Tomita, M. & Ishii, K. (2018) Genetic performance of the semidwarfing allele sd1 derived from a japonica rice cultivar and minimum requirements to detect its single-nucleotide polymorphism by MiSeq whole-genome sequencing. BioMed Research International. [Online] 2018, 4241725. Available from: [Accessed 6 July 2020].

Xu, T., Li, Y., Van Nostrand, J.D., He, Z. & Zhou, J. (2014) Cas9-based tools for targeted genome editing and transcriptional control. Applied and Environmental Microbiology. [Online] 80 (5), 1544–1552. Available from: [Accessed 19 February 2020].



  • There are currently no refbacks.

Copyright (c) 2020 Jurnal AgroBiogen

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

 Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.



P-ISSN : 1907-1094
E-ISSN : 2549-1547

Jurnal AgroBiogen

Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumber Daya Genetik Pertanian

Jl. Tentara Pelajar 3A, Bogor 16111
Jawa Barat, Indonesia
Telp.: (0251) 8339793, 8337975
Call Center: 08211181677
Faks.: (0251) 8338820

View My Stats