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Tolerance of T2 Generation ‘Kitaake’ Rice (Oryza sativa L.) CRISPR/Cas9-OsGA20ox-2 Mutant Strains to Drought Condition

https://doi.org/10.22146/ipas.37032

Husni Mubarok(1), Panjisakti Basunanda(2*), Tri Joko Santoso(3)

(1) Faculty of Agriculture Universitas Gadjah Mada, Yogyakarta
(2) Faculty of Agriculture Universitas Gadjah Mada, Yogyakarta
(3) Research and Development Center for Biotechnology and Genetic Resources (BB BIOGEN)
(*) Corresponding Author

Abstract


Rice (Oryza sativa L.) is important staple crop in Indonesia. Food demand that continues to rise while inadequate land could be managed by assembling superior cultivar using CRISPR-Cas9 system method. Editing the genome by mutating the GA20ox-2 gene could improve both crop yield and ability to thrive in marginal land (drought). This experiment aims to obtain non-transgenic mutant plants (non Cas9 and hpt genes), gain information on GA20ox-2 gene expression levels, and study the tolerance levels of the CRISPR /Cas9-OsGA20ox-2 mutant lines 'Kitaake' T2 generation against drought conditions. Planting material using a mutant gene GA20ox 2 ‘Kitaake’ (K23.1, K15, K29.1, K19.1) and wild-type comparison. From 20 plants, respectively the K23.1, K15, K29.1, and K19.1 lines have 50%, 50%, 0%, and 45% of non-transgenic plants. DNA mutations in the form of deletion 44 bases (K23.1, K29.1, K19.1) and insertion of two bases (K15) are transcribed into RNA. The transcription results in a number of lower amino acids compare to its wild type (389 amino acids). The lines K23.1, K29.1, K19.1 have 373 amino acids and the K15 line has 300 amino acids (frameshift). Differences in the number of amino acids result in different phenotypic expressions. K15 mutant line has lower plant height and leaf length than the other mutant lines and wild type. The decrease does not decrease the potential of the crop. Mutations in the K15 line did not indicate better tolerant response to drought stress than other mutant lines and wild type in both vegetative and generative phase.

 


Keywords


genome editing; non-transgenic; insersi; delesi; ekspression; frameshift

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References

Abdurachman, A., Sutono, S. 2005. Teknologi pengendalian erosi lahan berlereng. dalam Teknologi Pengelolaan Lahan Kering : Menuju pertanian produktif dan ramah lingkungan. Pusat Penelitian dan Pengembangan Tanah dan Agroklimat, Bogor.

Abdurachman, A., Dariah, A., Mulyani, A. 2008. Strategi dan teknologi pengelolaan lahan kering mendukung pengadaan pangan nasional. Jurnal Litbang 27(2):43-49.

Akram, H. M., Ali, A., Sattar, A., Rehman, H.S.U., Bibi, A. 2013. Impact of water deficit stress on various physiological and agronomic traits of three basmati rice (Oryza sativa L.) cultivar. The Journal Animal and Sciences 23(5):1415-1423.

Amang, B. 1995. Kebijaksanaan Pangan Nasional. PT. Dharma Karsa Utama, Jakarta.

Asanoa, K., Yamasakib, M., Takunoc, S., Miuraa, K., Katagirid, S, Itod, T., Doie, K., Wuf, J., Ebanag, K., Matsumotof, T., Innanc, H., Kitanoa, H., Ashikaria, M., Matsuokaa, M. 2011. Artificial selection for a green revolution gene during japonica rice domestication. University of Georgia, Georgia.

Badan Pusat Statistik. 2017. Impor Beras Menurut Negara Asal Utama 2000-2015. <https://www.bps.go.id/statictable/2014/09/08/1043/impor-beras-menurut-negara-asal-utama-2000-2015.html>. Diakses 12 Februari 2018.

Brenners, L., Barnettf, H., Crick, C., Orgel, A. 1061. The theory of mutagenesis. Journal Mol. Biol. 3:121-124.

Carsono, N. 2008. Peran Pemuliaan Tanaman dalam Meningkatkan Produksi Pertanian di Indonesia. Seminar on Agricultural Science, Tokyo.

Crick, F. H. C., Barnetts, L., Brenner, R. J., Tobin, J.W. 1961. General nature of the genetic code for proteins. Nature 192:1227-1232.

Fajriah, L. R. 2015. Mentan: 200 Ribu Hektare Lahan Kekeringan Tiap Tahun. http://ekbis.sindonews.com/read/1006877/34/mentan-200-ribu-hektare-lahan-kekeringan-tiap-tahun-1432900151. diakses pada 12 Desember 2016.

Hedden, P., Phillips, A.L. 2000. Gibberellin metabolism: new insights revealed by the genes. Trends in Plant Science 5: 523–530.

Kawahara,Y., Bastide, M. D. L., Hamilton, J.P., Kanamori, H., McCombie, W.R., Ouyang, S., Schwartz, D.C., Tanaka, T., Wu, J., Zhou, S., Childs, K.L., Davidson, R.M., Lin, H., Ocampo, L. Q., Vaillancourt, B., Sakai, H.,. Lee, S.S, Kim, J., Numa, H., Itoh, T., Buell C.R., Matsumoto, T. 2013. Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data. Rice 6(1) : 4.

Khush, G. S. 1999. Green revolution : preparing for the 21st century. Genome 42(4): 646-655.

Liana, P. D. 2012. Transgenik plant-advantages regarding their cultivation, potentially risks and legislation regarding GMO’s. University of Agriculture Science Veterinary Medicine Cluj-Napoca, Romania.

Peng, J., Richard, D.E., Hartley, N.M., Devos, K.M., Flintham, J.E., Beales, J., Fish, L.J., Worland, A.J., Pelica, F., Sudhakar, D., Christou, P., Snape, J.W., Gale, M.D., Harberd N.P. 1999. ‘Green revolution’ genes encode mutant gibberellin response modulators. Nature 400:256-261.

Qin, X., Liu, J.H., Zhao, W.S., Chen, X.J., Guo, Z.J., Peng, Y.L. 2012. Gibberellin 20-Oxidase gene OsGA20ox3 regulates plant stature and disease development in rice. Journal Molecular Plant-Microbe Interaction (MPMI) 26(2): 227-239.

Sakamoto, T., Miura, K., Itoh, H., Tatsumi, T., Ueguchi-Tanaka, M., Ishiyama, K., Kobayashi, M., Agrawal, G.K.,Takeda, S., Abe, K., Miyao, A., Hirochika, H., Kitano, H., Ashikari, M., Matsuoka M. 2004. An overview of gibberellin metabolism enzyme genes and their related mutants in rice. Plant Physiology 134: 1642–1653.

Santoso, T.J., Enggarini, A., Trijatmiko, K.R., Sitepu, M.B. 2016. Introduksi konstruk CRISPR-Cas9/Gen GA20 Ox-2 ke padi dan identifikasi mutan-mutan padi melalui analisis molekuler dan sekuensing. Laporan tahunan BB BIOGEN.

Santoso, T.J. 2015. CRISPR, teknologi pengeditan genom terarah untuk pengembangan tanaman non-transgenik. Warta Biogen 11(2): 9-12.

Silitonga, T.S., Risliawati, A. 2011. Pembentukan core collection untuk sumber daya genetik padi toleran kekeringan. Buletin Plasma Nutfah 17 (2) : 104-115.

Streisinger, G., Owen, J. E., 1984. Mechanisms of spontaneous and induced frameshift mutation in bacteriophage T4. Genetics Society of Anierica Genetic 109:633-659.

Sujinah, Jamil, A. 2016. Mekanisme respon tanaman padi terhadap cekaman kekeringan dan varietas toleran. Iptek Tanaman Pangan 11(1):1-8.

Tripathi, L. 2005. Techniques for detecting genetically modified crops and products. African Journal of Biotechnology 4 (13):1472-1479.

Vettakkorumakankav, N.N., Falk, D., Saxena, P., Fletcher, R.A. 1999. A crucial role for gibberellins in stress protection of plants. Plant Cell Physiol. 40:542-548.

White, P. T. 1994. Rice: the essential harvest. Journal National Geographic 185: 48-79.

Worldbank. 2016. Indonesian Population. <http://wdi.worldbank.org/table/2.1>. Diakses 8 November 2016.

Xu, R., Li, H., Qin, R., Wang, L., Li, L., Wei, P., Yang, J. 2014. Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice. SpringerOpen Journal 7:5.



DOI: https://doi.org/10.22146/ipas.37032

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