Assessment of zygosity and nitrogen used efficacy in Mekongga transgenic rice




Abstract. Aini MQ, Suharsono, Apriana A, Sisharmini A, Santoso TJ, Trijatmiko KR. 2022. Assessment of zygosity and nitrogen used efficacy in Mekongga transgenic rice. Biodiversitas 23: 4040-4046Nitrogen (N) is a vital macronutrient that can be a limiting factor for rice plant growth. Nitrogen-efficient plants can be assembled by modifying nitrogen-related metabolic pathways to improve Nitrogen Use Efficiency (NUE) in plants. Tgeneration of transgenic Mekongga rice plants containing the LeAlaAT gene was obtained. In transgenic plant research, zygosity test was desirable to ensure the integration and inheritance stability of transgene. This study aimed to assess zygosity and nitrogen use efficacy of two transgenic Mekongga rice lines, namely M41 and M50. This research involved three main activities, i.e. detection of flanking sequence for Tgeneration by TAIL-PCR method, zygosity test for Tgeneration, and efficacy test for Tgeneration of Mekongga transgenic lines in response to different N fertilizer rates of 0, 60, 90, and 120 kg ha?1. Zygosity analysis showed that six out of 14 M41 and three of 13 M50 transgenic lines were homozygous for LeAlaAT transgene. The efficacy test showed that homozygous transgenic lines had higher tiller numbers, grain numbers, and biomass compared to non-transgenic ones under the same nitrogen level. The notable discrepancies of the transgenic plants can also be recognized in the absorption, agronomical, and grain NUE of the studied plants.


Andrews M, Raven JA, Lea PJ. 2013. Do plants need nitrate? The mechanisms by which nitrogen form affects plants. Ann Appl Biol. 163(2):174–199. DOI:10.1111/aab.12045.
Apriana A, Sisharmini A, Aswidinnoor H, Trijatmiko KR, Sudarsono S. 2019. Promoter deletion analysis reveals root-specific expression of the alkenal reductase gene (OsAER1) in Oryza sativa. Funct Plant Biol. 46(4):376. DOI:10.1071/FP18237.
Beatty PH, Carroll RT, Shrawat AK, Guevara D, Good AG. 2013. Physiological analysis of nitrogen-efficient rice overexpressing alanine aminotransferase under different N regimes. Botany. 91(12):866–883. DOI:10.1139/cjb-2013-0171.
Congreves KA, Otchere O, Ferland D, Farzadfar S, Williams S, Arcand MM. 2021. Nitrogen use efficiency definitions of today and tomorrow. Front Plant Sci. 12. DOI:10.3389/fpls.2021.637108.
Dobermann AR. 2005. Nitrogen Use Efficiency – State of the Art". In: Plant Sci Com. Vol. 316. Lincoln: University of Nebraska Agronomy & Horticulture -- Faculty Publications. p. 1–16.
Galloway JN, Winiwarter W, Leip A, Leach AM, Bleeker A, Erisman JW. 2014. Nitrogen footprints: past, present and future. Environ Res Lett. 9(11):115003. DOI:10.1088/1748-9326/9/11/115003.
Good AG, Johnson SJ, de Pauw M, Carroll RT, Savidov N, Vidmar J, Lu Z, Taylor G, Stroeher V. 2007. Engineering nitrogen use efficiency with alanine aminotransferase. Canad J Bot. 85(3):252–262. DOI:10.1139/B07-019.
Huang S, Zhao C, Zhang Y, Wang C. 2018. Nitrogen Use Efficiency in Rice. In: Nitrogen in Agriculture - Updates. InTech.
Hung J-H, Weng Z. 2016. Designing polymerase chain reaction primers using Primer3Plus. Cold Spring Harb Prot. 2016(9):pdb.prot093096. DOI:10.1101/pdb.prot093096.
Masclaux-Daubresse C, Daniel-Vedele F, Dechorgnat J, Chardon F, Gaufichon L, Suzuki A. 2010. Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Ann Bot. 105(7):1141–1157. DOI:10.1093/aob/mcq028.
McAllister CH, Beatty PH, Good AG. 2012. Engineering nitrogen use efficient crop plants: the current status. Plant Biotech J. 10(9):1011–1025. DOI:10.1111/j.1467-7652.2012.00700.x.
Passricha N, Batra R, Behl RK, Sikka VK. 2015. Differential and temperature dependent regulation of ADP-glucose pyrophosphorylase by specific chromosome in wheat grains. Cereal Res Commun. 43(4):591–603. DOI:10.1556/0806.43.2015.020.
Passricha N, Saifi S, Khatodia S, Tuteja N. 2016. Assessing zygosity in progeny of transgenic plants: current methods and perspectives. J Biol Methods. 3(3):e46. DOI:10.14440/jbm.2016.114.
Rajeevkumar S, Anunanthini P, Sathishkumar R. 2015. Epigenetic silencing in transgenic plants. Front Plant Sci. 6. DOI:10.3389/fpls.2015.00693.
Samborski S, Kozak M, Azevedo RA. 2008. Does nitrogen uptake affect nitrogen uptake efficiency, or vice versa? Acta Physiol Plant. 30(4):419–420. DOI:10.1007/s11738-008-0164-4.
Selvaraj MG, Valencia MO, Ogawa S, Lu Y, Wu L, Downs C, Skinner W, Lu Z, Kridl JC, Ishitani M, et al. 2017. Development and field performance of nitrogen use efficient rice lines for Africa. Plant Biotech J. 15(6):775–787. DOI:10.1111/pbi.12675.
Shrawat AK, Carroll RT, DePauw M, Taylor GJ, Good AG. 2008. Genetic engineering of improved nitrogen use efficiency in rice by the tissue-specific expression of alanine aminotransferase. Plant Biotech J. 6(7):722–732. DOI:10.1111/j.1467-7652.2008.00351.x.
Sisharmini A, Apriana A, Kamila I, Poloroso A, Enggarini W, Santoso T, Hadiarto T, Husin B, Trijatmiko K. 2022. Construction and introduction of OsAER1::LeAlaAT cassette to improve the nitrogen use efficiency in rice cv. Mekongga. In: AIP Conference Proceedings. 2nd International Conference of Genetic Resource and Biotechnology. p. 1–9.
Sisharmini A, Apriana A, Khumaida N, Trijatmiko KR, Purwoko BS. 2019. Expression of a cucumber alanine aminotransferase2 gene improves nitrogen use efficiency in transgenic rice. J Genet Eng Biotechol. 17(1):9. DOI:10.1186/s43141-019-0010-7.
Stahl A, Vollrath P, Samans B, Frisch M, Wittkop B, Snowdon RJ. 2019. Effect of breeding on nitrogen use efficiency-associated traits in oilseed rape. J Exp Bot. 70(6):1969–1986. DOI:10.1093/jxb/erz044.
Triadiati A, Pratama A, Abdulrachman S. 2012. Pertumbuhan dan efisiensi penggunaan nitrogen pada padi (Oryza sativa L.) dengan pemberian pupuk urea yang berbeda. Bul Anatomi Fisiol. 20(2):1–14. DOI:10.14710/baf.v20i2.4767.
Xu Z, Ma J, Qu C, Hu Y, Hao B, Sun Y, Liu Z, Yang H, Yang C, Wang H, et al. 2017. Identification and expression analyses of the alanine aminotransferase (AlaAT) gene family in poplar seedlings. Sci Rep. 7(1):45933. DOI:10.1038/srep45933.
Yulita DS, Purwoko BS, Sisharmini A, Apriana A, Santoso TJ, Trijatmiko KR, Sukma D. 2021. Molecular characterization and n use efficiency of LeAlaAT ‘Mekongga’ transgenic rice lines. SABRAO J Breed Genet. 53(4):723–736. DOI:10.54910/sabrao2021.53.4.14.
Zhang H, Xu W, Feng Z, Hong Z. 2018. A low degenerate primer pool improved the efficiency of high-efficiency thermal asymmetric interlaced PCR to amplify T-DNA flanking sequences in Arabidopsis thaliana. 3 Biotech. 8(1):14. DOI:10.1007/s13205-017-1032-y.
Zhang QW, Yang ZL, Zhang AP, Wang M. 2013. Responses of rice (Oryza sativa L.) Nitrogen status indicators to Nitrogen rates during the different rice growth stages. Adv Mat Res. 726–731:4411–4417. DOI:10.4028/

Most read articles by the same author(s)