Expression of cytokinin responsive and ethylene biosynthesis genes in rice callus with different regeneration rates

Syafira Fatihatul Husna(1), Parawita Dewanti(2), Bambang Sugiharto(3), Wahyu Indra Duwi Fanata(4*)

(1) Department of Biotechnology Graduate School Program University of Jember, Jl. Kalimantan 37 Tegalboto, East Java 68121, Indonesia
(2) Department of Agrotechnology Faculty of Agriculture University of Jember, Jl. Kalimantan 37 Tegalboto, East Java 68121, Indonesia; Center for Development of Advanced Science and Technology (CDAST) University of Jember, Jl. Kalimantan 37 Tegalboto, East Java 68121, Indonesia
(3) Department of Biology Faculty of Mathematic and Natural Science University of Jember, Jl. Kalimantan 37 Tegalboto, East Java 68121, Indonesia; Center for Development of Advanced Science and Technology (CDAST) University of Jember, Jl. Kalimantan 37 Tegalboto, East Java 68121, Indonesia
(4) Department of Agrotechnology Faculty of Agriculture University of Jember, Jl. Kalimantan 37 Tegalboto, East Java 68121, Indonesia; Center for Development of Advanced Science and Technology (CDAST) University of Jember, Jl. Kalimantan 37 Tegalboto, East Java 68121, Indonesia
(*) Corresponding Author


This study aimed to investigate the correlation between callus regeneration rate and the expression of several genes responsible for cytokinin response and ethylene biosynthesis in the Ciherang, Mentik Wangi Susu, Hwayoung and Tarabas rice varieties. The callus regeneration rate of each rice variety was in vitro tested using N6 media, while the gene expression during the callus regeneration stages was examined using quantitative real‐time PCR (qRT‐PCR). Our results showed that the callus of Ciherang and Mentik Wangi Susu showed earlier green spot formation that then turned brown at a later stage, resulting in a low regeneration rate. While Hwayoung and Tarabas showed late green spot formation, high shoot regeneration was observed in both calluses. Gene expression analysis of regeneration media‐grown calluses showed that two cytokinin‐responsive genes, OsRR2 and OsRR6, were highly expressed in the Ciherang and Hwayoung callus, respectively. We also observed that ethylene biosynthesis genes such as OsACS1 and OsACO1 were highly expressed in the Mentik Wangi Susu and Hwayoung callus, respectively. Moreover, the expression of OsBBM1 was high in Hwayoung and Tarabas. Thus, the positive correlation between the expression of cytokinin‐responsive and ethylene biosynthesis genes with somatic embryogenesis activity likely depends on the induction level of OsBBM1.


rice callus; regeneration rates; ethylene biosynthesis; cytokinin response

Full Text:



Adkins SW, Shiraishi T, McComb JA. 1990. Rice callus physiology ­ Identification of volatile emissions and their effects on culture growth. Physiol. Plant. 78(4):526–531. doi:10.1111/j.1399­3054.1990.tb05237.x.

Amer IMB, Borner A. 1997. The relationship between green spot initiation and plantlet regeneration of wheat callus grown under short­term conditions. Plant Cell, Tissue Organ Cult. 50:67–69. doi:10.1023/A:1005855912655.

Arisandi DP, Paradisa FV, Sugiharto B, Avivi S, Fanata WID. 2020. Effect of ethylene inhibitor, type of auxin, and type of sugar on anther culture of local East Java aromatic rice varieties. J. Crop Sci. Biotechnol. 23(4):367–373. doi:10.1007/s12892­020­00045­6.

Boutilier K, Offringa R, Sharma VK, Kieft H, Ouellet T, Zhang L, Hattori J, Liu CM, van Lammeren AAM, Miki BLA, Custers JBM, van Lookeren Campagne MM. 2002. Ectopic Expression of BABY BOOM Triggers a Conversion from Vegetative to Embryonic Growth. Plant Cell 14(8):1737–1749. doi:10.1105/tpc.001941.

Chatfield SP, Raizada MN. 2008. Ethylene and shoot regeneration: hookless1 modulates de novo shoot organogenesis in Arabidopsis thaliana. Plant Cell Rep. 27(4):655–666. doi:10.1007/s00299­007­0496­-3.

Dewi IS, Purwoko BS. 2008. Role of polyamines in inhibition of ethylene biosynthesis and their effects on rice anther culture development. Indones. J. Agric. Sci. 9(2):60–67. doi:10.21082/ijas.v9n2.2008.p60­67.

Duan J, Yu H, Yuan K, Liao Z, Meng X, Jing Y, Liu G, Chu J, Li J. 2019. Strigolactone promotes cytokinin degradation through transcriptional activation of CYTOKININ OXIDASE/DEHYDROGENASE 9 in rice. Proc Natl Acad Sci USA 116(28):14319–14324. doi:10.1073/pnas.1810980116.

Fanata WID, Lee KH, Son BH, Yoo JY, Harmoko R, Ko KS, Ramasamy NK, Kim KH, Oh DB, Jung HS, Kim JY, Lee SY, Lee KO. 2013. N­glycan maturation is crucial for cytokinin­mediated development and cellulose synthesis in Oryza sativa. Plant J. 73(6):966– 979. doi:10.1111/tpj.12087.

Fanata WID, Qudsiyah DH. 2020. Daya regenerasi kalus dan tunas in vitro padi varietas Tarabas pada berbagai konsentrasi 2,4D. J. Bioteknol. Biosains. Indones. 7(2):250–258. doi:10.29122/jbbi.v7i2.4404.

Fraiture MA, Roosens NH, Taverniers I, De Loose M, Deforce D, Herman P. 2016. Current developments and future detection challenges in food and feed chain. Trends Food Sci. Technol. 52:66–79. doi:10.1016/j.tifs.2016.03.011.

Han Y, Broughton S, Liu L, Zhang XQ, Zeng J, He X, Li C. 2021. Highly efficient and genotype­independent barley gene editing based on anther culture. Plant Commun. 2(2):100082. doi:10.1016/j.xplc.2020.100082.

Hirose N, Makita N, Kojima M, Kamada­Nobusada T, Sakakibara H. 2007. Overexpression of a Type­A Response Regulator Alters Rice Morphology and Cytokinin Metabolism. Plant Cell Physiol. 48(3):523– 539. doi:10.1093/pcp/pcm022.

Inoue T, Higuchi M, Hashimoto Y, Seki M, Kobayashi M, Kato T, Tabata S, Shinozak K, Kakimoto T. 2001. Identifcation of CRE1 as a cytokinin receptor from Arabidopsis. Nature 409:1060–1063. doi:10.1038/35059117.

Jha P, Kumar V. 2018. BABY BOOM (BBM): a candidate transcription factor gene in plant biotechnology. Biotechnol. Lett. 40:1467–1475. doi:10.1007/s10529­018­2613­5.

Jiang C, Johkan M, Hohjo M, Tsukagoshi S, Maturo T. 2017. A correlation analysis on chlorophyll content and SPAD value in tomato leaves. Hortic. Res. 71:37– 42. URL https://opac.ll.chiba­ 1/S18808824­71­P037­JIA.pdf.

Juárez­González VT, López­Ruiz BA, Baldrich P, LujánSoto E, Meyers BC, Dinkova TD. 2019. The explant developmental stage profoundly impacts small RNAmediated regulation at the dedifferentiation step of maize somatic embryogenesis. Sci. Rep. 9(1):14511. doi:10.1038/s41598­019­50962­y.

Kakimoto T. 1996. CKll, a histidine kinase homolog implicated in cytokinin signal transduction. Science 274(5289):982–985. doi:10.1126/science.274.5289.982.

Khanday I, Santos­Medellín C, Sundaresan V. 2020. Rice embryogenic trigger BABY BOOM1 promotes somatic embryogenesis by upregulation of auxin biosynthesis genes. bioRxiv doi:10.1101/2020.08.24.265025.

Khanday I, Skinner D, Yang B, Mercier R, Sundaresan V. 2019. A male­expressed rice embryogenic trigger redirected for asexual propagation through seeds. Nature 565(7737):91–95. doi:10.1038/s41586­018­ 0785­8.

Khosroushahi AY, Hossein NM, Henrik TS. 2011. Effect of antioxidants and carbohydrates in callus cultures of Taxus brevifolia: Evaluation of browning, callus growth, total phenolics and paclitaxel production. BioImpacts 1(1):37–45. doi:10.5681/BI.2012.020.

Kieber JJ, Schaller GE. 2014. Cytokinins. The Arabidopsis Book 12:e0168. doi:10.1199/tab.0168. Kobayashi Y, Fukui H, Tabata M. 1991. Effect of carbon dioxide and ethylene on berberine production and cell browning in Thalictrum minus cell cultures. Plant Cell Rep. 9:496–499. doi:10.1007/BF00232104.

Lowe K, Wu E, Wang N, Hoerster G, Hastings C, Cho MJ, Scelonge C, Lenderts B, Chamberlin M, Cushatt J, Wang L, Ryan L, Khan T, Chow­Yiu J, Hua W, Yu M, Banh J, Bao Z, Brink K, Igo E, Rudrappa B, Shamseer P, Bruce W, Newman L, Shen B, Zheng P, Bidney D, Falco C, Register J, Zhao ZY, Xu D, Jones T, GordonKamm W. 2016. Morphogenic regulators Baby boom and wuschel improve monocot transformation. Plant Cell 28(9):1998–2015. doi:10.1105/tpc.16.00124.

Martínez MT, San­José MdC, Arrillaga I, Cano V, Morcillo M, Cernadas MJ, Corredoira E. 2019. Holm oak somatic embryogenesis: Current status and future perspectives. Front. Plant Sci. 10:239. doi:10.3389/fpls.2019.00239.

Ming NJ, Binte Mostafiz S, Johon NS, Abdullah Zulkifli NS, Wagiran A. 2019. Combination of plant growth regulators, maltose, and partial desiccation treatment enhance somatic embryogenesis in selected Malaysian rice cultivar. Plants 8(6):144. doi:10.3390/plants8060144.

Mostafiz SB, Wagiran A. 2018. Efficient callus induction and regeneration in selected indica rice. Agronomy 8(5):77. doi:10.3390/agronomy8050077.

Pais MS. 2019. Somatic embryogenesis induction in woody species: The future after OMICs data assessment. Front. Plant Sci. 10:240. doi:10.3389/fpls.2019.00240.

Ren B, Liang Y, Deng Y, Chen Q, Zhang J, Yang X, Zuo J. 2009. Genome­Wide comparative analysis of typeA Arabidopsis response regulator genes by overexpression studies reveals their diverse roles and regulatory mechanisms in cytokinin signaling. Cell Res. 19(10):1178–1190. doi:10.1038/cr.2009.88.

Tran TN, Sanan­Mishra N. 2015. Effect of antibiotics on callus regeneration during transformation of IR 64 rice. Biotechnol. Rep. 7:143–149. doi:10.1016/j.btre.2015.06.004.

Umar R, Wibisono Y, Ermawati N. 2017. Effect of medium compositions on the growth of rice (Oryza sativa L. cv. Ciherang) callus. UNEJ e­Proceeding p. 97–100. URL iding/article/view/4149.

Vercruyssen L, Tognetti VB, Gonzalez N, Van Dingenen J, De Milde L, Bielach A, De Rycke R, Van Breusegem F, Inzé D. 2015. Growth regulating factors Stimulates Arabidopsis chloroplast division, photosynthesis, and leaf longevity. Plant Physiol. 167(3):817– 832. doi:10.1104/pp.114.256180.

Wen CK, editor. 2015. Ethylene in Plants. Dordrecht: Springer Netherlands. doi:10.1007/978­94­ 017­9484­8. Yamburenko MV, Worthen JM, Zeenat A, Azhar BJ, Swain S, Couitt AR, Shakeel SN, Kieber JJ, Schaller GE. 2020. Functional analysis of the rice type­B response regulator RR22. Front. Plant Sci. 11:577676. doi:10.3389/fpls.2020.577676.

Yang C, Lu X, Ma B, Chen SY, Zhang JS. 2015. Ethylene signaling in rice and Arabidopsis: Conserved and diverged aspects. Mol. Plant 8(4):495–505. doi:10.1016/j.molp.2015.01.003.

Yang D, Peng S, Wang F. 2020. Response of photosynthesis to high growth temperature was not related to leaf anatomy plasticity in rice (Oryza sativa L.). Front. Plant Sci. 11:26. doi:10.3389/fpls.2020.00026.

Yasmin S, Mensuali­Sodi A, Perata P, Pucciariello C. 2013. Ethylene influences in vitro regeneration frequency in the FR13A rice harbouring the SUB1A gene. Plant Growth Regul. 72(1):97–103. doi:10.1007/s10725­013­9840­5.


Article Metrics

Abstract views : 813 | views : 825


  • There are currently no refbacks.

Copyright (c) 2022 The Author(s)

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