The effectivity of thidiazuron and 1‐naphthaleneacetic acid on somatic embryo induction in transgenic Dendrobium phalaenopsis Fitzg. carrying 35S::GR::AtRKD4

Muhammad Ilham(1), Fitriana Puspitasari(2), Endang Semiarti(3*)

(1) Department of Tropical Biology, Faculty of Biology, Universitas Gadjah Mada, Jl. Teknika Selatan, Sekip Utara, Yogyakarta 55281, Indonesia
(2) Department of Tropical Biology, Faculty of Biology, Universitas Gadjah Mada, Jl. Teknika Selatan, Sekip Utara, Yogyakarta 55281, Indonesia
(3) Department of Tropical Biology, Faculty of Biology, Universitas Gadjah Mada, Jl. Teknika Selatan, Sekip Utara, Yogyakarta 55281, Indonesia
(*) Corresponding Author


Dendrobium phalaenopsis Fitzg. (also known as the Larat orchid) is an endemic orchid from Larat Island, Eastern Indonesia. Its beautiful flowers mean that many plants are taken for commercial purposes, leading to the rapid decline of populations in their natural habitats. The objectives of this study were to determine which organs of the transgenic Larat orchid carrying the 35S::GR::AtRKD4 construct, together with which concentrations of the plant growth regulators (PGRs) auxin and cytokinin, are suitable for the induction of somatic embryos (SEs). In this study, the AtRKD4 gene in Larat orchids was confirmed using PCR with specific primers for the AtRKD4 and HPT genes. Thidiazuron (TDZ) (1, 3 and 5 mg/L) in combination with 1‐naphthaleneacetic acid (NAA) (0.5 and 1 mg/L) were used on new phalaenopsis (NP) medium to induce SEs from leaves, pseudobulbs and roots. The AtRKD4 transgenes were detected as being stably integrated into the DNA genome of transformant plants using specific primers for AtRKD4 and HPT genes, and positive results were obtained using actin gene primers as internal controls for PCR. Pseudobulbs produced 19 to 20 SEs from 108 pseudobulb explants (89–100%), a higher number than produced in explants of the other organs studied. Among the PGR treatments, the best results were obtained in NP medium supplemented with a combination of 1 mg/L TDZ and 1 mg/L NAA, 100% of the explants of which produced SEs (2.11 ± 1.36). No significant difference was found between the morphology of the SEs produced from the non‐transformant Larat orchid pseudobulb explants and the 35S::AtRKD4 carrier transformant.


Dendrobium phalaenopsis Fitzg; AtRKD4; Somatic Embryos ; NAA; TDZ

Full Text:



Balilashaki K, Ghehsareh MG. 2016. Micropropagation of Phalaenopsis amabilis var. ‘Manila’ by leaves obtained from in vitro culturing the nodes of flower stalks. Not. Sci. Biol. 8(2):164–169. doi:10.15835/nsb.8.2.9782.

Bayer M, Slane D, Jürgens G. 2017. Early plant embryogenesis — Dark ages or dark matter? Curr. Opin. Plant Biol. 35:30–36. doi:10.1016/j.pbi.2016.10.004.

Chardin C, Girin T, Roudier F, Meyer C, Krapp A. 2014. The plant RWP­RK transcription factors: Key regulators of nitrogen responses and of gametophyte development. J. Exp. Bot. 65(19):5577–5587. doi:10.1093/jxb/eru261.

de Melo Ferreira W, Barbante Kerbauy G, Elizabeth Kraus J, Pescador R, Mamoru Suzuki R. 2006. Thidiazuron influences the endogenous levels of cytokinins and IAA during the flowering of isolated shoots of Dendrobium. J. Plant Physiol. 163(11):1126–1134. doi:10.1016/j.jplph.2005.07.012.

Febryanti NLPK, Nurliana S, Gutierrez­Marcos J, Semiarti E. 2020. The expression analysis of AtRKD4 transgene in Dendrobium lineale rolfe transgenic orchid carrying 35S::GR::AtRKD4 for micropropagation. In: AIP Conf. Proc., volume 2260. p. 060021. doi:10.1063/5.0015876.

Feng JH, Chen JT. 2014. A novel in vitro protocol for inducing direct somatic embryogenesis in Phalaenopsis aphrodite without taking explants. Sci. World J. 2014:1–7. doi:10.1155/2014/263642.

Guo B, Abbasi BH, Zeb LL A ad Xu, Wei YH. 2011. Thidiazuron: a multidimension plant growth regulator. Afr. J. Biotechnol. 10(45):8984–9000. doi:10.5897/AJB11.636.

Islam MO, Ichihashi S, Matsui S. 1998. Control of growth and development of protocorm like body derived from callus by carbon sources in Phalaenopsis. Plant Biotechnol. 15(4):183–187. doi:10.5511/plantbiotechnology.15.183.

Ivakdalam LM, Pugesehan DJ. 2016. Keragaman jenis tanaman anggrek (Orchidaceae) di Cagar Alam Angwarmase, Kabupaten Maluku Tenggara Barat. J. Agroforestri 11(3):163–168.

Ji A, Geng X, Zhang Y, Wu G. 2011. Advances in somatic embryogenesis research of horticultural plants. Am. J. Plant Sci. 02(06):727–732. doi:10.4236/ajps.2011.26087.

Kim DH, Kang KW, Sivanesan I. 2019. Influence of auxins on somatic embryogenesis in Haworthia retusa Duval. Biologia (Bratisl). 74(1):25–33. doi:10.2478/s11756­018­0151­1.

Lee MH, Lee J, Choi SH, Jie EY, Jeong JC, Kim CY, Kim SW. 2020. The effect of sodium butyrate on adventitious shoot formation varies among the plant species and the explant types. Int. J. Mol. Sci. 21(22):8451. doi:10.3390/ijms21228451.

Li Z, He Y. 2020. Roles of brassinosteroids in plant reproduction. Int. J. Mol. Sci. 21(3):1–16. doi:10.3390/ijms21030872.

Mahadi I. 2017. Multifikasi tunas anggrek larat (Dendrobium phalaenopsis Fitzg.) dengan pemberian hormon IAA dan BAP terhadap pertumbuhan secara in vitro. Eksakta 2(XVII):1–6.

Mahendran G, Bai VN. 2016. Direct somatic embryogenesis of Malaxis densiflora (A. Rich.) Kuntze. J. Genet. Eng. Biotechnol. 14(1):77–81. doi:10.1016/j.jgeb.2015.11.003.

Meira FF, Luis ZG, de Araújo Silva­Cardoso IM, Everson Scherwinski­Pereira J. 2019. Developmental pathway of somatic embryogenesis from leaf tissues of macaw palm (Acrocomia aculeata) revealed by histological events. Flora Morphol. Distrib. Funct. Ecol. Plants 250:59–67. doi:10.1016/j.flora.2018.11.011.

Mose W, Daryono BS, Indrianto A, Purwantoro A, Semiarti E. 2020. Direct somatic embryogenesis and regeneration of an Indonesian orchid Phalaenopsis amabilis (L.) Blume under a variety of plant growth regulators, light regime, and organic substances. Jordan J. Biol. Sci. 13(4):509–518. URL jo/files/vol13/n4/Paper%20Number%2013.pdf.

Mose W, Indrianto A, Purwantoro A, Semiarti E. 2017. The influence of thidiazuron on direct somatic embryo formation from various types of explant in Phalaenopsis amabilis (L.) Blume orchid. HAYATI J. Biosci. 24(4):201–205. doi:10.1016/j.hjb.2017.11.005.

Murray MG, Thompson WF. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8(19):4321–4326. doi:10.1093/nar/8.19.4321.

Mursyanti E, Purwantoro A, Moeljopawiro S, Semiarti E. 2015. Induction of somatic embryogenesis through overexpression of ATRKD4 genes in Phalaenopsis “Sogo Vivien”. Indones. J. Biotechnol. 20(1):42–53. doi:10.22146/ijbiotech.15276.

Nolan T, Chen J, Yin Y. 2017. Cross­talk of Brassinosteroid signaling in controlling growth and stress responses. Biochem. J. 474(16):2641–2661. doi:10.1042/BCJ20160633.

Oliveira EJ, Koehler AD, Rocha DI, Vieira LM, Pinheiro MVM, de Matos EM, da Cruz ACF, da Silva TCR, Tanaka FAO, Nogueira FTS, Otoni WC. 2017. Morpho­histological, histochemical, and molecular evidences related to cellular reprogramming during somatic embryogenesis of the model grass Brachypodium distachyon. Protoplasma 254(5):2017–2034. doi:10.1007/s00709­017­1089­9.

Ouyang Y, Chen Y, Lü J, Teixeira Da Silva JA, Zhang X, Ma G. 2016. Somatic embryogenesis and enhanced shoot organogenesis in Metabriggsia ovalifolia W. T. Wang. Sci. Rep. 6:24662. doi:10.1038/srep24662.

Perdana NGA, Mose W, Lawrie MD, Gutierrez­Marcos J, Semiarti E. 2017. Stable transformant of Phalaenopsis amabilis somatic embryo carrying 35S::GR::AtRKD4 develops into normal phenotype of transgenic plant. J. Tropical Biodiversity Biotechnology 06(02):jtbb59210. doi:10.22146/jtbb.59210.

Picard D. 1993. Steroid­binding domains for regulating the functions of heterologous proteins in Cis. Trends Cell Biol. 3(8):278–280. doi:10.1016/09628924(93)90057­8.

Planas­Riverola, Gupta A, Betegon­Putze I, Bosch N, Ibanes M, Cano­Delgado AI. 2019. Brassinosteroid signaling in plant development and adaptation to stress. Development 146(5):dev151894. doi:10.1242/dev.151894.

Puspitasari F, Sutra CL, Sundari D, Wirajagat GC, Setiari N, Gutierrez­Marcos J, Semiarti E. 2020. Stability of AtRKD4 gene integration in the genome of Dendrobium phalaenopsis fitzg. Transformants induces somatic embryogenesis. Propag. Ornam. Plants. 20(4):129–138.

Semiarti E, Indrianto A, Purwantoro A, Machida Y, Machi C. 2011. Agrobacterium­mediated transformation of Indonesian orchids for micropropagation. London, United Kingdom: IntechOpen. doi:

Semiarti E, Mursyanti E, Suyoko A, Perdana FSW, Widyastuti CT, Subchan AN. 2018. Stability of T­DNA integration in Phalaenopsis “Sogo Vivien” transgenic orchid carrying 35S::Gal4::AtRKD4::GR. Biol. Med. Nat. Prod. Chem. 7(1):5–13. doi:10.14421/biomedich.2018.71.5­13.

Setiari N, Purwantoro A, Moeljopawiro S, Semiarti E. 2018. Micropropagation of Dendrobium phalaenopsis orchid through overexpression of embryo gene AtRKD4. Agrivita 40(2):284–294. doi:10.17503/agrivita.v40i2.1690.

Sherif NA, Franklin Benjamin JH, Senthil Kumar T, Rao MV. 2018. Somatic embryogenesis, acclimatization and genetic homogeneity assessment of regenerated plantlets of Anoectochilus elatus Lindl., an endangered terrestrial jewel orchid. Plant Cell. Tissue Organ Cult. 132(2):303–316. doi:10.1007/s11240­017­ 1330­4.

Sutikno. 2018. Plant Microtechnic Handbook for Plant Structure and Development Laboratory. Yogyakarta, Indonesia: Biology Faculty, Universitas Gadjah Mada.

Tang J, Han Z, Chai J. 2016. Q&A: What are brassinosteroids and how do they act in plants. BMC Biol. 14(113):1–5. doi:10.1186/s12915­016­0340­8.

Waki T, Hiki T, Watanabe R, Hashimoto T, Nakajima K. 2011. The arabidopsis RWP­RK protein RKD4 triggers gene expression and pattern formation in early embryogenesis. Curr. Biol. 21(15):1277–1281. doi:10.1016/j.cub.2011.07.001.

Zulkaidha Z, Muslimin M, Hapid A, Toknok B. 2019. Upaya Konservasi Tanaman Hias Anggrek Melalui Perbanyakan Secara Vegetatif dan Kultur Jaringan. In Seminar Nasional Hasil Penelitian dan Pengabdian Kepada Masyarakat (SNP2M) p. 217–221.

Zulwanis, Setiari N, Gutierrez­Marcos J, Semiarti E. 2020. The expression of AtRKD4 transgene during induction of somatic embryogenesis in transgenic Dendrobium phalaenopsis orchid carrying 35S::GR::AtRKD4. AIP Conf. Proc. 2260:060015– 1–060015–6. doi:10.1063/5.0015873.


Article Metrics

Abstract views : 368 | views : 540


  • 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.