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The Expression Analysis of Atrkd4 Transgene in Dendrobium Lineale Rolfe Transgenic Orchid Carrying 35S::GR::Atrkd4 for Micropropagation

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The Expression Analysis of Atrkd4 Transgene in Dendrobium Lineale Rolfe Transgenic Orchid Carrying 35S::GR::Atrkd4 for Micropropagation See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/345098402 The expression analysis of AtRKD4 transgene in Dendrobium lineale Rolfe transgenic orchid carrying 35S::GR::AtRKD4 for micropropagation Conference Paper in AIP Conference Proceedings · September 2020 DOI: 10.1063/5.0015876 CITATIONS READS 3 42 4 authors, including: Ni Luh Putu Kayika Febryanti Steffanie Nurliana Universitas Gadjah Mada Universitas Bengkulu 3 PUBLICATIONS 4 CITATIONS 6 PUBLICATIONS 8 CITATIONS SEE PROFILE SEE PROFILE Endang Semiarti Universitas Gadjah Mada 93 PUBLICATIONS 1,301 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: Biology Orchid Study Club (BiOSC) Strategic Researches View project Biology Orchid Study Club (BiOSC): Orchids Biodiversity and Conservation View project All content following this page was uploaded by Steffanie Nurliana on 28 February 2021. The user has requested enhancement of the downloaded file. The expression analysis of AtRKD4 transgene in Dendrobium lineale Rolfe transgenic orchid carrying 35S::GR::AtRKD4 for micropropagation Cite as: AIP Conference Proceedings 2260, 060021 (2020); https://doi.org/10.1063/5.0015876 Published Online: 16 September 2020 Ni Luh Putu Kayika Febryanti, Steffanie Nurliana, Jose Gutierrez-Marcos, and Endang Semiarti ARTICLES YOU MAY BE INTERESTED IN The expression of AtRKD4 transgene during induction of somatic embryogenesis in transgenic Dendrobium phalaenopsis orchid carrying 35S::GR::AtRKD4 AIP Conference Proceedings 2260, 060015 (2020); https://doi.org/10.1063/5.0015873 Agrobacterium-mediated transformation facillitates the CRISPR/Cas9 genome editing system in Dendrobium macrophyllum A. Rich orchid AIP Conference Proceedings 2260, 060016 (2020); https://doi.org/10.1063/5.0016200 Isolation and characterisation of putative embryo gene DlRKD4 from Indonesian orchid Dendrobium lineale rolfe AIP Conference Proceedings 2260, 060008 (2020); https://doi.org/10.1063/5.0015867 AIP Conference Proceedings 2260, 060021 (2020); https://doi.org/10.1063/5.0015876 2260, 060021 © 2020 Author(s). The Expression Analysis of AtRKD4 Transgene in Dendrobium lineale Rolfe Transgenic Orchid Carrying 35S::GR::AtRKD4 for Micropropagation Ni Luh Putu Kayika Febryanti1, Steffanie Nurliana2, Jose Gutierrez-Marcos3 and Endang Semiarti4, a) 1Post Graduate Program of Biology, Department of Tropical Biology, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia 2 Universitas Bengkulu, Faculty of Mathematics and Natural Sciences, Indonesia 3 University of Warwick, School of Life Science, England 4Biotechnology Laboratory, Faculty of Biology, Universitas Gadjah Mada, Jl. Teknika Selatan, Mlati, Sleman, Yogyakarta 55281, Indonesia a) Corresponding author: [email protected] Abstract. Dendrobium lineale Rolfe is an endemic orchid of Papua, Indonesia. Mass propagation of D. lineale has been performed through the induction of somatic embryogenesis by inserting AtRKD4 embryo gene from Arabidopsis thaliana. This research objective is to confirm whether the AtRKD4 in T-DNA that harbor 35S::GR::AtRKD4 can express properly in D. lineale transnsformant in order to induce somatic embryos (SEs). The leaves from 10 plantlets were cultivated in New Phalaenopsis (NP) medium added with 15 µM Dexamethasone (DEX) or 3 mg.L-1 Thidiazuron (TDZ) for SE induction and NP0 as control. The expression was analyzed in developmental stage of 1-, 3-, 5-days and 7-days after induction by DEX and TDZ. The expression of AtRKD4 in orchid genome was detected by Polymerase Chain Reaction (PCR) using AtRKD4 specific primers. The result showed that the transgene expression was detected as 382 bp fragment of cDNAs amplified from transformants after 1-, 3-, 5-and 7-days induction by DEX and TDZ, with the highest expression was after 5 days induction, and probably will induce somatic embryogenesis from leaves. In conclusion, AtRKD4 transgene can be activated by using DEX and TDZ after 1-7-days cultivation with the optimal induction in 5 days. INTRODUCTION The Orchidaceae is one of the largest and most diverse families of flowering plants, consists of 20,000-35,000 species. Dendrobium lineale Rolfe is one of endemic orchid of Papua, Indonesia [1]. At present, D.lineale orchids are increasingly rare in wild land, and threatened to be extinct because of overcollection, degradation or even total destruction of their natural habitats. It takes effort to multiply both in situ and ex situ to keep this orchid from the threat of extinction [2] Mass propagation by tissue culture technique is one method that can be used for the conservation of this orchid to produce high number of plantlets [3]. Tissue culture using orchid germination from seeds is difficult, as the seeds lack of endosperm and contain poorly differentiated embryos, rendering them ineffective for producing large quantities of seedlings [4]. Therefore, one of the promising methods is through production of somatic embryos (SEs). SE is embryos that developed from somatic cells but similar to zygotic embryo, including its growth patterns and the developmental stages [5]. SEs can be induced by using plant growth regulation (PGRs) such as Thidiazuron (TDZ) and through insertion of embryo gene AtRKD4 into orchid protocorms using Agrobacterium-mediated transfomation [6, 7]. According to [8] NP medium supplemented with 3 mgL-1 TDZ was the best medium to induce The 6th International Conference on Biological Science ICBS 2019 AIP Conf. Proc. 2260, 060021-1–060021-6; https://doi.org/10.1063/5.0015876 Published by AIP Publishing. 978-0-7354-2020-5/$30.00 060021-1 direct SEs formation from all various explants such as leaves, protocorm and stem of orchid P. amabilis. Besides that, AtRKD4 is one of the genes that can induce somatic cell changes into embryogenic cells. AtRKD4 gene encodes a protein with RWP-RK motif in Arabidopsis that required for embryonic pattern formation in a very early stage of embryo initiation [9]. Mass propagation of D.lineale orchids has been performed through the induction of somatic embryogenesis by inserting AtRKD4 embryo gene from Arabidopsis that carrying 35S::GR::AtRKD4 in plasmid pTA7002. Overexpression of the transgene in these systems could only be induced in transgenic plants when there is glucocorticoid response element (GRE) in the plant genome and the existence of glucocorticoid hormone in the medium [2]. Utilization of glucocorticoid regulation (GR) is a general tool for regulating gene expression in plant cells and whole plants [10]. GR has been well adopted for this purpose in plants to generate steroid-inducible plants. In orchid, [2] was successfully induced SEs in D. phalaenopsis after the AtRKD4 transgene in the genome was induced by using synthetic steroid hormone, DEX. In the absence of DEX, the complex of transcription factors and the hormone-binding domain of the glucocorticoid receptor (TF-GR) are held in an inactive state, due to their cytoplasmic localization [11]. In this paper we report the use of TDZ as PGRs and glucocorticoid selective inducible system DEX to express the AtRKD4 transgene in D. lineale orchid to induce somatic embryos. METHODS Plant Materials and Construct The leaves from 10 one year old Agrobacterium-mediated transformed plantlets that carrying T-DNA with the construct of 35S::GR::AtRKD4 (Fig.1) were used as plant materials. The T-DNA contains multiple strong promoters 35S, GVG, GAL 4 and VP16 with inducible glucocorticoid response element (GRE) for selective induction system with glucocorticoid hormone (DEX) (Fig. 2). The leaves from transformant plants carrying 35S::GR::AtRKD4 were cultivated in New Phalaenopsis (NP) medium added with 15 µM DEX or 3 mg.L-1 TDZ for SE induction and hormone free-NP (NP0) medium was used as control experiment. FIGURE 1. Plantlets of D.lineale Transformant carrying 35S::GR::AtRKD4. Two non-transformants (NT#1, NT#2) and 10 putative transformants (T#1-T#10:transformant lines no 1-10) of one year old plant. Bar = 1 cm 060021-2 FIGURE 2. The structure of T-DNA harboring 35S::GR::AtRKD4 in plasmid pTA7002/EHA 105 [7]. Detection of AtRKD4 Integration in The Genome of Orchid Transformant Detection of AtRKD4 in orchid genome was confirmed using 2 non-transformant and 10 putative transformants. DNA samples were analyzed by direct PCR using by MyTaq™ HS Red Mix (Bioline) with AtRKD4 specific primers that amplified 382 bp DNA band and Actin primers as positive control that amplified 545 bp DNA bands, respectively. The PCR reactions were as follows: an initial denaturation at 95 °C for 1 minutes, denaturation at 95 °C for 15 seconds, annealing at 58 °C (AtRKD4 and and 51 ˚C (Actin for 15 seconds, extension (polymerization) at 72 ˚C for 10 seconds, and cooling at 4 °C for 10 minutes. The PCR process was performed in 35 cycles. The PCR products were separated on 1 % agarose gel in TAE 1X buffer at 50 volts for 50 min and visualized under UV transilluminator (Bio-Rad). Induction of AtRKD4 Gene Expression in Transformant Plant The leaves originated from transformant and non-transformant plantlets, which grown on NP0 media were subsequently taken and cut into pieces, and then planted on NP medium containing 15 μM DEX or 3 mg.L-1 TDZ for 1-, 3-, 5-days and 7-days cultivation to induce AtRKD4 gene expression.The cultures were maintained under continues white light, at 250C incubation. Analysis of 35S::GR::AtRKD4 Gene Expression in D. lineale Transformant Leaves AtRKD4 gene expression was analyzed by isolating total RNA from leaves of two non transformant and ten transformant candidates. A population of mRNA was isolated from developmental stage of 1-days, 3-days, 5- days and 7-days after induction using 15 µM DEX or 3 mg.L-1 TDZ in vitro cultivated leaves by using TRIzol reagent (ambion). The cDNA was synthesized by iScript cDNA synthesis Kit (Bio-Rad). Synthesized cDNA were used to analyze the expression of AtRKD4 gene and the housekeeping gene Actin was used as an internal control for Reverse transcriptase-PCR (RT-PCR).
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