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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

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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

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© 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

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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). RT-PCR was conducted using a pair of specific primers of AtRKD4 gene and Actin primers with PCR method under amplification reaction the same as described above.

RESULTS AND DISCUSSION

Putative Transformant Confirmation

Transgene detection in samples of leaves from non transformant and transformant candidates was done by amplifying a target DNA fragment from AtRKD4 and HPT genes. The integration of HPT (545 bp) and AtRKD4 (382 bp) genes were detected in the genome of D. lineale transformant candidates (Fig. 3a and 3b). It was confirmed with the amplification of Actin (114 bp) as an internal control for PCR reaction (Fig. 3c). The detected AtRKD4 gene in the orchid genome has shown that the AtRKD4 gene was successfully integrated into the orchid genome. It is important to confirm that the AtRKD4 gene really integrated in the genome of D. lineale transformants. Hygromycin phosphotransferase (HPT) is an enzyme produced by Streptomyces hygroscopicus bacteria that is resistant to hygromycin, therefore it can be used as selection marker to obtain positive transformant [2]. Detection of the HPT gene in the orchid genome suggested that the plantlets of transformant candidates contained an integrated gene that was resistant to hygromycin antibiotics. The plantlets could grow well on medium containing hygromycin. Actin gene transcription (“house-keeping gene” as positive control) plays key roles in cell motility and cytoskeleton maintenance. Therefore, Actin is assumed to be constitutively expressed and widely used as reference gene [12].

060021-3 Actin appeared in all samples both transformant and non-transformant samples (Fig. 3c). This indicated that the Actin gene was expressed in all plant samples, both transformants and non-transformants but AtRKD4 and HPT genes were expressed only in transformant plants.

FIGURE 3. Detection of HPT and AtRKD4 transgenes integration in the genome of D. lineale transformants. a). 545 bp DNA fragment of HPT gene; b). 382 bp DNA fragment of AtRKD4 transgene; c). 114 bp fragment of Actin gene. Lane 1-2 Non- transformant plants; Lane 3-12 Transformant plants

The expression of AtRKD4 transgene after induction by DEX and TDZ

The expression of AtRKD4 using DEX is necessary due to the T-DNA construct in pTA7002 plasmid contained glucocorticoid response element (GRE) which can be activated after being treated with steroid hormone such as DEX. Leaves were cultivated on NP medium containing 15 M DEX and 3 mgL-1 TDZ for 4 time points i.e 1, 3, 5, and 7-days. It is necessary to use several time points because the gene expression changes is dependent on the dose and duration of the DEX treatment [11]. In this study, TDZ which plays role in somatic embryo induction was also being used as an alternative agent to activate AtRKD4 gene expression. The result showed that the AtRKD4 gene expression detected as 382 bp fragment of cDNAs after 1-days, 3-days, 5- days and 7-days of induction by DEX and TDZ (Fig. 4).

FIGURE 4. Expression of AtRKD4 induced by DEX and TDZ in D.lineale transformants. a). One-day after induction ; b). 3-days after induction ; c). 5-days after induction ; and d). 7-days after induction.

According to [2] gene expression analysis of AtRKD4 using protocorms and [7] using leaves was planted on NP medium containing 15 μM DEX in order to induce somatic embryo formation for 7 days. However we found that the expression of AtRKD4 have been detected in 1-day since induction with either DEX or TDZ (fig. 4a) with the thickest bands were found on 5-days induction with either DEX or TDZ. We assumed that the highest rate of

060021-4 AtRKD4 expressions occured at 5-days induction (fig. 4c) and gradually reduced the expression up to 7-days induction (fig. 4d). This data suggest that activation of AtRKD4 gene in transformant could be induced rapidly by DEX just shortly in one-day, then gradually increased and optimal on 5-days induction, then gradually become lower again as detected after 7-days induction, indicating the dynamic of AtRKD4 gene expression occurrence in transformant plants. In this study, it is needed to activate the AtRKD4 gene using GVG inducible system which responds to the synthetic glucocorticoid DEX. DEX works traverses the plasma membrane by diffusion and binds to the glucocorticoid receptor (GR), which exists in the unliganded state, as a cytoplasmic complex with the 90kDa heat shock protein (HSP90). Dissociation of HSP90 leads by the ligand binding to the rapid localization of the liganded form of the receptor into the nucleus. Inside the nucleus, the GR binds to specific DNA sequences (GREs) and activates the expression of AtRKD4 gene [13]. The result also shows that TDZ as a synthetic cytokinin can act as an alternative agent to induce the expression of AtRKD4 gene. Expression of AtRKD4 with TDZ 3 mg.L-1 also showed similar dynamic pattern with the use of DEX. Based on [13], in an ideal condition, there is expectantly a high expression level in the presence of an steroid inducer in inducible promoter and a low level of gene expression in the absence of inducer. In this case, TDZ is not a group of glucocorticoid hormone. TDZ is a substituted phenylurea compound which has a highly efficacious bioregulant of morphogenesis in the tissue culture and have cultural responses such as induction of callus and formation of somatic embryos [14]. It can be assumed that the presence of TDZ increases the amount of HSP90 as a co-chaperon in signaling of plant cytokinins. As a co-chaperon, HSP90 stimulate the increasement of HSP90, therefore the assosiaction of HSP90 and GVG will be disturb and the GVG can enter the nucleus to activate the AtRKD4 gene. HSP90s are essential for housekeeping functions and formed as chaperon or co-chaperon in cytoplasm which can interact with various plant hormones. As a chaperon, HSP90 could interact with F-box proteins that participate in plant hormone signaling. In Giberellin signaling, HSP90 can be associated with SCF (Skp, Cullin, F-box-containing) and is able to recognize negative regulators in Giberellin signaling known as DELLA protein and direct DELLA to the ubiquitin-dependent degradation pathway [15]. On the other hand, it also can be assumed that TDZ has capacity to stimulate Brassinosteroids. Brassinosteroids are plant natural products with the same function with TDZ and have similar structure as animal steroid hormones. Brassinosteroids contain Brassinolide which is the most bioactive form of the growth promoting plant steroids [16]. According to [17] brassinosteroids generally acts via a soluble receptor–ligand complex that binds to nuclear sites to regulate the expression of specific genes and these facts as well as animal steroid hormones that suggest brassinosteroids may be acts as one of the critical signals controlling plant growth and development. It strongly suggested that TDZ can be used as an alternative agent to activate the expression of AtRKD4 transgene on 35S::GR::AtRKD4. Further experiments using transformant plants that activate glucocorticoid receptors are needed at both RNA and protein levels to find out how long the induction of embryo gene AtRKD4 activation can be performed on D. lineale orchid transformants.

CONCLUSION

The expression of AtRKD4 transgene in D. lineale orchid plant transformant carrying 35S::GR::AtRKD4 occurred 1 day after being induced with Dexamethasone and Thidiazuron, and the highest expression was achieved on the fifth day to induce somatic embryos (SEs).

ACKNOWLEDGEMENT

This reseaerch was supported by grant from Ministry of Research and Technology Higher Education RI on the Scheme of Rekognisi Tugas Akhir (RTA) 2019 Contract No. 2129/UN1/DITLIT/DIT-LIT/LT/2019 to Endang Semiarti as PI.

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