In Vitro Propagation and DNA Barcode Analysis of the Endangered Silene Schimperiana in Saint Katherine Protectorate

Ghareb et al. Journal of Genetic Engineering and Biotechnology (2020) 18:41 Journal of Genetic Engineering https://doi.org/10.1186/s43141-020-00052-8 and Biotechnology RESEARCH Open Access In vitro propagation and DNA barcode analysis of the endangered Silene schimperiana in Saint Katherine protectorate Heba El-Sayed Ghareb1, Shafik Darwish Ibrahim2 and Ghada Abd El-Moneim Hegazi1* Abstract Background: Anthropogenic activity, climate change, pollution, and exploitation of natural resources are some reasons that cause threatening of plant diversity. Silene schimperiana is an endangered plant species in Egypt and is endemic to the high mountain of Saint Katherine Protected Area in southern Sinai. The purpose of the study was the ex situ conservation of Silene schimperiana through in vitro propagation and DNA barcode analysis. Results: To develop an efficient ex situ conservation program of the plant, in vitro propagation protocol has been achieved from shoot tip and stem nodal segment explants of in vitro germinated seedlings. Explants were established in vitro on Murashige and Skoog (MS) medium supplemented with 2.89 μM gibberellic acid (GA3), 1.08 μM α-naphthaleneacetic acid (NAA), and 1.16 μM kinetin (Kin). The highest number of axillary shoots (9.27) was obtained when they were transferred to MS medium supplemented with 4.48 μM 6-benzyl adenine (BA). Hundred percent of multiple axillary shoots were rooted on quarter-strength MS medium supplemented with 4.92 μM indole-3-butyric acid (IBA) and 10.75 μM NAA. Rooted plants were transferred to pots containing a soil-peat mixture (1: 2 v/v) and successfully acclimatized in the greenhouse. Plant identification is a crucial aspect to understand and conserve plant diversity from extinction. DNA barcode analysis of Silene schimperiana was carried out using two chloroplast DNA markers (cpDNA): 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) and RNA polymerase subunit (rpoC1) and a nuclear ribosome DNA marker (ncDNA), internal transcribed spacer (ITS). Phylogenetic analysis revealed a successful identification of Silene schimperiana on the species and genus levels and supported the inclusion of Silene schimperiana in genus Silene. Conclusions: In this study, a relevant in vitro propagation method was established to facilitate the recovery of Silene schimperiana, in addition to DNA barcoding of the plant as a tool for effective management and conservation of plant genetic resources. Keywords: Caryophyllaceae, Micropropagation, DNA barcoding, Southern Sinai, Egypt * Correspondence: [email protected] 1Tissue Culture Unit, Department of Genetic Resources, Desert Research Center, 1 Mathaf El-Matareya Street, Cairo, El-Matareya 11357, Egypt Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Ghareb et al. Journal of Genetic Engineering and Biotechnology (2020) 18:41 Page 2 of 15 Background Studying the molecular phylogeny of plants depends Saint Katherine Protectorate is one of the largest pro- mainly on the sequencing of the chloroplast genome, be- tected areas in Egypt with the highest mountains. It sup- cause of its simple and stable genetic structure. Univer- ports surprising biodiversity and a high proportion of sal primers are used for these target sequences endemic and rare plants. The flora of the mountains dif- amplification, such as 1,5-bisphosphate carboxylase/oxy- fers from the other areas, due to its unique geological, genase large subunit (rbcL) and RNA polymerase sub- morphological, and climatic aspects [1]. The protection unit (rpoC1) have been heavily relied upon for the and conservation of endemic plant species is a world- development of markers for plant DNA barcoding [15]. wide need; especially under the threat of climate change. The Consortium for the Barcode of Life (CBOL) [16] Endemic species are the most defenseless, because of plant working group evaluated the efficacy of the chloro- their unique evolutionary history and low population es- plast DNA marker (cpDNA), rbcL among the recom- timate [2]. mended markers. The rbcL gene is the most published Silene schimperiana Boiss. is a perennial herb, belong- plastid barcode due to its high amplification success rate ing to the largest genus from family Caryophyllaceae. Its and is considered a core DNA barcoding marker for the Arabic name is Wesbi or Losseiq [3]. The plant is a determination of plant diversity [13, 17, 18]. Besides, the hemicryptophyte, growing on rocky wadi beds. It is po- nuclear ribosome DNA marker (ncDNA), internal tran- tentially an ornamental plant with attractive white scribed spacer (ITS) and cpDNA, rpoC1 were frequently flowers [4] and has economic importance in grazing pro- evaluated as plant barcodes [12, 19]. DNA barcoding cesses as a pastoral plant [5]. markers are universal and the choice of the correct loci Silene schimperiana is endangered in Egypt and is is challenging [20]. endemic to the high mountain of Saint Katherine To date, there has been no published researches on Protected Area in southern Sinai. The plant popula- the in vitro culture or DNA barcoding of Silene schim- tions are severely fragmented with a continuing de- periana and therefore, the aim of the present study was cline in habitat quality [5]. Drought is the major the ex situ conservation of Silene schimperiana as an en- threat affecting the distribution of Silene schimperi- dangered plant, endemic to Saint Katherine, through ana, in addition to climate change. The plant has low in vitro propagation and identification by DNA barcode viability due to destructive overgrazing, causing loss analysis using two cpDNA genes: rpoC1 and rbcL and a of reproductive organs. With drought, the effect of ncDNA gene, ITS. overgrazing is more harmful and the wild populations of this species could be in extreme danger in the near Methods future [5]. Ex situ conservation by in vitro propaga- Plant material tion and molecular identification are important ac- Leaf specimens and seeds of Silene schimperiana were tions needed to conserve the plant [5]. collected from shrubs grown in Gebel Tennia, Saint In vitro propagation of other species of Silene have Katherine, South of Sinai E: 33.90090 N: 28.57364 Alt: been achieved previously, such as S. leucophylla [6], S. 1810 (Figs. 1 and 2). Identification of plant was carried fetissovii, S. obovata, S. sussamyrica and S. ladyginae [7], out by Dr. Omran Ghaly, Head of Plant Taxonomy Unit, Silene cretacea [8], Silene bolanthoides [9]andSilene Desert Research Center, Egypt, given the voucher num- fabaria subsp. domokina [10]. ber CAIH-1003-R and voucher specimens were depos- Identification of rare and endemic plant species is an ited in the Herbarium of Desert Research Center important base for evolutionary and phytogeographic (CAIH). studies as well as for the determination of conservation priorities [11]. In vitro experiments DNA barcoding is a molecular marker-based tech- In vitro germination nique, in which short fragments of DNA from a stan- Collected seeds of Silene schimperiana were washed with dardized genome position are utilized. This technique running tap water and detergent, then surface sterilized identifies plant species more definite than the traditional by soaking in commercial bleach containing sodium taxonomic tools, with unrecognizable plant parts and hypochlorite (5.25%) under laminar airflow cabinet (Hol- without requiring taxonomic experience [12, 13]. DNA ten LaminAir HVR 2448, USA), at different concentra- barcoding has applications in biodiversity monitoring, tions (0.5, 1.0, and 1.5% sodium hypochlorite solution). conservation impact assessment, forensic botany, moni- After 15 min, seeds were taken out and thoroughly toring illegal trading, etc. Also, DNA is stable and is washed thrice with sterilized distilled water. Seeds were found in all tissues; therefore, DNA barcoding of medi- cultured on half-strength Murashige and Skoog (1/2 cinal plants can be used for identifying powdered or MS) [21] medium (Duchefa, Haarlem, the Netherlands) processed plant materials [14]. supplemented with 3% (w/v) sucrose and 5.77 μM Ghareb et al. Journal of Genetic Engineering and Biotechnology (2020) 18:41 Page 3 of 15 Fig. 1 A map showing the location of Gebel Tennia, the collection site of Silene schimperiana gibberellic acid (GA3; Sigma Cell Culture, min. 90%, St. min. 90%, St. Louis, USA). MS medium without plant Louis, USA) (0.45 μm filter sterilized) for germination. growth regulators (PGRs) served as a control. The pH of The pH was adjusted to 5.7 ± 0.1, then medium was the medium was adjusted to 5.7 ± 0.1 and autoclaved at gelled with 0.3% (w/v) phytagel (Duchefa, Haarlem, the a pressure of 1.06 kg/cm and 121 °C for 15 min. Cultures Netherlands) before autoclaving at a pressure of 1.06 kg/ were incubated at 25 ± 1 °C at a photoperiod of 16/8 h cm, and 121 °C for 15 min (Harvey Sterilemax autoclave, light/darkness under cool white fluorescent tubes of Thermo Scientific, USA). Cultures were incubated under 2500-3000 lux. The survival and growth percentage (%), cool fluorescent tubes at day-night regime of 16-h the mean number and length (cm) of axillary shoots/ex- photoperiod with the light intensity of 2500-3000 lux plant were recorded after 3 weeks of culture.

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