In vitro propagation and DNA barcode analysis of the endangered Silene schimperiana in Saint Katherine protectorate

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 (GA 3) , 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.

rapid and efficient DNA extraction method from high oily content seeds:

“…Despite the fact that the DNA isolated using the current approach had a slightly higher A260/280 ratio, the DNA isolated was effectively employed for the downstream application, PCR. The extracted DNA was used as a template for PCR amplification of the ITS region of the rRNA encoding gene, which is commonly used for molecular identification and assessment of the molecular diversity of eukaryotic cells (Cheng et al, 2016;Yang et al, 2018;Ghareb et al, 2020;Soliman and A., 2021). PCR amplification of the extracted DNA samples yielded the expected product size of approximately 700 bp, as shown in Figure 3.…”

Dynamics of acid phosphatase production by cell suspension system and its further characterization

“…Before embarking on the study, two barcode loci, ITS and rbcL of in vitro regenerated plant, were amplified, sequenced and subjected to NCBI database analysis as described by (Ghareb et al, 2020)…”

Molecular identification and in vitro propagation of the endangered medicinal Cleome droserifolia plant of Saint Katherine

“…Interestingly, IBA proved to be more effective than NAA in this regard. IBA, being a natural auxin with heightened stability and low oxidative propensity, showcases notable root-inducing properties [67]. Among the tested media, the most effective one for prompting root formation in in vitro multiplied shoots of Cleome droserifolia was found to be ¼ MS medium fortified with 7.42 µM IBA.…”