In vitro propagation and DNA barcoding of the rare near endemic Plantago sinaica (Barnéoud) plant in Saint Katherine, Sinai

Abstract

Plantago sinaica is a rare perennial shrub near-endemic to Egypt and found in Saint Katherine Protectorate in Sinai. The first successful in vitro propagation protocol was conducted to protect the plant outside its natural reserves. Shoot tip, stem node section, cotyledonary node, and root explants separated from in vitro germinated seedlings were cultured in vitro on Murashige and Skoog (MS) medium enriched with different concentrations and types of cytokinins. It was found that 6-benzyl adenine (BA) is the most efficient cytokinin. MS medium containing 3.33 µM BA and 0.54 µM α-naphthalene acetic acids (NAA) produced 10.25 and 11.30 shoots/explant using shoot tip and stem node section, respectively. Conversely, MS medium + 2.22 µM BA + 0.54 µM NAA produced 13.25 shoots from root explants. Surprisingly, the cotyledonary node explants favored MS medium free from plant growth regulators (PGRs), which produced only 4.25 shoots/explant. The multiplied shoots were rooted successfully with a 100% rooting percentage on half MS medium containing 1.23 or 2.46 µM indole-3-butyric acid (IBA). In vitro, rooted plantlets were efficiently transferred to the greenhouse with a 90% survivability. Finally, the plant was identified using three DNA barcodes; 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL), plastid photosystem II protein D1 intergenic spacer region (psbA–trnH), and Internal Transcribed Spacer (ITS) barcodes. Additionally, psbA–trnH and ITS were novel and submitted to the GenBank databases for the first time for Plantago sinaica. Our study supports the United Nations Sustainable Development Goal number 15, which is to preserve, restore and reinstate sustainable usage of terrestrial ecosystems and to stop biodiversity loss.