# Conservation of Verbascum sinaiticum Benth using innovative tissue culture technique and DNA barcoding

## TL;DR

This paper presents a new method to conserve a rare Egyptian medicinal plant using tissue culture and DNA barcoding for genetic identification.

## Contribution

The study introduces the first optimized tissue culture protocol and molecular authentication for Verbascum sinaiticum.

## Key findings

- An efficient in vitro regeneration system was established for V. sinaiticum using optimized tissue culture techniques.
- HPLC analysis showed the highest rutin and vanillin levels in ethanolic extracts, and highest gallic and chlorogenic acid in aqueous extracts.
- SDS-PAGE identified five protein bands ranging from 240 kDa to 28 kDa in V. sinaiticum.

## Abstract

Egypt has diverse medicinal plants, many of which are endangered because of environmental and human pressures. Verbascum sinaiticum Benth (V. sinaiticum) is a medicinal plant from the Scrophulariaceae family and a locally rare species endemic to Sinai. This study introduces an innovative in vitro regeneration protocol for V. sinaiticum, developed for the first time using optimized tissue culture techniques. Additionally, the incorporation of molecular identification by DNA barcoding and biochemical profiling (via HPLC and SDS-PAGE) presents a complete strategy to conservation and genetic documentation, to support future biotechnological applications. An efficient in vitro regeneration system for V. sinaiticum was established using tissue culture techniques. The study investigated the effects of three different shoot induction media on growth, The first medium was BA medium, a combination of 0.5 mg L− 1 benzyl adenine (BA), 5 mg L− 1 thiamine HCl, 0.5 mg L− 1 nicotinic acid, and 0.5 mg L− 1 pyridoxine was added to the initial medium. The second medium was MD medium supplemented with 3 mg L− 1 BA, 0.2 mg L− 1 naphthaleneacetic acid (NAA), 10 mg L− 1 thiamine HCl, 1 mg L− 1 nicotinic acid, and 1 mg L− 1 pyridoxine. The third medium was AB medium formulated with 1 mg L− 1 Adenine sulfate, 1 mg L− 1 benzyl adenine, 10 mg L− 1 thiamine HCl, 1 mg L− 1 nicotinic acid, and 1 mg L− 1 pyridoxine, and three media for rooting, The first medium was IB medium supplemented with 0.4 mg L− 1 indol butyric acid, 5 mg L− 1 thiamine HCl, 0.5 mg L− 1 nicotinic acid, and 0.5 mg L− 1 pyridoxine. The second medium was IA medium containing 5 mg L− 1 thiamine HCl, 0.5 mg L− 1 nicotinic acid, and 0.5 mg L− 1 pyridoxine. The third medium was NA media formulated with 0.4 mg L− 1 naphthaleneacetic acid, 5 mg L− 1 thiamine HCl, 0.5 mg L− 1 nicotinic acid, and 0.5 mg L− 1 pyridoxine. All media were prepared using Murashige and Skoog (MS) basal salts 4.4 g L− 1 supplemented with 30 g L− 1 sucrose, and solidified with 2.8 g L− 1 gelrite. Various propagation protocols were tested based on the availability of plant materials. We have devised flexible methods for large-scale micropropagation that offer a sustainable and quick production strategy, even in the face of resource constraints. The surface sterilization technique was refined, resulting in up to 75% contamination-free cultures. The highest germination rate was observed with 20% Clorox for 15–20 min, but extended exposure (25–30 min) resulted in decreased germination.HPLC analysis revealed that the ethanolic extract had the highest quantities of rutin (19.07 µg/mL) and vanillin (6.29 µg/mL), while the aqueous extract had the highest levels of gallic acid (7.02 µg/mL) and chlorogenic acid (6.02 µg/mL). SDS-PAGE examination revealed five protein bands ranging in molecular weight from 240 kDa to 28 kDa. This study introduces an innovation and preservation strategy for V. sinaiticum, a locally rare species in Egypt. DNA barcoding was used for molecular authentication, and the acquired sequence was matched to GenBank database sequences using the BLAST tool.