Document Type : Research Paper
Authors
1 M.Sc. graduated, Department of Biology, Faculty of Sciences, Gonbad Kavous University, Gonbad-e Kavus, Iran
2 Department of Biology, Faculty of Sciences, Gonbad Kavous University, Gonbad-e Kavus, Iran
3 Department of Biology, Faculty of Sciences, Gonbad Kavous University, Gonbad, Iran.
4 Assistant professor, Department of Biology, Faculty of Science, Gonbad Kavous University, Gonbad, Iran
Abstract
Background and objectives: As a member of the Lamiaceae family native to Europe, Asia, and North Africa, Vitex agnus-castus is a popular medicinal plant. Despite much research, it is always essential to verify the safety of this valuable species, as it is among the world's best-selling plants. Several species in the genus Vitex do not exhibit recognizable morphologies, confusing their identification. The medicinal use of V. agnus-castus differs from that of other species. For effective pharmaceutical performance, the identification of this species is essential. This study used morphological, micromorphological, and molecular approaches to identify this medicinal plant.
Methodology: A total of 17 populations of the target species were investigated in Maraveh Tappeh, a city in the Golestan province located in the eastern region. Pollen samples were collected directly from the natural habitat of the target area. After acetolysis, 30-40 pollen grains were photographed from the polar and equatorial views with a light microscope and 40 and 100 magnification and with the help of a Canon digital camera. In order to study the seed morphology, fully ripe fruits were collected from each studied population during the fruiting season. Twenty seeds from each population were kept for photography with light microscopy and Scanning electron microscopy (SEM). Electron micrographs were prepared from suitable seeds and pollen at the Razi Metallurgical Research Center (RMRC) using an SEM electron microscope. Leaf cells from herbarium samples were extracted from DNA using a DNA extraction kit. Plastid trnL-trnF sequences and nrDNA ITS region sequences were used as barcodes. Polymerase chain reaction (PCR) was performed in 20 microliters with desired primers and a specific temperature program in a thermocycler. After performing the polymerase chain reaction, in order to ensure the amplification of fragments, the final product was electrophoresed. Strong single bands were sent to Codon Genetics Company in Tehran for sequencing.
Results: Pollen grains of all species are small (12-28 micrometers). According to Ertman's classification, Ghazan ghayeh pollen grains are prolate spheroidal (elongated spherical), and Ghoshe Tappeh pollen grains are subprolate (semi-elongated). All pollen grains are tricolporate. Ghazan ghayeh ornamentation is micro-perforated, while Ghoshe Tappeh ornamentations are reticulated-micro-perforated. With an average length of 3.84 mm and width of 1.52 mm, Ghazan ghayeh seeds are the largest. They are all almond-shaped, but in the Ghazan Ghayeh population, the outer surface of the seed is hollow; in the Ghoshe Tappeh population, the surface is wrinkled and striated. The trnL-trnF gene locus was found to have a multiplication success rate of 85% in the examined plants. There were 516 nucleotides in the amplified fragment in this species. For final registration, the sequence was sent to the GenBank. A 675 nucleotide fragment amplified from this species' ITS marker was sequenced, and the chromatograms were compared with the NCBI database. The gene bank has been notified of the sequence determination. The results showed the highest similarity (98%) with the Vitex agnus-castus species reported from America.
Conclusion: Local herbal medicines are gaining popularity in different countries and play a significant role in treating diseases today. In order to make medicinal plants more accessible, we need to pay more attention to trust, marketing, and consumption. Barcoding and molecular approaches serve this purpose effectively. To compare plant samples available on the market with natural medicinal plants using DNA barcoding of correctly identified medicinal species. Hence, the sequences used in this study are essential for barcoding Vitex agnus-castus. The medicinal species has been correctly identified and can be used as a standard for evaluating the plants available on the market.
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