Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop Genetic stability analysis using DNA barcoding and molecular markers and foliar micro-morphological analysis of in vitro regenerated and in vivo grown plants of Artemisia vulgaris L. Phanikanth Jogam a , Dulam Sandhya a , Mahipal S. Shekhawat b , Anshu Alok c , Manokari M d , Sadanandam Abbagani a , Venkateswar Rao Allini a, * a Department of Biotechnology, Kakatiya University, Warangal, Telangana, 506009, India b Department of Botany, Kanchi Mamunivar Center for Postgraduate Studies, Puducherry, 605008, India c University Institute of Engineering and Technology, Punjab University, Chandigarh, 160014, India d Siddha Clinical Research Unit, Central Council for Research in Siddha, Tamil Nadu, 636401, India ARTICLE INFO Keywords: Artemisia vulgaris DNA barcoding Ex vitro rooting Genetic fidelity Micro-morphological studies ABSTRACT The current study provides an enhanced and reproducible micropropagation system in Artemisia vulgaris L. using nodal explants of mature plants. The explants were cultured on basal Murashige and Skoog’s (MS) medium augmented with 6-benzylaminopurine (BAP) and Kinetin (Kin) concentrations individually. Direct regeneration of shoots (7.7 ± 0.36 shoots per explant) from nodal meristems was attained on MS medium combined with 1.5 mg L -1 BAP alone. Sub-culturing of these cultures on MS medium supplemented with 0.5 mg L -1 BAP and 0.5 mg L -1 inddole-3 acetic acid (IAA) helped in further multiplication of shoots (75.8 ± 1.00 shoots per culture vessel). Elongated shoots were transferred to half strength MS medium contained 1.0 mg L -1 IBA for roots formation. Rooting was also stimulated from the sliced ends of the shoots via ex vitro rooting method by pulse treating the shoots with 300 mg L -1 IBA for 5 min. Well rooted plants were shifted into small paper cups having manure: sand: soil in 1: 1: 1 proportion for hardening. The acclimatized plants were shifted to the natural field conditions for complete plant growth. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) markers and universal DNA barcoding (rbcLa gene primers) were applied to analyze the genetic stability of micropropagated plants with the mother plant. The banding patterns of regenerants were found to be mono- morphic in nature, representing the similarity with the mother plant and no somaclonal variations were ob- served. Similarly, foliar micro-morphological characters like stomata, trichome and vein pattern of leaves were studied from in vitro and acclimatized plants to focus on the developmental adaptation of micropropagated plantlets towards survival in field conditions. This study explores the highly reproducible procedure for A. vulgaris through nodal propagation without any somaclonal variations. It is the first report on foliar micro- morphological analysis at subsequent stages of in vitro propagation and genetic homogeneity of micropropagated plants with the mother plant. The current study paves the way for the development of genetically uniform A. vulgaris plants regenerated from nodal shoot segments and successful survival in field. 1. Introduction Artemisia vulgaris L. (family Asteraceae) is a perennial herb, native to Europe but distributed worldwide except Antarctica (Weston et al., 2005). It is commonly referred as mugwort and has a long history for medicinal and aromatic properties (Gruenwald et al., 2008; Bora and Sharma, 2011). The whole plant possesses diverse bioactive compounds with astonishing pharmacological actions. The major constituents are flavonoids, essential oils, artemisinin, sesquiterpenes, alkaloids, quinones, sterols, coumarins, glycosides, monoterpenes, saponins, etc. (Khan and Gilani, 2009; Tajadod et al., 2012). The reported secondary metabolites are responsible for various vital pharmacological properties such as anti-malarial, anti-viral, anti-oxidant, anti-inflammatory, anti- carcinogenic, anti-rheumatic, larvicidal, insecticidal, CNS-stimulant, anti-spasmodic, hypoglycemic, anti-fertility and hepatoprotective ac- tivities (Wang et al., 2006; Afsar et al., 2013; Lian et al., 2018). Tra- ditional and modern systems of medicines imply its uses in the treat- ment of rheumatism, gastric ulcers, asthma, epilepsy, liver disorders, https://doi.org/10.1016/j.indcrop.2020.112476 Received 4 January 2020; Received in revised form 22 February 2020; Accepted 11 April 2020 ⁎ Corresponding author. E-mail address: vrao.alleni@gmail.com (V.R. Allini). Industrial Crops & Products 151 (2020) 112476 0926-6690/ © 2020 Elsevier B.V. All rights reserved. T