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.

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 monomorphic in nature, representing the similarity with the mother plant and no somaclonal variations were observed. 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.