In Vitro Mass Multiplication and Assessment of Genetic Stability of In Vitro Raised Artemisia absinthium L. Plants Using ISSR and SSAP Molecular Markers
The present investigations were made attempting to develop a rapid, reliable, and reproducible in vitro regeneration protocol for Artemisia absinthium L., a medicinal plant of Kashmir Himalayas. Out of several auxin-cytokinin combinations tested, Murashige and Skoog’s (MS) medium supplemented with 0.5？mgL？1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5？mgL？1 kinetin (Kn) was found to be the best for the callus induction. On the other hand, 4.5？mgL？1 6-benzylaminopurine (BAP) and 0.5？mgL？1 1-α-naphthaleneacetic acid (NAA) in the medium resulted in maximum shoot induction from the callus. Similarly, BAP and NAA at a concentration of 1.5？mgL？1 and 0.5？mgL？1, respectively, proved to be the best for the multiple shoot induction from nodal explants. Numerous shoots were obtained from nodal explants after third subculture. In vitro rooting was maximum on medium containing indole-3-butyric acid (IBA) at 0.5？mgL？1. The genetic stability of the in vitro raised plants of Artemisia absinthium was assessed using the intersimple sequence repeat (ISSR) and sequence-specific amplification polymorphism (SSAP) molecular markers. Both markers were able to detect the somaclonal variations in the callus regenerated plants, while no variation was detected in the plants regenerated from the nodal explants. SSAP has been found to be more useful in detection of variability as compared to ISSR molecular marker. The results of present study concluded that the direct regeneration protocol will be useful for the production of true to type plants of this medicinally important plant. This will go a long way in reducing the pressure on the natural populations for the secondary metabolite production, especially for extraction of essential oils. 1. Introduction Artemisia is a large genus based on >500 species, distributed primarily in temperate and subtropical regions of Asia, Europe, and North America. Central Asia is known to be its center of diversification, while the Mediterranean region and North West America are two derived speciation areas [1]. Artemisia plants are mostly used for medicinal purposes, although some of these have been put to other uses like food, forage, ornamentals, or soil stabilizers in disturbed habitats. Some taxa are known to be toxic or allergenic while some others are invasive weeds, which can adversely affect crop yield [2]. Artemisia absinthium L., commonly known as wormwood, is a perennial herb. This plant is medicinally very important and has been used in traditional ways because of its antihelminthic, insecticidal, antiseptic, and antibacterial
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