Annals of Plant Sciences 7.5 (2018) pp. 2239-2246 * Corresponding Author: Shiwani Kaushal, Assistant Professor, Department of Biosciences, Asian Educational Institute, Patiala, Punjab, India. E-mail: shiwanikaushal23@gmail.com http://dx.doi.org/10.21746/aps.2018.7.5.6 Page | 2239 Research Article Somatic embryogenesis and plant regeneration from cell suspension cultures of Gentiana kurroo Royle. Shiwani Kaushal 1, 2* and Arushdeep Sidana 1 1 Department of Biotechnology, Shoolini University, Solan, Himachal Pradesh, 173229, India. 2 Department of Biosciences, Asian Educational Institute, Patiala, Punjab 147001, India. Received: 4/10/2018; Revised: 4/20/2018; Accepted: 4/27/2018 Abstract: Gentiana kurroo Royle, native to North- Western Himalayas, is one of the critically endangered perennial herbs, which is being overexploited due to its multiple medicinal uses. The goal of this work was to develop a protocol for somatic embryogenesis and efficient plant regeneration through cell suspension culture. Selected ranges of plant growth regulators were experimented for somatic embryogenesis. Light micrographs of the established suspension cell cultures of somatic embryos at different developmental stages were also observed under light microscope. Somatic embryogenesis was induced from the leaf explants and the maximum induction frequency (97.2%) was obtained on Murashige and Skoog (MS) medium supplemented with 0.5 mg/l each of NAA, BAP and TDZ. Cell suspension cultures were established in MS liquid medium containing IAA (0.5 mg/l) + BAP (1.0 mg/l) with 89-93% viable cells. Transfer of cotyledonary somatic embryos to the MS agar medium containing 2, 4-D (0.4 mg/l) and KN (1.5 mg/l) resulted in somatic embryogenesis at high frequencies (80%) with an average of 28 ± 0.2 embryos/ callus piece. After transfer onto the half strength MS medium without growth regulators approximately 80-90% somatic embryos developed into complete plantlets. The protocol described here could be used for somatic hybridization, genetic transformation, isolation of protoplasts and large-scale propagation of G. kurroo. Keywords: Cell suspension, Gentiana kurroo, Murashige and Skoog (MS), Somatic embryogenesis Introduction Gentiana kurroo Royle (Family: Gentianaceae) is an important native Indian species used for medicinal purposes. It is a rosette forming small perennial herb also known as Indian Gentian, Neelkanth, karu and chireta. It is mainly found in Kashmir and Himachal Pradesh with adjoining hills of North-Western Himalayas at altitudes of 1500-3400 m. In traditional and modern medicine, roots and rhizomes of this plant are valued as a bitter tonic, antiperiodic, antibilious, anthelmintic, astringent, antipsychotic, sedative, stomachic and carminative. The roots of this plant are a source of iridoid glycosides like gentiopicrine and gentiamarin and the alkaloid, gentianin (Raina et al., 2003). Unfortunately, the pharmaceutical industries are largely dependent on natural population of G. kurroo to fulfill their demands, which is depleting the wild stands of this plant. Therefore, this plant has been listed as critically endangered by the Government of India (Sharma et al., 1993). Many in vitro studies have been carried out on propagation of G. kurroo using shoot tips, nodal segments, seedlings, petioles, leaves and apical meristem as explants (Sharma et al., 1993; Fiuk et al., 2003; Sharma et al., 2014; Kaushal et al., 2014). It was found that G. kurroo can be propagated through rhizome cuttings; shoot nodal segments, seeds and somatic embryogenesis. Suspension culture studies have been previously established from seedling explants for various Gentiana species such as G. tibetica, G. cruciata and G. pannonica (Mikuła and Rybczynski, 2001; Mikuła et al., 2005). Up to the present study very few workers have established the suspension cultures with the use of embryogenic callus derived from seedling explants (Fiuk and Rybczynski, 2008 a). Hence, in the present study, we have described the light micrographs showing stages in embryo development of G. kurroo Royle suspensions and plant regeneration through cell suspension cultures. Materials and Methods Plant Material Two-week-old authentic aseptic cultures of Gentiana kurroo were collected from Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan and was maintained under controlled temperature (25°C), humidity (70-75%) and light (10 h dark and 14 h light) conditions in a growth chamber. Culture media and growth conditions For initiating the tissue culture, leaf cuttings were used as explants for somatic embryogenesis. MS medium (Murashige and Skoog, 1962) containing 3% sucrose gelled with 0.8 % agar supplemented with varying concentrations and combinations of BAP (0.5-1.0 mg/l), TDZ (0.5-1.0 mg/l), NAA (0.5-1.0 mg/l) and 2, 4-D (0.5-2.0 mg/l) were used as shown