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A Rapid Micropropagation Protocol of Atropa Acuminata Royle Ex Lindl.―A Threatened Medicinal Plant Species of Kashmir Himalaya

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A Rapid Micropropagation Protocol of Atropa Acuminata Royle Ex Lindl.―A Threatened Medicinal Plant Species of Kashmir Himalaya Indian Journal of Biotechnology Vol 15, October 2016, pp 576-580 A rapid micropropagation protocol of Atropa acuminata Royle ex Lindl.―A threatened medicinal plant species of Kashmir Himalaya Farhana Maqbool*, Seema Singh, Zahoor A Kaloo and Mahroofa Jan Plant Tissue Culture Laboratory, Department of Botany, University of Kashmir, Hazratbal, Srinagar 190006, India Received 12 June 2015; revised 10 November 2015; accepted 16 November 2015 In the present study, different explants, viz., petiole and nodal explants, of Atropa acuminata Royle ex Lindl. were used to develop an efficient micropropagation protocol for the conservation of this medicinally important plant. The petiole explants produced the maximum amount of callus on MS medium supplemented with BAP (3 mg/L) within 18 d in 80% cultures. Further, shoot regeneration was obtained when these calli were subcultured onto MS medium supplemented with BAP (5 mg/L), with a mean shoot length of 2.2±0.19 cm in 40% cultures within 48 d. Similarly, from nodal explants, the maximum amount of callus was achieved on MS medium supplemented with BAP (2 mg/L) in 80% cultures within 17 d. When these calli were transferred onto MS medium supplemented with BAP (2 mg/L), shoot regeneration was obtained with a mean shoot length of 2.0±0.20 cm in 80% cultures within 14 d. Root differentiation with 100% response was obtained within 18 d in in vitro grown shoots on MS medium augmented with IBA (0.5 mg/L) with a mean number of 21.6±6.9 roots. The in vitro raised plantlets were then successfully acclimatized and hardened in compost under greenhouse conditions within 3 wk with 80% response. The hardened plants were successfully transferred to the field conditions. Keywords: Atropa acuminata, callus, explants, micropropagation, root regeneration, shoot regeneration Introduction important source of medicinally important tropane Atropa acuminata Royle ex Lindl. (Family: alkaloids, including atropine, scopolamine and Solanaceae), commonly known as Indian Belladonna, is hyoscyamine9. The drugs atropine and hyoscyamine a perennial plant that grows about 1.6 m tall. It has extracted from the plant act as stimulants to the simple leaves, which are ovate with entire margins. The sympathetic nervous system and are employed as flowers are solitary, bell-shaped and yellowish white in antidotes to opium6. The plant also contains highly colour. They are hermaphrodites and are pollinated by oxygenated oleanane triterpenes, such as, 2α,3α,24 insects1. Flowering period is from June to July and the trihydroxyolean-12-ene-28,30-dioic acid and seeds ripen from August to October. The black fruits are 2α,3α,24,28-tetrahydroxyolean-12-ene10. Monoterpene, berries. It is found in the Western Himalayan ranges, sesquiterpene, phenylpropanoid, flavonoid and quinine extending from Kashmir at the altitude of 1800-3600 m are present as main constituents11. asl to the adjoining hills of the Himachal Pradesh up to Ranking at the top of medicinal plant inventory from 2500 m asl. In North West Himalaya, it is distributed in North West Himalaya, A. acuminata figures among 59 Kashmir, Muzzafarabad and Chakrata2. The rhizome of critically endangered taxa and negative list of exports in this plant has been traditionally used as a sedative India, and has been prioritized for immediate antidote in cases of mushroom or toadstool poisoning, conservation and large scale multiplication12. Unabated analgesic, antispasmodic, hallucinogenic, mydriatic, as the plant extraction continues to be, far are not days narcotic, diuretic and anodyne; arthritis related when this precious legacy will be lost forever. It is inflammatory disorders, muscle and joint pain, muscle indeed a crisis situation for the species, which calls for spasms, sore throat and ulcerative colitis3-7. Aerial parts the salvage of whatever is left. It is, therefore, that the of the plant have been used to treat innumerable present study for its in vitro propagation and ailments, such as, acute infections, anxiety and chicken conservation has been taken up. 8 pox . A. acuminata plants serve as one of the most Materials and Methods ――――― *Author for correspondence: A. acuminata plants was collected from Gulmarg [email protected] and Daksum of Kashmir Himalaya and were MAQBOOL et al: MICROPROPAGATION OF A. ACUMINATA 577 transplanted at the Kashmir University Botanical IAA13. Petiole derived callus when subcultured on MS Garden (KUBG), Srinagar. The specimen was medium supplemented with BAP (5 mg/L) (Fig. 3a) and collected and processed for herbarium preparation and BAP (3 mg/L) in combination with IAA (2 mg/L) (Fig. latter deposited at Kashmir University Herbarium 3b), it regenerated shoots with a mean shoot length of (KASH) under Voucher Specimen No. 1913 2.2±0.19 and 1.99±0.19 cm in 40 and 20% cultures [Ref.No.F1 (Specimen vouchers. CBT) KU/2013]. within 48 and 53 d, respectively (Table 2). Shoots Different explants were collected from plants grown obtained from MS medium supplemented with BAP (5 at KUBG (Fig. 1). Petiole and nodal segments mg/L) was healthier and with greater length as (explants) were first thoroughly washed under compared to shoots achieved from BAP (3 mg/L) in running tap water in order to remove dirt and dust, combination with IAA (2 mg/L) and also in terms of per followed by washing with detergent labolene (1% v/v) cent culture response. and surfactant Tween-20 (1% v/v). The detergent was removed by washing the explants with double Nodal Explants Nodal segments were inoculated on MS medium distilled water. Then they were treated under laminar containing different growth hormones (BAP, Kn, air flow hood with chemical sterilant 2 or 4% sodium IAA, NAA, IBA & 2, 4-D) individually or in different hypochlorite for 8-10 min. This was followed by washing with autoclaved double distilled water and finally the explants were inoculated on sterilized nutrient medium. Medium and Culture Conditions Murashige and Skoog’s (MS) medium, gelled with 8% agar was supplemented with different concentrations of auxins and cytokinins, both individually and in combination. Auxins like 2, 4-D, IAA, NAA and IBA, and cytokinins like BAP and Kn, were used in concentration range of 0.1-5 mg/L. The pH of the media was adjusted to 5.8 before autoclaving at 121°C and 15 lb. The cultures were Fig. 1―A. acuminata mother plant. incubated at 22±4°C and exposed to a 12 h light and 12 h dark. Results and Discussion Callus Production and Shoot Regeneration Petiole Explants Callus production was achieved from petiole explants by using different growth hormones (BAP, Kn, IAA, NAA, IBA & 2, 4-D) individually as well as in different combinations. However, optimal callus regeneration was induced when MS medium was supplemented with BAP Fig. 2―Callus production from petiole explant on MS medium (3 mg/L) (Fig. 2a) and BAP (3 mg/L) in combination containing: (a) BAP (3 mg/L); & (b) BAP (3 mg/L)+IAA with IAA (2 mg/L) (Fig. 2b). The callus was compact (2 mg/L). and light green in colour in both the combinations (MS+ Table 1―Effect of plant growth regulators on callus production AP & MS+BAP+IAA), and was obtained in 80 and 60% from petiole explant cultures within 18 and 38 d, respectively (Table 1). Petiole explant produced maximum amount of callus on Treatments No. of days taken for % culture response callus production MS medium supplemented with BAP (3 mg/L) as compared to BAP (3 mg/L) in combination with IAA MS basal - - (2 mg/L) and also in terms of per cent culture response MS+BAP (3 mg/L) 18 80 and number of days taken for callus production. Similar MS+BAP (3 mg/L) 38 60 results were also obtained from the petiole explant of +IAA (2 mgL) Salvia canariensis L. but they used NAA instead of (10 replicates per treatment) 578 INDIAN J BIOTECHNOL, OCTOBER 2016 combinations. MS medium fortified with BAP (2 combinations with cytokinins like BAP. About mg/L) (Fig. 4a) and BAP (3 mg/L)+IAA (2 mg/L) 21.6±6.9 and 6.1±0.87 mean number of roots were (Fig. 4b) proved effective in differentiating compact obtained in 100 and 60% cultures within 18 d on full and cream coloured callus in 80 and 60% cultures strength MS medium supplemented with IBA within 17 and 21 d, respectively (Table 3), and (0.5 mg/L) and IBA (1 mg/L), respectively (Figs 6a). maximum amount of callus was obtained on MS Roots were also regenerated on half strength MS medium supplemented with BAP (2 mg/L). Callus medium supplemented with IBA (0.5 mg/L). Roots obtained from nodal explants when subcultured on regenerated from full strength MS medium enriched MS medium supplemented with BAP (3 mg/L) with IBA (0.5 mg/L) was greater in number as (Fig. 5a) and BAP (2 mg/L) (Fig. 5b) regenerated compared to IBA (1 mg/L) and ½ MS+IBA (0.5 shoots with a mean shoot length of 2.0±0.20 and mg/L), and also in terms of per cent culture response. 1.9±0.23 cm in 80 and 70% cultures within 14 and 15 However the per cent cultures showing rooting as d, respectively (Table 4). Shoots regenerated on MS medium augmented with BAP (3 mg/L) was healthier and with greater length as compared to BAP (2 mg/L) and also in terms of number of days taken for shoot regeneration. Similar results were obtained from the shoot regeneration of Solanum nigrum L. on MS medium supplemented with BAP (3 mg/L)14. Rooting of Regenerated Shoots For induction of roots, in vitro cultured shoots were placed onto both full and half strength MS medium Fig.
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