Indian Journal of Biotechnology Vol 11, July 2012, pp 295-299 In vitro propagation and conservation of Swertia bimaculata Hook.f. & Thoms. Arpita Dafadar and Timir Baran Jha* Plant Biotechnology Laboratory, Department of Botany, Presidency University, Kolkata 700 073, India Received 15 April 2011; revised 30 October 2011; accepted 10 December 2011 Swertia bimaculata Hook.f. & Thoms., a less bitter species of the genus Swertia, is phytochemically important for its bioactive compounds (xanthones). Flower buds and immature capsules were collected from in vivo donor plants from South Sikkim during the month of September 2009. Three to five per cent (3-5%) seeds within the intact or split capsule germinated aseptically in full strength Murashige and Skoog’s (MS) medium with low concentration of either benzylaminopurine (BA; 2.22 µM) or kinetin (Kn; 2.32 µM). In vitro grown shoot tips were used as primary explants. Shoot multiplication was obtained on half-strength MS medium (BM) with 3% (w/v) sucrose and with different combinations and concentration of BA (2.22-4.44 µM), Kn (2.32-4.65 µM) and 1-napthalene acetic acid (NAA; 0.54 µM). However, the best response with 15.6 shoots per explants was obtained on BM with 2.22 µM BA, 2.32 µM Kn and 0.54 µM NAA. The number of shoots was further increased to 20.6 on addition of 10 mM KNO3 in the medium. About 100% healthy root induction on isolated shoots was achieved on BM devoid of any plant growth regulator (PGR) within 5 wk. Complete rooted plantlets were successfully hardened and transplanted in the soil with 80-90% survival rate. Meiotic study from flower buds of donor plants and mitotic chromosome study from regenerated plants revealed n=13 bivalents and 2n=26 chromosomes respectively. The protocol of in vitro propagation and chromosomal database developed for the first time in an unexplored S. bimaculata is highly efficient, repeatable and can be used for conservation of germplasm. Keywords: Chromosomal database, Swertia bimaculata, shoot multiplication Introduction It has been reported long back that S. bimaculata The genus Swertia is used in the traditional system contains medicinally important bioactive compounds4, of medicine since prehistoric times. It comprises of such as, gentiananine, a chemical known for about 170 known species worldwide1 and about hypothermic activity, and xanthones, an antioxidants 40 species are reported to be available in eastern and that can combat cancer and help to reduce cholesterol western parts of Indian Himalayas at high altitudes, and hardening of arteries. However, no systematic ranging between 1,200-3,000 m. Among the effort has been made to formulate alternative methods different species available in India, S. chirata is for its propagation and germplasm conservation. considered the most elite one in terms of medicinal Chromosome analysis in any plant species is carried properties, although it is very bitter in taste. Several out at the somatic and gametic level primarily to reports on phytochemical analysis of S. chirata are determine the stability of the plant at the cellular level available in the literature2,3. However, most of the and also for genetic and biotechnological studies. other species did not receive much attention Earlier chromosomal analyses5,6 reported 22 and 26 concerning conservation and chemical analysis. somatic chromosomes (2n=22/26) and 11 gametic Even well documented morphological comparisons chromosomes (n=11) in S. bimaculata, which are not available in the genus. S. bimaculata Hook.f. necessitates the reassessment of chromosomal studies. & Thoms. (syn. Ophelia bimaculata Siebold & A review of literature reveals that maximum Zucc.) is an unexplored, less bitter species of the published reports on micropropagation are available genus and is frequently mixed with S. chirata as a in S. japonica, S. pseudo-chinensis, and S. chirata7-15. substitute and/or adulterant. Continuous However, no published work on in vitro propagation indiscriminate harvesting has enormously reduced its and conservation is available for Indian S. bimaculata. population strength in the natural habitats. Therefore, the present study primarily focuses on in vitro propagation and conservation of S. bimaculata _______________ using shoot tip explants from in vitro grown seedlings *Author for correspondence: Tel: +91-33-22198647, Fax: +91-33-22412738 and simultaneous preparation of chromosomal E-mail: [email protected] database from gametic (n) and somatic (2n) tissues. 296 INDIAN J BIOTECHNOL, JULY 2012 Materials and Methods Chromosomal Analysis Plant Material Healthy root-tips, randomly taken from Green capsules (0.5-1.0 cm) of S. bimaculata plants micropropagated plants growing on BM, were pre- were collected during the month of September, 2009 treated with 2 mM hydroxyquinolene for 3½ h at from South Sikkim (2000 m). Capsules were washed 12-14ºC temperature, followed by overnight fixation thoroughly in running tap water and treated with 5% in acetic alcohol (1:3). Hydrolysis of the root tips was (v/v) Teepol (commercial bleach) for 25-30 min, performed with 1 N HCl for 12-15 min at room temperature (RT). Hydrolyzed root tips were stained followed by surface sterilization with 0.1% HgCl2 for 25-30 min. Sterilants were removed by 4-5 times in 2% aceto-orcein for 3 h and squashed in 45% acetic washing with sterilized distilled water. Intact capsule acid to obtain well-scattered metaphase plates. containing large number of immature seeds as well as Chromosome numbers per cell were recorded and isolated individual seeds were placed on Murashige photographs were taken under Ziess and Skoog’s (MS) basal medium with plant growth photomicroscope. Meiosis was studied in flower buds regulator. collected from the in vivo donor plant. Dissected anthers were smeared with 2% aceto-carmine to Culture Media and Culture Conditions obtain the meiotic metaphase I plates. Chromosome MS medium with 3% (w/v) sucrose, 0.75% a numbers per pollen mother cell (PMC) were manually gar-agar and 2.22 µM benzyl aminopurine (BA) or counted and photographs were taken under Ziess 2.32 µM kinetin (Kn) were used to induce germination photomicroscope. of seeds. On the other hand, half-strength MS medium (BM) with different combinations and concentrations Statistical Analysis of BA (2.22-4.44 µM), Kn (2.32-4.65 µM), All experiments were repeated no less than three 1-napthalene acetic acid (NAA; 0.54 µM) and times. The experiments were set up in a randomized 10 mM KNO3 were used for multiplication of design. Data were analyzed by analysis of variance shoots. The number of shoots developed per (ANOVA) to detect significances between means. explants was recorded after 6-8 wk. The pH of Means differing significantly were compared using culture medium was adjusted to 5.6-5.8 and Duncan’s multiple range test (DMRT) at a 5% sterilized at a 1.06 kg cm2 pressure, 121°C probability level. Variability of data has also been temperature for 15-17 min. All the aseptic cultures expressed as the mean±standard error (SE). were incubated at 22±2ºC under a photoperiod of 16 h light/8 h dark cycles. All experiments were Results and Discussion repeated more than three times. Germination of 3-5% seeds was obtained from About 4-5 cm long in vitro regenerated intact or half splitted capsules (Figs 1ai & ii) in MS shoots were placed in BM with/without different medium with either BA or Kn but not from any concentrations of indole-3-acetic acid isolated seeds. In vitro grown shoot tips were used as (IAA; 5.71-11.42 µM) or indole-3-butyric acid (IBA; primary explants and the shoot multiplication was 4.90-9.80 µM) for induction of roots in 16 h light/8 h achieved on BM with different levels of BA dark cycles. Frequency of root induction and number (2.22-4.44 µM), Kn (2.32-4.65 µM) and 0.54 µM of roots developed per shoots were recorded in NAA within 10 wk of culture. However, the best between 2-5 wk. response was obtained with 2.22 µM BA, 2.32 µM Kn and 0.54 µM NAA, where about 15.6 shoots Hardening of Plants were obtained (Table 1, Fig. 1b). The number of Complete plants with well-developed roots were shoots was further increased to nearly 20.6 when taken out carefully from the culture flasks and additional 10 mM KNO3 was added in the medium. thoroughly washed in tap water and transplanted in Also in our earlier work, positive roles of KNO3 earthen pots containing a mixture of were reported on shoot multiplication of soil+sand+compost (1:1:1). Initially plants were S. chirata 11. Similar findings were also reported by covered by polythene bags and kept inside the plant other workers in many other plants like Fragaria growth chamber for 2-3 wk under 80-90% humidity. ananassa15, Camellia sinensis16, Helianthus annuus17 Covers were gradually withdrawn from the hardened and Nicotiana tabacum18. It can be thus concluded plants and they were grown normally. that higher number of shoots can be induced in both DAFADAR & JHA: IN VITRO PROPAGATION OF S. BIMACULATA HOOK.f. & THOMS. 297 Fig. 1 (a-g)—In vitro propagation of Swertia bimaculata: a (i & ii). Intact capsule containing large number of immature seeds and germinated seeds with seedlings in MS medium with either 2.22 µM BA or 2.32 µM Kn (bar=0.2 cm); b. Multiplication of shoots on ½ MS medium with 2.22 µM BA, 2.32 µM Kn and 0.54 µM NAA after 10 wk of culture (bar=1 cm); c. Induction of roots in excised shoots on ½ MS basal medium without any growth regulator (bar=1 cm); d.