71 J. Hortl. Sci. Vol. 12(1) : 71-77, 2017 In vitro regeneration and conservation of an endangered medicinal plant sarpagandha (Rauvolfia serpentina L.) Subhendu S. Gantait*1, Koushik Dutta2 and Jayoti Majumder1 1Associate Professor,Department of Floriculture & Landscaping, Faculty of Horticulture, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia-741252, West Bengal, India 2Formerly Assistant Professor, Department of Floriculture, Medicinal and Aromatic Plants, Uttar Banga Krishi Viswavidyalaya, Cooch Behar, West Bengal, India *Email : ssgflori@gmail.com ABSTRACT An investigation on in vitro plant regeneration of endangered medicinal plant Rauvolfia serpentina L. was carried out. The newly emerging leaves were used as explants, which were transferred to half strength MS medium along with various combinations of growth regulators for callus regeneration. The half strength MS medium fortified with 5.0 mgl-1 2,4-D and 2.0 mgl-1 NAA was found most suitable for qualitative (light green colour) and quantitative callus production (86%). The shoot regeneration (81.67 %) and elongation (5.67 cm) was highest in full MS medium supplemented with 6.0 mgl-1 BAP and 2.0 mgl- 1 GA3 and consequently, the root initiation was highest (55%) in half strength MS medium containing 3.0 mgl-1 IBA. The regenerated plantlets were preserved for nine months in good condition through repeated subculturing on full strength MS media. The callus were kept in storage by repeated sub-culturing in in vitro condition on MS medium fortified with 0.2 mgl-1 2,4-D and the preservation could be extended for nine months in its best condition. It was evident that, the regeneration capacity of callus was reduced as the time of callus storage was increased. Key words: In vitro, regeneration, Rauvolfia, callus, medicinal plant INTRODUCTION Rauvolfia serpentina L. commonly known as sarpagandha is a woody perennial medicinal plant belonging to family Apocynaceae. The roots of this shrub have been used in ayurvedic medicines from ancient time of Indian medical therapy (Singh et al., 2015). This shrub is highly effective for high blood pressure control. According to Ayurveda, it is the best among all anti-hypertensive drugs. It has been stated that, the drug is useful in mental disease, epilepsy, sleeplessness and several other ailments (Ojha and Mishra, 1985). But due to increasing anthropogenic activities and rapidly eroding natural ecosystem, the natural habitat of this species is decreasing. Rauvolfia is threatened in India due to its indiscriminate collection a nd over exploitation of natur a l resour ces for commercial purposes to meet the requirements of pha r ma ceutica l industr y, coupled with limited cultivation (Singh et al., 2009). IUCN has kept this plant under endangered status (Anonymous, 2009; Jain et al., 2003). In the present study, an attempt has been made to preserve the plant and to maintain its essence for future generation, as we know the plant is an endangered species. Seeds of Rauvo lfia ha s er r a t ic a nd low germination percentage and if the seeds stored more than 7-8 months practically don’t germinate at all. More over, the germination percentage of seed is very poor and variable ranging from 25-50 percent (Mitra, 1976). Hence, tissue culture technique in this plant is expected to be helpful to ex situ conservation by reducing the risk due to natural vagaries. Micropropagation is a unique method for Original Research Paper 72 J. Hortl. Sci. Vol. 12(1) : 71-77, 2017 Gantait et al the production of homozygous and disease free plants. This technique would also facilitate to obtain large number of plants irrespective of season and an alternative source of secondary metabolites in colossal quantity. The process of micropropagation u s ing va r i ou s p hyt ohor mones in dif f er ent concentr ation for Rauvolfia ha s been r epor ted earlier (Roy et al., 1994; Vanisree et al., 2004; Baksha et al., 2007; Rahman et al., 2008, Mallick et al., 2012). But, the conservation of callus for months through indirect regeneration and mass replication of secondary metabolite in hassled free cultivation method is a new invention and there is also a need to apply in vitro methods for the regeneration and conservation of this valuable endangered plant. Hence, a n effort has been made to develop an efficient protocol for the recovery of plants through or ganogenesis of Rauvolfia serpentina. It was hypothesised that, use of suitable plant growth regulators (PGR) as supplement of basic MS media could improve the in vitro plantlet regeneration and conservation potentiality of Rauvolfia. Different growth regulators as supplement of MS medium were optimized for rapid in vitro multiplication and conservation of Rauvolfia serpentina callus using leaf explant as an initial plant material. MATERIAL AND METHODS Plant material and explant preparation The present investigation was carried out at the Tissue Culture Laboratory of the Department of Floriculture, Medicinal and Aromatic Plants, UBK V, West Benga l, India . T he disea se f r ee juvenile leaf explants, used for callus formation and r egener a tion of Rauvolfia serpentina L. wer e collec ted fr om the gr een house of r esp ective department of the university. They were washed three times with de-ionized wa ter. Rest of the sterilization process was carried out in the horizontal laminar flow cabinet. The leaf was carefully made into 1 cm2 incision along with the midrib. The exc is ed ex pla nt s wer e tr ea t ed with diff er ent concentration of various disinfectants (4% Bavistin for 10 min and 0.1% HgCl2 for 1 min.) and then cultured on the suitable medium. During inoculation, the basal portions of the leaves were kept in the contact with media. Culture medium and experimental treatments The medium comprised of macro and micro elements according to Murashige and Skoog (1962) with and sucrose of 30gl-1 (Himedia® Mumbai), solidified with 0.6% agar (Himedia® Mumbai). The Plant growth regulators used were 2,4-D, BA, NAA and IBA (Sigma Eldritch® USA). All experiments were carried out in 100 ml culture tubes and jam bottles (Borosil, Kolkata). The pH of media was adjusted to 5.7 prior to autoclaving at 1210C at 15 lbs/sq inch pressure for 20 minute. Culture was incubated under 1600 lux light intensity for 16 h/8h light and dark cycles. The leaf was placed on standard and ½ MS medium supplemented with various concentrations of 2,4-D (2.0 and 5.0 mgl-l) and NAA (2.0 mgl-l) for callus induction, growth and development (Table 1) . After 28 days of culture, the inducted in vitro calli were cut into small pieces and transferred to the medium for shoot regeneration. MS medium containing different concentration and combination of BAP (2.0, 4.0 and 6.0 mgl-l) with GA3 (2.0 mgl - l) wer e used for shoot regener a tion a nd shoot multiplication (Table 2). The shoot regeneration was observed a fter 3-4 weeks of subculturing. For initiation of roots 4-5 weeks old shoots (2-4 cm length) were cultured on half strength MS medium supplemented with different concentrations of IBA (1.0, 2.0 and 3.0 mgl-l) (Table 3). Callus conservation The developed callus and regenerated plantlets calli were kept in in vitro condition under a foresaid temperature, light and humidity ideal for the plant development for a period of 9 months. The inducted calli were subcultured at 1½ month interval repeatedly on MS medium supplemented with 2,4-D (0.1, 0.2 or 0.3 mgl-l) for regeneration of callus in in vitro (Table 4). Wherein, the in vitro regenerated plantlets were kept by subculturing at 1½ month intervals on different strengths of MS medium (full, ½ and ¼ MS) without any plant growth regulators (Table 5). The well developed callus were kept at 20±3C under 1600 lux light intensity of 16h/8h day-night photoperiodic conditions and maintained as such for nine months by repeated sub culturing. 73 J. Hortl. Sci. Vol. 12(1) : 71-77, 2017 In vitro regeneration in sarpagandha Table 1. Effect of 2, 4-D and NAA on callus induction in Rauvolfia serpentina *”Arc sin transformed MS - Murashige & Skoog Table 2. Effect of BAP and GA3 on shoot regeneration in Rauvolfia serpentina MS - Murashige & Skoog Table 3. Effect of IBA on root initiation in Rauvolfia serpentina MS - Murashige & Skoog 74 Table 4. Effect of 2, 4-D on in vitro callus regeneration in Rauvolfia serpentina MS - Murashige & Skoog Gantait et al J. Hortl. Sci. Vol. 12(1) : 71-77, 2017 Table 5. Effect of media strength on in vitro regeneration of plantlet in Rauvolfia serpentina MS - Murashige & Skoog Acclimatization The well rooted in vitro regenerated plantlets wer e t r a ns f er r ed t o p la s t ic c u p s c ont a ining autoclaved vermiculite and vermicompost (1:1) for four weeks in culture room. Then the pr ima ry hardened plantlets were transferred to poly cups containing garden soil, sand, vermicompost (1:1:1) along with biofertilizers and were maintained under shed net with natural day length and temperature condition. Experimental design and data analysis The experiments were designed in completely randomized design (CRD). In each treatment 10 explants (n=10) were inoculated inside jam bottles separately. The statistical analysis was done by O.P. S t a t s of t w a r e p a c ka ges a nd t he mea n wer e compared using Duncan’s multiple range test at the 5 % probability level. RESULTS AND DISCUSSION Callus regeneration The callus grew within a month from inoculation on the callus formation medium containing different combinations of plant growth regulators viz. 2,4-D and NAA (Table 1). Among the different combinations ½ MS media supplemented with 5.0 mgl-l 2,4-D and 2.0 mgl-l NAA was found to be the best for maximum callus initiation (86.0%) of green coloured compact callus and took minimum days (18.33 days) to callus formation compared to others. It was also evidenced by Mallick et al. (2012) that, the high-frequency callusing induced in leaf and stem explant of Rauvolfia serpentina on modified MS medium supplemented with 2.5 mgl-l 2,4-D. MS medium containing 2.0 mgl-l of BAP plus 1.0 or 2.0 mgl-l 2,4-D resulted into copious callus induction observed by Ilahi et al. (2007). Shoot regeneration from callus It was obtained from MS medium supplemented with different combinations of BAP and GA3 (Table 2). MS medium supplemented with 6.0 mgl-l BAP and 2.0 mgl-l GA3 gave highest level of shoot regeneration (81.67%) and number of shoots (4.0) per explant with a mean shoot length of 5.67 cm followed by the MS medium supplemented with 4.0 mgl-l BAP and 2.0 mgl- l GA3 resulted better shoot length (4.67 cm) and higher number of shoots per explant (3.33). The data also reveals that, a combination of BAP and GA3 is 75 In vitro regeneration in sarpagandha J. Hortl. Sci. Vol. 12(1) : 71-77, 2017 Table 6. Regeneration capacity of the callus in Rauvolfia serpentina (Figures in the parenthesis are angular transformation values) necessary for shoot regeneration and elongation and also for early shoot development (22.00 days). Mallick et al. (2012) also r eported that, the ma ximum r egener ation of shoots fr om callus (90%) wa s observed in MS medium supplemented with 0.2 mgl-l NAA and 1.5 mgl-l BA. Similar result was obtained by Panwar et at. (2011) with maximum shoots (25.4) per culture from the callus in shooting medium containing 2.0 mgl-l BAP + 0.5 mgl-l NAA. Susila et al. (2013) showed that the best shoot proliferation (92%) was in MS medium containing 0.1 mgl-1 NAA and 2.5 mgl-1 BA. Root regeneration Out of different IBA concentrations tested, IBA 3.0 mgl-l was the best for root initiation (Table 3). T he r oot initia tion wa s 55% a t t he s a me concentration. The total number of roots per explant was 3.0 with root length of 4.67 cm within 12 days of transferring. MS medium supplemented with IBA (2.0 mgl-l) also induced better rooting (46.0%) with 2.43 roots per explant. The present results are in accordance with the result reported by Rahman et al. (2008) that, the adventitious shoots best rooted on half strength MS medium supplemented with 1.0 mgl-l each of IBA and IAA. Rooting of the young shoots was obtained with 1.0, 2.0 or 4.0 mgl-l of IBA (Ilahi et al., 2007). Susila et al. (2013) reported that half strength MS medium supplemented with 0.4 mgl-1 NAA a nd 0. 1 mgl-1 IBA caused for maximum root formation (91%). Callus conservation The calli were best conserved by repeated sub-culturing on MS medium fortified with 0.2 mgl- 1 2,4-D evidenced with light green callus turn brown, increase in size and shape and the preservation could be extended for nine months in its best condition (Table 4 & Fig. 1D). The regeneration capacity of the stored callus was tested at three months interval by tra nsferring the ca llus on best regeneration media. The maximum regeneration (72.0%) of the stored callus was noticed after three months of preservation with minimum days (13.67) taken for regener ation (Table 6). After nine months, the regeneration capacity of the repeated sub-cultured callus was reduced drastically with average of only 4 3 . 0 % r egen er a t ion a nd t he t ime t a ken f or regeneration was longer (24.33 days). Significant results were found during callus conservation in ter ms of the c a pa cit y of ca llus r egener a tion. Furthermore, the plantlets regenerated from calli were survived successfully for nine months in good condition on full strength MS media comparing to ½ or ¼ strength media through repeated subculturing at 1½ month interval (Table 6 & Fig. 1E). This technique provides a large number of disease free true to type plantlets throughout the year which will confer a large scale cultivation of this endangered species for commercial cultivation. Therefore, this micropropagation protocol and in vitro conservation of R . s e r p e n t i n a will b e u s ef u l f or f u r t her improvement of this important medicinal shrub. 76 Fig. 1: In vitro regeneration of R. serpentina. A. Callus regenerated in ½ MS medium supplemented with 2,4-D 5.0 mgl-1 and NAA 2.0 mgl-1. B. Multiple shoots induction n MS media containing BAP 6.0 mgl-1 and GA3 2.0 mgl -1. C. Plantlets initiate roots in ½ MS medium containing IBA 3.0 mgl-1. D. Conservation of callus in MS medium containing 2,4-D 0.2 mgl-1. E. Conservation of plantlets in MS medium Gantait et al J. Hortl. Sci. Vol. 12(1) : 71-77, 2017 CONCLUSION From the present study it can be concluded that, in R. Sepentina most qualitative and quantitative callus formation is possible in half strength MS medium supplemented with 5.0 mgl-1 2,4-D and 2.0 mgl-1 NAA. The plant multiplication was highest in full strength MS medium fortified with 6.0 mgl-1 BAP and 2.0 mgl-1 GA3. The calli were preserved for nine months in their best condition by repeated sub-culturing in in vitro condition on MS medium fortified with 0.2 mgl-1 2,4-D. It was also evident from the study that, the regeneration capacity of callus was reduced as the time of callusing increased. ACKNOWLEDGEMENT T he a uthor s a r e tha nkful to Mr. Rocky Thockchom and Mr. Kamal Das for assistance in experimental works in the laboratory and greenhouse. 77 In vitro regeneration in sarpagandha J. Hortl. Sci. Vol. 12(1) : 71-77, 2017 REFERENCES Anonymous. 2009. Rauvolfia sepentina L. Germplasm Resources Information Network. United States Department of Agriculture. 2003-03-14. Retrieved 2009-11-11. http://en.wikipedia.org/wiki/Rauwolfia. Baksha, R., Ara, M., Jahan, A., Khatun, R. and Munshi, J. L. 2007. In vitro Rapid Clonal Propagation of Rauwolfia serpentina. L. Bangladesh J. Sci. Ind. Res., 42: 37- 44. Ilahi, I., Rahim, F. and Jabeen, M. 2007. 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F. and Nalawade, S. M. 2004. Studies on the production of some important secondary metabolites from medicinal plants by plant tissue cultures. Bot. Bull. Acad. Sinica., 45: 1-22. (MS Received 12 April 2016, Revised 04 May 2017, Accepted 28 June 2017) 00 Content JH June 2017.pdf 00 SPH-Journal November new 12.pdf 01 SPH-Journal November new 01.pdf 02 SPH-Journal November new 04.pdf 03 SPH-Journal November new 05.pdf 04 SPH-Journal November new 07.pdf 05 SPH-Journal November new 09.pdf 06 SPH-Journal November new 10.pdf 07 SPH-Journal November new 11.pdf 08 SPH-Journal November new 13.pdf 09 SPH-Journal November new 03.pdf 10 SPH-Journal November new 06.pdf 11 SPH-Journal November new 08.pdf