International Journal of Pharma and Bio Sciences

Transcription

1 Research Article Biotechnology International Journal of Pharma and Bio Sciences ISSN EFFECT OF SUCROSE AND AUXIN CONCENTRATION ON INDUCTION OF In Vitro ADVENTITIOUS ROOTS OF Withania somnifera D. PRADEEPA, R. KALAISELVI, T. PANKAJAVALLI AND KALAISELVI SENTHIL * Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore, India. ABSTRACT The genus Withania is an important member of the family Solanaceae, Twenty-three species of the genus Withania have been reported. Of these, only two species namely Withania somnifera and Withania coagulans have been reported from India Withania somnifera, popularly known as "Indian Ginseng". Roots of this plant contain important secondary metabolite withanolide, which have many pharmacological activities. In the present study we induced in vitro adventitious root from the leaf explants of Withania somnifera, was achieved on full strength Murashige and Skoog s medium (MS medium) supplemented with 45g/ L sucrose, and different concentration of auxins. Basal medium supplemented with 1mg/ L indole butyric acid (IBA) and 1mg/ L indole acetic acid (IAA) achieved the maximum number of roots with % response. The roots were transferred to full strength MS suspension medium supplemented with the above combination of auxins and incubated on an orbital shaker at rpm at 25±2 C. Adventious roots were harvested after 10 th and 25 th day of inoculation, and growth index was found to be 6 and respectively. KEY WORDS: Withania somnifera, Adventitious root, root induction, Sucrose, IBA, IAA. KALAISELVI SENTHIL Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute For Home Science and Higher Education for Women, Coimbatore, India. B - 5

2 INTRODUCTION Withania somnifera, popularly known as "Ashwagandha", is one among the top ranking medicinal plants of India and is highly valued for its medicinal and neutraceutical properties 1. The roots and leaves of ashwagandha contain various alkaloids, viz., withanolides, withaferins and withanone 2. Roots, leaves and preparations of the plant are traditionally used as a tonic, hypnotic, sedative and diuretic 3. Plant roots are characterized as rasayanas, that are believed to endorse health and longevity by enhancing the defence against diseases, arresting the ageing process, revitalising the body in debilitated conditions, increasing the capability of the individual to resist adverse environmental factors and by creating a sense of mental well being 4. Generally, the roots are considered to be enriched with bioactive withanolides and used in polyherbal preparations 5. Withanolide A is an important secondary metabolite in Withania somnifera, which is having a high medicinal value and possesses potent anti-tumor and antioxidant properties 6. In vitro root culture has become an alternative method for the production of valuable secondary metabolites on a commercial scale. Adventitious roots are natural, grown vigorously in phytohormone supplemented medium and have shown tremendous potentialities of accumulation of valuable secondary metabolites 7. Adventitious root induced by in vitro methods showed a high rate of proliferation and active secondary metabolism 8. Rooting is an energy requiring process thus an adequate level of carbon source is desired 9. Sucrose has been used as common carbon source for plant tissue and cell cultures, serving as the principal energy source and a component for secondary metabolite biosynthesis 10. MS media supplemented with 1 mg/l IBA+mg/L IAA with 3% sucrose concentration in light was found to be the suitable media for highest percentage of root induction (standardized in our lab). In the present study, an attempt was made to observe the maximum root induction by increasing sucrose concentration along with various combinations of auxins (IAA and IBA). MATERIALS AND METHODS (i)plant material The leaves of W. somnifera were used as explants for root induction. Fresh and healthy leaves from plantlets grown on full strength MS basal media were selected from plants of one-two months of age. These plants have been maintained in MS basal medium for at least two generations. This was done in order to rule out any residual hormonal effect. (ii)root induction media I The leaf explants were inoculated on MS medium supplemented with varying concentration of sucrose ranging from 3%,3.5%,4.0%,4.5%,5.0%,5.5% and 6% along with 1 mg/l IBA+mg/L IAA were added. The ph of the medium was adjusted to 5.6 ± 0.2 before sterilization. (iii)root induction media II From the above experiment 4.5% sucrose along with 1 mg/l IBA+mg/L IAA gave the maximum number of roots, so root induction media II was prepared by adding different combinations of IBA and IAA to the MS basal media with 4.5% sucrose. Hormone combinations tried were given in Table 1. The leaf explants were inoculated on the appropriate medium and the culture was kept under 16/8h light/dark photoperiod at 25 ± 2 C. After four weeks of initiation, roots were separated from the explants aseptically and were sub cultured into 50 ml liquid medium with the same hormone combination. B - 597

3 Table 1 Hormone combination used in root induction media II S.NO IBA mg/l IAA mg /L S.NO IBA mg/l IAAmg /L T0 0 0 T T1 0 T T2 0 T12 T T13 T T T5 0 T T6 0 T16 T7 0 T17 T8 T T9 T (iv) Determination of growth index Roots were separated from the medium by passing through a 1mm stainless steel sieve. Root fresh weight (FW) was measured after rinsing once with sterile water, and root dry weight (DW) was recorded after roots were dried to a constant weight at 60ºC for several days. The growth index was calculated using following equation 11. RESULTS AND DISCUSSION Main aim of this study was to identify the new media for adventitious root induction. From the experiments earlier conducted in our lab, MS media supplemented with 1 mg/l IBA+ mg/l IAA with 3% sucrose concentration in light was found to be the suitable media for highest percentage of root induction. In the present study, the concentration of sucrose was varied keeping the concentration of hormones as reported earlier. The results are presented in Table 2. A significant variation in the number of root induced per explant was observed ranging from a minimum of 9 roots per explant to 16 roots per explants during a period of 12 days. On prolonged culturing the number of roots increased enormously. The result showed the highest response in medium supplemented with 4.5% sucrose (15.75± 4.) followed by 3.5% and 5.5% sucrose concentrations as shown in (Figure 1). Not much difference was observed in 3%, 4%, 5% and 6% sucrose concentration supplemented with 1mg/L IBA + mg/l IAA. Supplementation of sucrose in growth medium meets the energy demands for growth and physiological function, also helps in the maintenance of osmotic potential of cells and conservation of water 9. Our study corresponds to Nagella and Murthy 2, where it was observed that the sucrose was the principle source of secondary metabolite biosynthesis in the shoot culture of Withania somnifera and the biomass growth rate is directly associated with sugar utilization. With the various concentrations of sucrose (1 8%) tested 4% sucrose was best for biomass accumulation and 3% sucrose in case of withanolide A. B - 598

4 Table 2 Effect of sucrose concentration on adventitious root induction S. No Sucrose concentration No of roots initiated per explants Percentage of Root Induction % 9.37± % 13.5± ± % 15.75± ± % 12.87± ± *Data represents mean ± SE, percentage of root induction of 3 replicates with 4 explants, experiment were repeated thrice. Figure 1 Adventitious root induction under the influence of varying sucrose levels a Adventitious roots grown on MS medium supplemented with 3.0% sucrose with b Adventitious roots grown on MS medium supplemented with 3.5% sucrose with c Adventitious roots grown on MS medium supplemented with 4.5% sucrose with d Adventitious roots grown on MS medium supplemented with 5.5% sucrose with 1 mg/l IBA + 1mg/L IAA 1 mg/l IBA + 1mg/L IAA 1 mg/l IBA + 1mg/L IAA 1 mg/l IBA + 1mg/L IAA The variations in root induction might be due to differential signaling levels of sucrose concentration 10 and also donor tissues have been an important role to play in biochemistry of subsequent cultures line 12,13,14. In Panax ginseng, 3% sucrose favoured for ginsenoside production 15, whereas in Bacopa monnieri shoot culture, 2% sucrose favored for bacoside A 16. Thus, the rate of biomass growth is usually directly correlated with sugar consumption 10. Wadegaonkar et al., reported that combination of IAA and IBA was effective in the induction of adventitious roots from leaf explants of W. somnifera. In the present study, maximum root induction was observed with the media supplemented with 4.5% sucrose. So the next part of the work was carried out to study the effect of IBA and IAA in 19 different combinations (one was kept as control without adding any hormone) supplemented with 4.5% sucrose concentration on root induction in W.somnifera under 16/8 h light/dark photoperiod. Different combinations of IAA and IBA showed varied response for adventitious root induction (Figure 2). B - 599

5 Int J Pharm Bio Sci 2014 April ; 5 (2) : (B) Figure 2 Influence of auxins on in vitro adventitious root induction in MS media supplemented with 4.5% sucrose Figure 2. a). MS media without auxin supplementation, b). MS media+mg/l IBA, c). MS media + 1mg/L IBA, d). MS media + 2mg/L IBA, e). MS media + 4mg/L IBA, f). MS media + mg/l IAA, g). MS media + mg/l IBA+mg/L IAA, h). MS media +1mg/L IBA+mg/L IAA, i). MS media + 2mg/L IBA+mg/L IAA, j). MS media +4mg/L IBA+ mg/l IAA, k). MS media mg/l IAA, l). MS media + mg/l IBA+mg/L IAA, m). MS media+1mg/l IBA+mg/L IAA, n). MS media +2mg/LIBA+ mg/l IAA, o). MS media + 4mg/L IBA+mg/L IAA, p). MS media + 1mg/L IAA, q). MS media + mg/l IBA+1 mg/l IAA, r). MS media +1mg/L IBA+ 1 mg/l IAA, s). MS media +2 mg/l IBA+ 1 mg/l IAA, t). MS media + 4 mg/l IBA+ 1 mg/l IAA Table 3 Response of explants to variation in hormone concentration and root induction S.No IBA mg/l IAA mg/l T0 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T Percentage of Root Induction After After After 12 days 25 days 40 days T ±1.45 T13 T14 T15 T16 T17 T18 T ± ±1.76 5±1 5.67±0.33 6±0 5±8 5.33±2.03 No of roots initiated per explants after 12 days 1±8 5±8 4±8 3.67± ± ± ± ±1.86 5±0 3± ±0.67 4±1.53 *Data represents mean±se, percentage of root induction of 3 replicates with 3 explants, experiment were repeated thrice. B - 600

6 The results showed that auxin is essential to induce rooting in the W. somnifera leaf explants. Among the different treatment groups, MS media supplemented with 1mg/L IBA+1mg/L IAA in 4.5% sucrose showed the % of root induction and quicker response (Figure 2r), whereas MS media supplemented with 4mg/L IBA+1mg/L IAA and 2 mg/l IBA+1mg/L IAA showed 88% and.3% respectively, during a period of 12 days. The results are shown in Table 3. Hundred percent root induction was observed in both 1mg/L IBA+1mg/L IAA and 2mg/L IBA+1mg/L IAA, whereas 88% root induction was observed in 4mg/L IBA+1mg/L IAA during a period of 25 days. Hence it is also clear from the results that rooting percentage showed the tendency to increase with increasing concentration of IBA, however, the higher concentration (>2.0 mg/l) than the optimal exerted its inhibitory effect on the number of roots. At lowest concentration of IAA (<mg/l), the percentage of root induction found to be decreased. Sugiyama 18, reported that the effect of an auxin on rooting is a promotory at low concentrations and inhibitory at supraoptimal concentrations. Shilpashree and Rai 19, also reported that the efficient rooting was observed in MS medium supplemented with mg/l IAA in Hypericum mysorense, and also the study clearly indicates the use of BA and IAA play an important role in the biosynthesis of secondary metabolites in in vitro grown cultures. Adventitious roots were transferred to suspension in order to calculate the root mass. For the establishment of roots in suspension, the roots obtained from MS supplemented with 1 mg/l IBA+1 mg/l IAA in 4.5% sucrose was taken, which was chosen as the best auxin combination for root induction (Figure 3). After 10 days and 25 days of root cultures in suspension, the fresh weight of the root was noted. Finally Growth index was calculated 11 that was presented in Table 4. At the end of the 10 th day, the growth index was found to be 6, whereas at the end of 25 th day the growth index was found to be Figure 3 Growth of roots in suspension a Growth of roots after 10 days in suspension medium b Growth of roots after 20 days in suspension medium c Growth of roots after 30 days in suspension medium Table 4 Growth index of roots in suspension culture Media Inoculated fresh weight(g) Harvested fresh weight(g) Growth index (After 10 days) MS+1mg/L IBA+1mg/L IAA (After 25 days ) These results suggest that adventitious root cultures of W. somnifera are promising for large-scale biomass production in suspension cultures. Similarly, they are proved to be efficient for biomass accumulation in P. notoginseng 20 and Echinacea purpurea 11. Further, the adventitious roots can be transferred to bioreactor for mass cultivation. Thus, for the large scale production of bioactive secondary metabolites applicable for pharmaceutical industry, in vitro mass cultivation of roots will be an efficient method. B - 601

7 CONCLUSION From the present study, it can be concluded that MS medium supplemented with 1mg/L IBA+ 1 mg/l IAA along with 4.5% sucrose was found to be optimum concentration for maximum induction of adventitious roots. The roots grown in the above medium showed good growth index when transferred to suspension medium. ACKNOWLEDGEMENT The authors would like to thank the University Grants Commission (F.No \2008 (SR) dt. 2 Jan 2009), for establishment of the work. REFERENCES 1. Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, Sharma R, Sangwan NS, Suri KA, Qazi GN and Tuli R, Phytochemical Variability in Commercial Herbal Products and Preparations of Withania somnifera (Ashwagandha). Current Science, 86: , (2004). 2. Nagella P and Murthy HN, Establishment of cell suspension cultures of Withania somnifera or the production of withanolide A. Bioresource Technology, 101: , (2010). 3. Sumithradevi S, Pradeepa D and Senthil K, A simple method to purify withanolide from the roots of Withanaia somnifera Dunal. International Journal of Pharma And Bio Sciences, 2(1): , (2011). 4. Gupta GL and Rana AC, Withania somnifera (Ashwagandha): A Review. Pharmacological Reviews, 1: , (2007). 5. Asthana R and Raina MK, Pharmacology of Withania somnifera (L.) Dunal-a review. Indian Drugs, 26: , (1989). 6. Praveen N, Naik PM, Manohar SH and Murthy HN, Distribution of Withanolide A content in various organs of Withania somnifera (L.) Dunal. International Journal of Pharma And Bio Sciences, 1(3): 1-5, (2010) 7. Murthy HN, Dijkstra C, Anthony P, White DA, Davey MR, Power JB, Hahn EJ and Paek KY, Establishment of Withania somnifera Hairy root cultures for the production of Withanolide A. Journal of Intergrative Plant Biology, 50(8): ,(2008). 8. Hahn EJ, Kim YS, Yu K, Jeong CS and perk KY, Adventitious root cultures of Panax ginseng C.A mayer and ginsenoside production through large scale bioreactor system. Journal of plant biotechnology, 5:1-3, (2003). 9. Hazarika BN, Acclimatization of tissue cultured plants. Current science, 85: , (2003). 10. Rao S and Ravishankar GA, Plant cell cultures: Chemical factories of secondary metabolites. Biotrchnology advances, 20: , (2002). 11. Wu CH, Murthy HN, Hahn EJ and Paek KY, Establishment of adventitious root co-culture of Ginseng and Echinacea for the production of secondary metabolites. Acta physiol. Plant,30(6): 891-8, (2008). 12. Nigra HM, Caso OH and Ginilietti AM, Production of solasodine by calli from different parts of Solanum eleagnifolium Cav. plants. Plant Cell Rep, 6: , (1987). 13. Banerjee, S, Ahuja PS, Pal A, Gupta,MM and Naqvi AA, Solasodine production by calli from different explants of Solanum sarrachoides. Fitoterapia, 64: , (19). 14. Mischenko NP, Fedoreev SA, Glazunov VP, Chernoded GK, Bulgakov VP and Zhuravle YN, Anthraquinone production by callus cultures of Rubia cordifolia. Fitoterapia, 70: , (1999). 15. Lian ML, Chakrabarty D and Paek KY, Effect of plant growth regulators and B - 602

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