Stevia rebaudiana (Bertoni) is an economically important medicinal plants act as an option for artificial sweetening agents. The plant regeneration protocol under in vitro conditions has been developed for this natural sweetener herb. Callus was efficiently induced when leaf segments were cultured on MS medium supplemented with 1.0 mg/l 2, 4-D + 1.0 mg/l Kinetin. The above callus was sub cultured on MS medium + 0.5 mg/l BAP + 0.3 mg/l NAA for root differentiation and on MS medium + 0.5 mg/l BAP + 0.1 mg/l NAA for shoot differentiation. Biochemical changes occurring during the callus differentiation were adopted to quantify the metabolites. Metabolites like starch, total soluble sugars and total phenols decreased while total soluble proteins increased in callus culture during the process of root and shoot differentiation.Key words: Callus, differentiation, in vitro, metabolites, Stevia rebaudianaAbbreviations:Ã‚Â  BAP Benzyl amino purine; 2,4-D 2,4-Dichlorophenoxyacetic acid; MS Murashige and Skoog (1962) basal medium; NAA ÃŽÂ±-naphthalene acetic acid; IBA Indole-3-butyric acid; IAA Indole acetic acidNarender Singh et al. Metabolic Changes during Differentiation in Callus Cultures of Stevia rebaudiana (Bertoni). J Phytol 3/3 (2011) 63-67

Kumari and Renu, Narender Singh*, Kuldeep Yadav, Suman

Journal of Phytology

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Journal of Phytology 2011, 3(3): 63-67                                                                                                     ISSN: 2075-6240  Tissue Culture                                                                                     Available Online: www.journal-phytology.com  REGULAR ARTICLE METABOLIC CHANGES DURING DIFFERENTIATION IN CALLUS CULTURES OF STEVIA REBAUDIANA (BERTONI)  Narender Singh*, Kuldeep Yadav, Suman Kumari and Renu Plant Tissue Culture Laboratory, Department of Botany, Kurukshetra University, Kurukshetra,                       Haryana, India-136119   SUMMARY Stevia rebaudiana (Bertoni) is an economically important medicinal plants act as an option for artificial sweetening agents. The plant regeneration protocol under in vitro conditions has been developed for this natural sweetener herb. Callus was efficiently induced when leaf segments were cultured on MS medium supplemented with 1.0 mg/l 2, 4-D + 1.0 mg/l Kinetin. The above callus was sub cultured on MS medium + 0.5 mg/l BAP + 0.3 mg/l NAA for root differentiation and on MS medium + 0.5 mg/l BAP + 0.1 mg/l NAA for shoot differentiation. Biochemical changes occurring during the callus differentiation were adopted to quantify the metabolites. Metabolites like starch, total soluble sugars and total phenols decreased while total soluble proteins increased in callus culture during the process of root and shoot differentiation. Key words: Callus, differentiation, in vitro, metabolites, Stevia rebaudiana Abbreviations:  BAP Benzyl amino purine; 2,4-D 2,4-Dichlorophenoxyacetic acid; MS Murashige and Skoog (1962) basal medium; NAA α-naphthalene acetic acid; IBA Indole-3-butyric acid; IAA Indole acetic acid Narender Singh et al. Metabolic Changes during Differentiation in Callus Cultures of Stevia rebaudiana (Bertoni). J Phytol 3/3 (2011) 63-67. *Corresponding Author, Email: nsheorankuk@yahoo.com; kuldeep0608@gmail.com  1. Introduction Stevia rebaudiana Bertoni, belongs to the Asteraceae family is a natural sweetener perennial herb commonly known as “Sweet Weed”, “Sweet Leaf”, “Sweet Herbs” and        “Honey Leaf”. The leaves of this plant are estimated to be 300 times sweeter than sucrose and the sweetness is due to glycosides of which the most abundant is stevioside (Kinghorn, 1992). The increasing consumption of sugar (sucrose) has resulted in several nutritional and medical problems, such as obesity. Therefore, low caloric sweeteners have been investigated to substitute sugar. The refined extracts of leaves of this plant are officially used as high potency natural-source, low calorie (nonsucrose) sweetener in processed foods, artificial diets and pharmaceuticals (Mizutani & Tanaka, 2002). The sweet compounds pass through the digestive process without chemically breaking down, making it safe for diabetic and obese people.  The plants grow better in the temperature range of 0-40°C (Carneiro et al., 1997). The seeds of this plant show a very low germination percentage and vegetative propagation is limited by lower number of individuals. So, Tissue culture is the only rapid process for the mass propagation of this plant because the species is becoming rare in natural habitat condition due to habitat destruction and over exploitation. The process of growth and morphophysiological specialization of cells from unorganized mass of callus cells i.e. differentiation, is a prerequisite for the application of biotechnology for crop improvement. Differentiation of organized structures in tissue culture is controlled by growth regulators such as cytokinins and auxins along with other components of the culture medium. Differentiation through callus cultures involves changes in some of the biochemicals (Kumar et al., 2009). But very little is known about the biochemical Narender Singh et al./J Phytol 3/3 (2011) 63-67  events occurring in the cultured cells. Analysis of various cellular metabolites and enzyme activities provides a reasonable and promising approach towards an understanding of the biochemical basis of the developmental pathway (Singh et al., 2009). There are few reports available on biochemical studies related to shoots differentiation in callus cultures of another plant species (Singh et al., 2006).The present study was therefore, undertaken to determine the changes in the levels of metabolites during shoot and root differentiation from callus cultures of S. rebaudiana.  2. Materials and Methods MS basal medium was used without additives and supplemented with different concentrations of BAP, KIN, 2,4-D, IAA or NAA alone or in different combinations with different concentrations for callus induction. The pH of the medium was adjusted to 5.8, prior to addition of agar and autoclaving at 121˚C, 1.2 kg cm-2 pressure for 15 min.  Plant material and callusing  The leaf explants were obtained from mature plant of S. rebaudiana growing in Herbal Garden of Department of Botany, Kurukshetra University, Kurukshetra. These were washed with liquid detergent under running tap water to remove dust particles. The explants were then treated with 0.1% (w/v) mercuric chloride for 3-5 minutes under aseptic conditions. These explants were then thoroughly washed 4-5 times with sterilized double distilled water   to remove the traces of mercuric chloride. Surface sterilized explants were inoculated onto the medium aseptically. Two explants were placed in each culture bottles.  The cultures were incubated at a temperature of 25±2ºC and a photoperiod of 16hrs light (intensity of 2000 lux) and 8hrs of dark.  Differentiation After four-five weeks, the developed calli from leaf tissues were separated from explants and transferred to fresh medium of the same composition as induction medium (MS + 1.0 mg/l 2, 4-D + 1.0 mg/l Kn) thus using it also as ‘callus proliferation medium’. For biochemical studies during root and shoot differentiation, the calli were further subcultured for root differentiation on MS medium + BAP (0.5 mg/l) + NAA (0.3 mg/l) and for shoot differentiation on MS medium+ BAP (0.5 mg/l) + NAA (0.1 mg/l). The content of various metabolites, viz. starch, total soluble sugars, total soluble proteins and total phenols were studied on day 0, 5, 10, 15 and 20 after inoculation in callus on root induction medium, as well as on shoot induction medium.  Estimation of metabolites  Extraction of metabolites was done by the modified method of Barnett and Naylor (1966). For each sample, 100 mg of dry callus was homogenized in 80 per cent ethanol (v/v). The homogenate was refluxed for 15 min on a water bath at 60°C and centrifuged for 10 min at 5000 rpm. This procedure of refluxing and centrifuging was done three times with residue and collect supernatant each time. The supernatants were pooled and used for estimation of starch, total soluble sugars, total soluble proteins and total phenols by using methods of Yemm and Willis (1954), Hassid and Neufeld (1964), Bradford (1976), Amorim et al. (1977) respectively. Various metabolites (starch, total soluble sugars, reducing sugars,  total soluble proteins and total phenols) was assayed during roots differentiation as well as shoots differentiation at 5th , 10th , 15th   and 20th   day after inoculation of callus. The visual appearance of root formation was observed after 10th day of subculture. Before the appearance of shoot formation, green patches were formed. The visual appearance of shoots were observed after 15th day of subculture. The callus was harvested on 5th, 10th, 15th   and 20th day after inoculation. The undifferentiated callus before keeping on differentiating media was served as control.     Narender Singh et al./J Phytol 3/3 (2011) 63-67  Statistical data analysis  For the above experiments, 24 replicates were used for each treatment and each experiment was repeated thrice. The results are expressed as a Mean ± S.E. of three independent experiments. Statistical analysis was done by using the formula: SE = ± ( )( )1nn X2−  SE = Standard error X = deviation of mean n = number of replicates (Snedecor, 1956).  3. Results and Discussion The basal medium without growth regulators failed to respond for callus induction from leaf explant. Supplementation of growth regulators to MS basal medium resulted in callus induction but the frequency varied with the type and concentration of growth regulators used. MS basal medium supplemented with auxins (IBA and NAA) alone failed to induce callus.  However, when supplemented with 2, 4-D, 26-87% callusing was observed at different concentrations (0.5 - 2.0 mg/l) with poor growth of callus. Presence of 2,4-D has been shown to be essential for callus formation in Momordica charantia (Agrawal and Kamal, 2004). On adding different concentrations of Kn to MS medium, the callus growth was poor On MS medium supplemented with 2,4-D (0.5 mg/l) + Kn  (0.5 - 1.5 mg/l),  per cent callus induction was upto 100% but visual growth of callus was poor to moderate. Good callus growth was observed when concentration of 2, 4-D was increased i.e. 1.0 mg/l along with Kn (0.5 and 1.0 mg/l). Best growth of callus was observed on 2, 4-D (1.0 mg/l) along with Kn (1.0 mg/l). So, this combination was selected for callus maintenance and for biochemical studies during root and shoot differentiation. The above callus was subcultured for root differentiation on MS medium + BAP (0.5 mg/l) + NAA (0.3 mg/l) and for shoot differentiation on MS medium+ BAP (0.5 mg/l) + NAA (0.1 mg/l). The role of auxins and cytokinins in callus induction was also advocated by Kumar and Singh (2009) in Stevia rebaudiana, Goel and Singh (2009) in Peganum harmala, Kumar and Singh (2009) in Prosopis cineraria, Lal and Singh (2010) in Celastrus paniculatus and Yadav and Singh (2010) in Spilanthes acmella. To understand the differentiation events in callus tissues on regeneration medium, metabolites associated with organ differentiation in callus tissue were studied.  Metabolites: Starch  In the undifferentiated calli, starch content was high but on 5th and 10th day after subculture to rooting medium, the content increased further. The starch content decreased during root formation   (10th and 15th days) but a little increase was observed on 20th day (Fig. 1). The status of starch content in the calli during shoot differentiation showed a zig-zag trend i.e. the content increased on 5th day, then it decreased on 10th and 15th day but again it increased on shooting. Thus, it was observed that the undifferentiated calli contained more starch content than the differentiated ones (Fig. 1). In the present study, it was found that starch content in callus declined during the process of root and shoot differentiation and increased only after shoot initiation. These results concide with the findings in Chlorophytum borivilianum (Singh et al., 2006). The decrease in starch content could be due to decrease in the activity of synthesizing enzymes or increase in hydrolyzing enzymes.  Fig. 1. Changes in the concentration of starch at different intervals on root and shoot differentiating medium             Narender Singh et al./J Phytol 3/3 (2011) 63-67  Fig. 2. Changes in the concentration of total soluble sugar at different intervals on root and shoot differentiating medium          Fig. 3. Changes in the concentration of total soluble proteins at different intervals on root and shoot differentiating medium           Fig. 4. Changes in the concentration of total phenols at different intervals on root and shoot differentiating medium           Total soluble sugars  A steady rise in total soluble sugars was observed from undifferentiating callus i.e. 0 day to 10th day old calli kept on root differentiation medium. But after that, a marked decrease was observed in total soluble sugars content in callus from root initiation to root appearance i.e. 15th and 20th day (Fig. 2). Unlike root differentiating calli, marked increase in total soluble sugar content was observed only upto 5th day then the sugar content in callus decreased steadily upto shoot differentiation. Overall results show that the differentiated calli had less soluble sugars than undifferentiated calli (Fig. 2). The decline in total soluble sugar content was associated with utilization of sugars for growth and differentiation process. Similar trend was also reported in Cardiospermum halicacabum (Jeyaseelan and Rao, 2005). Total soluble proteins In root differentiating calli, a steady increased in protein content was observed upto 15th day, but the content decreased a little after 15th day (Fig. 3). While in case of shoot differentiating calli, the content increased slowly upto 5th day but a sharp increase was observed by 10th day. Thereafter, the protein content decreased on 15th day. There was again an increase in content on 20th day (Fig. 3). It was observed that total soluble proteins in root and shoot forming calli were higher during root and shoot differentiation than in controlled callus. Since during differentiation the cells are quantitatively changing their activities, new proteins have to be synthesized, thus the protein concentration is high before differentiation. Similar observation was also reported by Mohapatra and Rath (2005). Total phenols  The phenol content followed a zig-zag path from non-differentiated callus to differentiated one i.e. increased on 5th day, decreased during 10th and 15th day and then again increased on 20th day (Fig. 4). The same trend was followed in shoots differentiating calli. Total phenols increased upto 5th day, decreased on 10th and 15th day and then again increased on 20th day. Thus, it showed that the amount of total phenols was higher in non-differentiated calli than in differentiated ones (Fig. 4). Present investigation showed gradual decrease in phenolic content during differentiation. Phenols participate in formation of cross-linking of cell wall constituents which is catalyzed by peroxidase (Mader and Fussel, 1982). Changes in the levels of metabolites and enzymes during differentiation from callus culture may be helpful in our understanding of the biochemical basis of developmental pathway of S. rebaudiana.  Narender Singh et al./J Phytol 3/3 (2011) 63-67  Acknowledgements The authors are grateful to University Grants Commission, New Delhi and Kurukshetra University, Kurukshetra for providing financial assistance and laboratory facilities to carry out the work. References Agarwal M, Kamal R (2004) In vitro clonal propagation of Momordica charantia L.   Indian  Journal of Biotechnology 3:426-430 Amorim H A, Dougall D K, Sharp W R (1977) The Effect of Carbohydrate and Nitrogen Concentration on Phenol Synthesis in Paul's Scarlet Rose Cells Grown in Tissue Culture. Physiologia Plantarum 39: 91-95 Barnett N M, Naylor A W (1966) Amino acid and protein metabolism in Bermuda grass during water stress. Plant Physiology 41: 1222-1230 Bradford M (1976) "A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding" Ann. Biochemistry. 72:248-254 Carneiro J W P, Muniz A S, Guedes T A (1997) Green house bedding plant production of Stevia rebaudiana (Bert.). Canadian Journal of Plant Science. 77: 473-474. Fors A (1995) A new character in the sweetener scenario. Sugar Journal 58:30  Goel N, Singh N, Saini R (2009) Efficient in vitro multiplication of Syrian Rue (Peganum harmala L.) Using 6- benzylaminopurine pre-conditioned seedling explants. Nature and Science 7:129-134 Hassid WZ, Neufeld EF (1964) Quantitative determination of starch in plant tissues. In: Methods of carbohydrate chemistry. Ed. Whistler, M B, New York, Academic Press. 4:33-36 Jeyaseelan M, Rao M V (2005) Biochemical studies of embryogenic and non-embryogenic callus of Cardiospermum halicacabum L. Indian Journal of Experimental Biology 43: 555-560 Kinghorn A D (1992) Food Ingredient Safety Review: Stevia rebaudiana Leaves. Herbal Research Foundation, Boulder, CO USA Kumar S, Singh N, Mangal M (2010) Biochemical changes during shoot differentiation in callus cultures of Simmondsia chinensis (Link) Schneider. Journal of Plant Biology 36(1): 1-6 Kumar S, Singh N (2009) In vitro propagation   of   Stevia rebaudiana Bertoni: An important medicinal sweet herb. Environment Ecology 27(IA): 459-464  Kumar S, Singh N (2009) Micropropagation   of Prosopis cineraria(l.) Druce – a multipurpose desert tree. Researcher 1:28-32  Lal D, Singh N (2010) Mass Multiplication of Celastrus paniculatus Willd – An Important Medicinal Plant Under In vitro Conditions using Nodal Segments. Journal of American Science 6: 55-61 Mader M, Fussel R (1982) Role of peroxidase in lignification of tobacco cells. Plant Physiology 70: 1132-1134 Mizutani K O, Tanaka (2002) Use of Stevia rebaudiana sweeteners in Japan. In: Stevia, the Genus Stevia. Medicinal and aromatic plants-industrial profiles. Taylor and Francis: London and New York, Vol. 19: 178-195 Mohapatra H P, Rath S P (2005) In vitro studies of Bacopa monnieri- An important medicinal plant with reference to its biochemical variations. Indian Journal Experimental Biology 43: 373-376  Singh S R, Singh R, Dhawan A K (2009) Biochemical changes related to shoot differentiation in callus cultures of Tylophora indica Wight and Arn. Journal of Indian Botanical Society 88(3&4): 49-53. Singh R, Singal H R, Goyal S C (2006) Biochemical changes during shoot differentiation in callus cultures of Chlorophytum borivilianum Sant. Et Fernand. Indian Journal Plant Physiology 11: 130-135  Snedecor G W (1956) Statistical method applied to experiments in agriculture and biology. Iowa State College Press, Ames Yadav K, Singh N (2010) Micropropagation of Spilanthes acmella Murr. – An Important Medicinal Plant. Nature and Science 8(9):5-11  Yemm E W, Willis A J (1954) The estimation of carbohydrates in plant extracts by anthrone. Biochemistry Journal 57:508-514. 

Metabolic Changes during Differentiation in Callus Cultures of Stevia rebaudiana (Bertoni)

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Metabolic Changes during Differentiation in Callus Cultures of Stevia rebaudiana (Bertoni)

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