High Frequency Clonal Multiplication of Stevia rebaudiana Bertoni, Sweetener of the Future Sharma Shiwali1, Shahzad Anwar1,* 1Plant Biotechnology Section, Department of Botany, Aligarh Muslim University, Aligarh-202 002, U.P., India *shahzadanwar@rediffmail.com, ashahzad.bt@amu.ac.in
Abstract The present study describes an efficient, rapid clonal propagation protocol for a natural sweetener herb, Stevia rebaudiana. Shoot tips and nodal segments were inoculated on Murashige and Skoog's medium [MS 1962] containing different cytokinin and auxin alone or in combinations. Shoot tips were proved to be better in comparison to nodal segments having higher rate of shoot induction and multiplication. Best shoot multiplication from both the explants was obtained on MS medium supplemented with 5.0 µM 6-benzyladenine (BA) and 1.0 µM á-naphthalene acetic acid (NAA). A maximum of 19.60 shoots per shoot tip and 16.00 shoots per nodal segment with an average shoot length of 4.70 and 4.28 cm were obtained. Subculturing of the regenerating tissue on the optimized treatment showed further enhancement in shoot proliferation without any decline in multiplication rate. The regenerated microshoots showed the most efficient rooting on half strength MS medium augmented with 2.5 µM indole-3-butyric acid (IBA). Plantlets went through a hardening phase prior to ex vitro transfer and established in earthen pots containing garden soil and green manure (1:1). The established plantlets were uniform and identical to mother plant with respect to growth characteristics and vegetative morphology. Top Keywords Micropropagation, Natural Sweetener, Stevia rebaudiana, Stevioside. Top |
Introduction Stevia rebaudiana Bertoni is a perennial herb belongs the family Asteraceae. It is a natural sweetener plant known as “Sweet Weed”, “Sweet Leaf”, “Sweet Herb”, “Honey Leaf” etc. It is native of certain regions of South America particularly in Paraguay and Brazil. |
The leaves of Stevia are the rich sources of diterpene glycosides, such as steviolbioside, rubsoside, rebaudioside A, B, C, D, E and F, dulcoside and stevioside [Starratt et al., 2002]. Among them stevioside stands first and is non-caloric, thermo-stable, intense sweetener which imparts about 300 times higher sweetness than sucrose. Now a day's use of this sweetening compound has increased dramatically due to health concerns related to sucrose usage, such as dental caries, obesity and diabetes. This sweetening compound passes through the digestive process without chemically breakdown, making safe to control sugar level [Strauss, 1995]. Refined extract of Stevia leaves are officially approved as food additives in Brazil, Korea and Japan [Choi et al., 2002; Mizutani and Tanaka, 2002]. Japan was the first country in Asia to market stevioside as a sweetener in food and drug industries as a substitute for synthetic sweetener, aspartame. Since then cultivation of the plant has expanded to several countries in Asia including India, China, Malaysia, Singapore, South Korea, Taiwan and Thailand. By all the means, recently S. rebaudiana has attracted economic and scientific interest. |
Generally, propagation of S. rebaudiana is done by stem cutting but main problem involved in the cultivation of this species is its heterozygous and self-incompatibility natures which lead to the lack of fertilization [Miyazaki and Wanteabe, 1974]. The seeds of Stevia show very less vigor and propagation and do not allow the production of homogenous population which leads to variability in sweetening level and composition [Felippe and Lucas 1971; Miyagawa et al., 1986]. Poor seed germination percentage is the limiting factor to large scale cultivation of this species. Vegetative propagation is also limited by the low number of individuals obtained from single plant. Therefore, to overcome all these obstacles, micropropagation or in vitro culture technique can play a vital role for mass propagation and the production of genetically identical plants of S. rebaudiana. As far as the literature is concerned, few reports of micopropagtion of S. rebaudiana have been cited [Sivaram and Mukundan 2003, Ahmed et al., 2007; Rafiq et al., 2007; Anbazhagan et al., 2010] but none of them are proved to be satisfactory in term of industrial requirements as raw material. In this way, the present study was aimed at the establishment of an efficient protocol for high frequency in vitro mass propagation of S. rebaudiana. |
Top Materials and Methods Source of Explant and Surface Sterilization The twigs (about 5.0–6.0 cm) of pot grown S. rebaudiana plant were collected from the Department of Botany, A.M.U., Aligarh. The twigs with shoot tip and 3–4 nodes were washed in running tap water for 30 min to remove the superficial dust particles. They were then immersed in 1% (w/v) Bavistin (Carbendazim Powder, BASF India Ltd), a broad spectrum fungicide followed by 5% (v/v) Labolene (Qualigens, India) each for 15 min. After each step of sterilization, the explants were washed with sterilized double distilled water (DDW). Further, sterilization procedure were carried out in laminar air flow chamber by using 0.1% (w/v) HgCl2 (Qualigens) for 3 min. The explants were then rinsed five times with sterilized DDW. From these sterilized twigs, shoot tips (0.5–1.0 cm) and nodal segments (1.0 -1.5 cm) were excised aseptically and implanted vertically on regeneration media. Shoot Regeneration and Multiplication Murashige and Skoog's medium (MS) fortified with specific concentrations of plant growth regulators (PGRs) (BA, kinetin Kn, IAA, IBA and NAA) singly or in combinations was used for shoot regeneration and multiplication with 3% (w/v) sucrose (Qualigens) and 0.8% (w/v) agar (Qualigens). All the PGRs were purchased from Duchefa, Netherland. Basal MS medium without any PGR was used as control. For further shoot proliferation optimized treatment was used. Subculturing was regularly done at four week of interval. In Vitro Rooting For in vitro root induction the regenerated microshoots (3.0–4.0 cm) were transferred to half-strength MS medium supplemented with NAA and IBA (1.0, 2.5 and 5.0 µM). Half strength basal MS nutrient medium devoid of auxin was treated as control. Culture Conditions All the cultures were raised in 25 × 150 mm culture tubes (Borosil, India). The pH of the medium was adjusted to 5.8 with 1N NaOH and 1N HCl before autoclaving at 1.06 kg cm2 and 121ºC for 15 min. After inoculation all the cultures were maintained at 25 ± 2ºC in 16/8 h light/dark cycle with 50 µmol m −2 s−1 irradiance provided by cool fluorescent tubes (Philips, India, 40 W). Ex vitro Hardening Plantlets with well developed shoots and roots were removed from the culture media, washed gently under running tap water and transferred to thermocol cups (expanded polystyrene) containing sterilized soilrite (75% Irish peat moss and 25% horticulture grade expanded perlite) (Keltech Energies Ltd., India) under diffused light (16/8 h photoperiod) conditions. Plantlets were covered with transparent polythene membranes to ensure high humidity and watered every third day with normal tap water. Polythene membranes were opened progressively after two weeks in order to harden plants to culture room conditions. After four weeks, these plantlets were transferred to earthenware pots (12 cm diameter) containing normal garden soil and green manure (1:1) and maintained in a greenhouse under normal day length conditions. Top Statistical Analysis All the experiments were repeated thrice with 20 replicates for each treatment. The data were analyzed using SPSS Version 12 (SPSS Inc., Chicago, USA) and mean were compared using Tukey's test at 5% level of significance. |
Top Results and Discussion In this study experiments were conducted to standardize the explant source and culture media for multiple proliferation of shoot and result in mass propagation of homogenous elite plantlets of S. rebaudiana. Shoot tips and nodal segments from field grown plant of S. rebaudiana were inoculated on MS medium supplemented with various cytokinins alone or in combination with auxin at different concentrations. Mean number of shoots per explant and mean shoot length varied considerably with the type of explant and treatment used (Table 1). |
The proliferation efficiency of shoot tips was significantly higher than that of nodal segments when evaluated up to four weeks. Shoot tips and nodal segments remained green and elongated into single shoot on hormone free MS medium. However, supplementation of cytokinin (BA and Kn) and auxin (NAA and IAA) to basal MS medium induced direct shoot multiplication as a result of split in the pre existing meristem. |
Shoot tips exhibited their initial response with the appearance of new leaves within 3–4 days of incubation. Apical bud started to sprout in multiple buds after one week of culture (Figure 1 A). In the present study, BA was more potent when compared with Kn for apical bud break. A linear correlation was achieved with the increase in BA/Kn concentration from 1.0 to 5.0 mM and shoot proliferation efficiency Supplementation of 5.0 µM BA resulted in maximum proliferation as a solitary cytokinin. Shoot tips produced higher number of shoots 7.40 per explant with the mean length of 3.92 cm after four week of culture (Figure 1 B). However, nodal segments produced 5.60 shoots per explant with 3.40 cm shoot length on similar treatment. This discrepancy in result might be due to the difference in endogenous hormonal balance in different explant types. When BA concentration was increased beyond the optimum level (5.0 µM), the rate of shoot multiplication and elongation was reduced in both the explant types due to callogenesis. According, to Preece et al., [1991], the formation of callus at the basal cut ends of explant on cytokinin enriched medium is frequent in species with strong apical dominance. Superiority of BA over other Kn for axillary bud promotion in Stevia rebaudiana has also been reported by Sivaram and Mukundan [2003], Rafiq et al., [2007], Ahmed et al., [2007] and Anbazhagan et al., [2010]. Our observations on the suppression of bud break at higher concentration of cytokinin was in conformity with the reports of Ahuja et al., [1982] in Ocimum gratissimum and O. viride and Patnayak and Chand [1996], in Ocimum americanum and Ocimum santum. |
Addition of auxin to the optimized concentration of BA (5.0 µM) showed stimulatory effect in the enhancement of multiple shoot induction. Among different cytokinin-auxin combinations attempted, enhanced proliferation has been achieved in BA (5.0 µM) + NAA (1.0 µM) for both the explant types, thus exhibiting similar type of hormonal requirement for morphogenesis. On this treatment a maximum number of 19.60 and 16.00 shoots per explant with an average shoot length of 4.70 and 4.28 cm have been obtained through shoot tip and nodal segments respectively (Figure 1 C, D, E). Thus, incorporation of NAA to BA supplemented medium exerted rapid enhancement of shoot length with higher number of nodes per shoot which make them sturdy as compared to shoots induced on BA alone. On further increasing NAA concentration to (1.5 µM) morphogenic potential of shoot tips and nodal segments was reduced. The results substantiate with the earlier findings of several workers where the addition of low level of NAA with cytokinins promoted shoot proliferation as reported in Ceropegia bulbosa [Britto et al., 2003], Salvia nemorosa [Ska3a and Wysokiñska,2004] and Spilanthes mauritiana [Sharma et al., 2009]. However, some studies advocated the use of BA and IAA for satisfactory axillary bud multiplication in Stevia rebaudiana [Sivaram and Mukundan 2003, Anbazhagan, 2010] Ocimum gratissimum [Gopi et al., 2006] and Scoparia dulcis [Karthikeyan et al., 2009]. |
Effect of Subculture Passage on Shoot Proliferation As it has been observed in the present study, MS medium supplemented with BA (5.0 mM) and NAA (1.0 mM) was found to be most effective for axillary bud multiplication and proliferation from shoot tips and nodal segments, therefore this treatment has been selected as optimal and used for further shoot proliferation. By repeated subculturing of regenerating tissue, a prolific shoot culture was established (Figure 1F). The number of shoots per explant increased significantly after every subculture passage as clear from Figure 2 A. Besides, subculturing has a significant promotive effect on shoot length also Figure 2 B. Enhanced shoot multiplication in subsequent subculture was in accordance with earlier published reports on Eclipta alba by Borthakur et al., [2000] and Husain and Anis [2006]. Subculturing had a more significance effect on shoot proliferation from shoot tips as compared to nodal segments/tissue. In Vitro Rooting Microshoots excised from in vitro proliferated cultures were implanted to half strength MS medium with or without different concentrations of auxin, NAA and IBA (1.0, 2.5 and 5.0 mM) for in vitro rooting. Microshoots showed similar responses regarding in vitro rooting irrespective of explants source. Hormone free nutrient medium failed to induce in vitro rooting. However, auxin supplemented media induced rooting within one week of culture Table 2. Among the treatments tried, 2.5 mM IBA was found to be the best which resulted in the induction of an average of 8.00 roots per shoot with an average root length of 6.36 cm in 84% of culture within four week of incubation (Figure 3 A). Similar hormonal requirement for in vitro rooting has also been reported by Sivaram and Mukundan [2003], in same plant species. However, they reported a slightly higher number of roots but the roots formed in the present study were good enough for their successful establishment in soil. On the other hand Ahmed et al., [2007] and Anbazhagan et al. [2010], suggested the requirement of IAA for best rooting in S. rebaudiana while Rafiq et al. [2007], found optimum rooting in NAA supplemented nutrient medium. Ex Vitro Hardening Plantlets with well developed roots were acclimatized and hardened off in solirite condition as described in materials and method. The use of sufficiently porous substratum that allows adequate drainage and aeration has been recommended for fast acclimatization of in vitro regenerated plants [Dunstan and Turner 1984]. After four weeks under greenhouse, the potted plants were transferred to natural field conditions for better establishment and at the end of this study, around 96% of plants were thrived well in natural field environment (Figure 3 B). Top Figures | Figure 1: (A) Induction of multiple shoots on MS + BA (5.0 µM) from shoot tip after two week of incubation (B, C) High frequency of shoot multiplication on MS + BA (5.0 µM) + NAA (1.0 µM) from shoot tip after two and four week of incubation (D) Nodal segments placed on MS + BA (5.0 µM) + NAA (1.0 µM) (E) Shoot multiplication and elongation on MS + BA (5.0 µM) + NAA (1.0 µM) from nodal segment after four week of incubation (F) Culture showing shoot proliferation during subculturing
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| | Figure 2: (A, B) Effect of subculture passages on shoot proliferation and elongation efficiency through shoot tip and nodal segments
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| | Figure 3: (A) In vitro rooting in half strength MS + IBA (2.5 µM) (B) An acclimatized plant of S. rebaudiana under field condition
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Tables | Table 1:: Effect of different concentrations of growth regulators on multiple shoot formation through shoot tip and nodal segments after 4 weeks of culture
| | Growth regulator (µM) | Shoot tip | Nodal segment | | No. of shoots per explant (Mean ± SE) | Shoot length (cm)(Mean ± SE) | No. of shoots per explant (Mean ± SE) | Shoot length (cm) (Mean ± SE) | | control | 1.00 ±. 00g | 2.50 ±. 80h | 1.00 ±. 00g | 2.38 ±. 05g | | BA (1.0) | 2.40 ±. 24fg | 2.86 ±. 11gh | 1.80 ±. 20fg | 2.62 ±. 05fg | | BA (2.5) | 3.60 ±. 24ef | 3.38 ±. 05ef | 3.00 ±. 31f | 3.02 ±. 06de | | BA (5.0) | 7.40 ±. 40d | 3.92 ±. 10bc | 5.60 ±. 24e | 3.40 ±. 04cd | | BA (7.5) | 4.80 ±. 37e | 3.22 ±. 08fg | 3.20 ±. 37f | 2.90 ±. 08ef | | Kn (1.0) | 1.80 ±. 20fg | 3.16 ±. 06fg | 1.40 ±. 24fg | 2.78 ±. 05ef | | Kn (2.5) | 2.80 ±. 20efg | 3.50 ±. 03ef | 2.00 ±. 31fg | 3.40 ±. 07cd | | Kn (5.0) | 4.60 ±. 24e | 3.62 ±.05cde | 3.00 ±. 31f | 3.78 ±. 11bc | | Kn (7.5) | 2.80 ±. 20efg | 3.90 ±. 04bc | 2.40 ±. 24fg | 3.10 ±. 04de | | BA (5.0) + NAA (0.5) | 13.80 ±. 58b | 4.04 ±. 05b | 10.60 ±. 40c | 3.80 ±. 07b | | BA (5.0) + NAA (1.0) | 19.60 ±. 50a | 4.70 ±. 10a | 16.0 ±. 31a | 4.28 ±. 08a | | BA (5.0) + NAA (1.5) | 11.60 ±. 50c | 3.74 ± 10bcde | 9.2 ±. 58cd | 4.02 ±. 06ab | | BA (5.0) + IAA (0.5) | 11.60 ±. 50c | 3.52 ±. 03def | 8.4 ±. 50d | 3.78 ±. 08bc | | BA (5.0) + IAA (1.0) | 15.00 ±. 31b | 3.88 ±. 05bcd | 12.8 ±. 73b | 4.08 ±. 08ab | | BA (5.0) + IAA (1.5) | 9.20 ±. 86d | 3.24 ±. 08f | 9.0 ±. 44cd | 3.38 ±. 10d |
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| | | Table 2:: Effect of different concentrations of auxins supplemented to half strength MS nutrient medium on in vitro root induction after 4 weeks of culture
| | Auxin (µM) | % of microshoots rooted | No. of roots per shoots (Mean ± SE) | Root length (cm) (Mean ± SE) | | Control | - | 0.00 ±. 00e | 0.00 ±. 00f | | IBA (1.0) | 75 | 4.80 ±. 37bc | 5.24 ±. 08cd | | IBA (2.5) | 84 | 8.00 ±. 31a | 6.36 ±. 06a | | IBA (5.0) | 73 | 5.60 ±. 24b | 5.68 ±. 05b | | NAA (1.0) | 68 | 3.00 ±. 31d | 4.50 ±. 08e | | NAA (2.5) | 75 | 6.00 ±.31b | 5.46 ±. 07bc | | NAA (5.0) | 70 | 3.60 ±. 24cd | 5.00 ±. 06d |
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