ABSTRACT
Stevia rebaudiana is a non-caloric sweetening herb, which has a high potential for commercialization. During this study low cost media sterilization, seesap (CSUP) technique which applies sodium hypochlorite was used as an alternative to autoclaving. In vitro cultures were established using nodal segments collected from stevia mother plants. These in vitro shoots were cultured on Murashige & Skoog (MS) medium supplemented with 16 combinations of 6- benzyl amino purine (BAP) and kinetin (KN) with 3% sugar at 5.8 pH, to investigate the in vitro shoot multiplication. The developed shoots were transferred to half MS medium supplemented with 0, 1 and 2 mg/L Indole butyric acid (IBA) with 3, 4 and 5% sugar in nine combinations at 5.8 pH, to investigate the rooting. The well rooted stevia plantlets were transferred to Jiffy™ bags for hardening. The results showed that the combination of 1.0 mg/L BAP and 1.5 mg/L KN resulted in the highest number of shoots (11.8 shoots/shoot). The combination of 2.0 mg/L IBA with 5% sugar in half MS medium resulted in the highest root induction (7.2 roots / plant) and root elongation (1.02 cm). The plants grown in culture medium containing 5% sucrose gave 100% survival rate at acclimatization.
Key words: Stevia, micropropagation, shoot induction, root induction, acclimatization, low cost seesap (CSUP).
Stevia (Stevia rebaudiana Bert.), the popular family member of Asteraceae, is a sweet, medicinal herb of Paraguay, containing a non-caloric natural sugar, alternative to artificially produced sugar substitutes. The stevia leaf is 300 times sweeter than sugar (sucrose) obtained from sugar beet, sugarcane etc., with a zero-calorie value (Richman et al., 1999). The stevia leaves are the significant resource of diterpene glycoside like rubsoside, steviolbioside, dulcoside, rebaudiosides and stevioside (Starratt et al., 2002). Stevioside ranks top in dramatically accelerated use in health concerns related to dental cares, diabetes and obesity. The sweetness is due to stevioside, the most abundant glycoside (Kinghorn, 1992). The sweet compounds pass through the digestive track without chemically breaking down, making stevia safe for diabetic and obese people.
Stevia can be propagated using the seeds, or stem cuttings. However, propagation by the seed is not efficient due to low fertility (Tadhani et al., 2006). Propagation using the seed also causes great variability on stevioside level and composition (Nakamura and Tamura, 1985). Seed germination is often poor and rates below 10% (Miyazaki and Wantabe, 1974). The stem cutting method has limitations such as low number of new plants, destruction of the donor plant and requires a significant amount of labor. Therefore, the conventional methods of cultivation and propagation of stevia are time consuming, unpredictable, unreliable and less productive.
Micropropagation or in vitro culture appears to be the best method to overcome those problems and has the potential to produce large quantity of stevia plantlets in a short time. Uddin et al. (2006), established in vitro propagation from the leaf, nodal and internodal segments of S. rebaudiana by using certain plant growth regulators in the medium. Stevia can form multiple shoots from the nodal explants and appears to be suitable for large- scale production. Furthermore stevia in vitro cultures can be grown in bioreactors and stevioside can be extracted continuously (Ramírez-Mosqueda et al., 2016).
The ability of in vitro micropropagation to produce high quality planting material in large quantities is hindered by their high cost of production. Hence, the price of planting material becomes high, making them unaffordable to growers. To overcome this barrier, use of the seesap (CSUP) technique has to be investigated on stevia micropropagation (Peiris et al., 2012). CSUP technique is a low cost method, where sodium hypochlorite is used to sterilize glassware and culture media. When glassware is sterilized by rinsing with 10% (v/v) sodium hypochlorite solution and the culture medium is poured into the glassware, the medium becomes sterilized and even the transferring of axenic cultures can be performed without the use of a laminar flow cabinet.
This technique can be adopted to replace the use of an autoclave and the laminar air flow cabinet in the major micropropagation activity of in vitro multiplication. Hence this method saves the capital cost by Rs. 1.5 to 2 Million, which will encourage entrepreneurs to have small scale laboratories (Peiris et al., 2012). In connection with that, this study was carried out to develop an efficient low-cost method of micropropagation of stevia. Hence, the objective of this research was to optimize the stevia micropropagation protocol using the low cost CSUP technique.
All the experiments were conducted at the Plant Tissue Culture Laboratory, Faculty of Science, Horizon Campus, Malabe, Sri Lanka.
Plant material
Stevia rebaudiana plants used in this study were procured from the Sepra Natural Pvt. Ltd, Godapola, Imbulgasdeniya, Polgahawela, Sri Lanka, and maintained in the plant protected house of Faculty of Science, Horizon Campus, Malabe, Sri Lanka.
Surface sterilization
Stevia nodal segments excised from three months old mother plants were placed under running tap water for 15 to 30 min to remove the dust particles. Then, the explants were soaked in a detergent (Teepol™) for 5 min, rinsed with distilled water and soaked in 70% (v/v) ethanol for a few seconds. The explants were further sterilized by immersing in 10% (v/v) sodium hypochlorite (common house hold bleaching solution) for 10 min and rinsed with sterile distilled water three times in the laminar flow cabinet.
Sterilization of glass jars/plastic tubes and culture medium
Seesap (CSUP) technique was used for sterilization of glassware and culture media (Peiris et al., 2012) used in this study. Under this technique 10% concentration of sodium hypochlorite was identified as the suitable concentration to sterilize glassware/plastic tubes and culture media in a preliminary study.
Multiple shoot induction
The in vitro shoots generated in the establishment stage were transferred to semisolid MS medium in 16 treatment combinations of BAP (0, 0.5, 1.0 and 1.5 mg/L) with KN (0, 0.5, 1.0 and 1.5 mg/L) for shoot multiplication. The number of shoots and shoot length were recorded after four weeks. 20 samples were used per treatment combination and the experiment was repeated twice.
Root induction
The in vitro shoots were transferred onto semisolid half MS medium supplemented with nine combinations of IBA (0, 1.0 and 2.0 mg/L) with sugar (3, 4 and 5%) to study the effects on root induction. Number of roots and root length was recorded after four weeks. 20 samples were used per treatment combination and the experiment was repeated twice.
All in vitro cultures were kept in a growth room at 25 ± 2°C temperature with around 75% humidity and under illuminated (60 µmoles/m-2/s-1, 12 h/day) condition.
Acclimatization
In vitro rooted plantlets, on ½ MS medium supplemented with 3, 4 and 5 % of sugar were removed gently from the medium, washed in running tap water to remove all the traces of the rooting medium and soaked in 0.2 g/L fungicide (Daconil™) solution for 1 min. Plantlets were transferred to JiffyTM bags for acclimatization. Survival percentage of the acclimatized plantlets was recorded after three months.
Statistical analysis
The experiments were arranged in a Completely Random Design (CRD). Each experiment was repeated twice. All the values were expressed as the mean ± standard deviation (sd), calculated using Minitab Statistics software. Data from in vitro cultures were subjected to analysis using one-way analysis of variance (ANOVA) and means were compared using the Tukey’s significance difference test.
Shoot Induction
There were significant differences in the mean values of number of shoots (P = 0.0009) and shoot length (P = 0.0009) of the 16 different combinations after 4 weeks (28 days) of culturing of S. rebaudiana in vitro generated micro shoots.
The highest number of shoots (11.80 ± 0.4) was observed on the MS medium supplemented with the growth regulator combination of 1.0 mg/L BAP and 1.5 mg/L KN and the least number of shoots (1.00 ± 0.0) was found on the MS medium supplemented with 1.5 mg/L KN with 0 mg/L BAP after four weeks of culture. The maximum shoot length (2.20 cm ± 0.1) was observed on the MS medium without any growth regulators (control). However, the second shoot length (1.09 ± 0.1) was recorded on the MS medium supplemented with 1.0 mg/L BAP and 1.5 mg/L KN. The least shoot length (0.43 ± 0.1) was recorded on the MS medium supplemented with 1.0 mg/L BAP with 0 mg/L KN (Table 1).
Root induction
There were significant differences in the mean values of number of roots (P = 0.015) and root length (P = 0.006) of the 9 different treatments after 4 weeks (28 days) of culturing of S. rebaudiana in vitro generated shoots.
The maximum number of roots (7.20 cm ± 0.4) and the longest root length (1.07 cm ± 0.1) were observed on the half MS medium supplemented with 2.0 mg/L of IBA and 5% sugar. The medium without IBA did not exhibit any root induction. The results clearly showed that the number of roots as well as the root length increased with increasing the sugar level. Out of the three different sugar percentages (3, 4 and 5%) used along with IBA, 5% sugar produced the highest shoot number and highest shoot length in both the IBA concentrations (Table 2).
Acclimatization
The results were recorded after three months of acclimatization, and the effect of different sugar concentrations used in pre-transplant stage for survival rate of the acclimatized stevia plantlets were analyzed.
The results showed that the plants grown on culture medium containing 5% sucrose produced the best response and showed 100% survival rate even after 3 months of acclimatization (Figure 1). From Figure 2, it was evident that the survival rate of the stevia plants assessed after acclimatization are all directly proportional to the concentration of sugar used in the culture medium in the in vitro pre-transplant stage in agar gelled medium.
In the present study higher concentrations of BAP or KN resulted in less multiple shoot induction. Sivaram and Mukudan (2003) reported that the combination of low concentrations of BAP and KN (0.5 mg/L) induced high number of multiple shoots and higher concentration of BAP resulted in decreased multiple shoots formation of stevia. However, Tadhani et al. (2006) showed that the maximum number of shoots was achieved on MS medium supplemented with 0.6 mg/L of BAP. According to the results shown in Table 1, the highest number of shoots was produced by 0.5 mg/L BAP concentration out of 3 different concentration of BAP alone and 3 different concentrations of KN alone. This is the best result recorded when BAP and KN growth regulators were used independent from each other. The large number of shoots produced in the presence of BAP was due to the fact that BAP greatly reduced apical dominance and released lateral buds from dormancy and enhanced shoot formation (George and Sherrington, 1984). Das et al. (2011) also reported the longest shoot length of stevia when KN was present in the medium. Although the maximum shoot length of this study was recorded in the control, the second maximum shoot length was recorded in growth regulator combination of 1.0 mg/L BAP and 1.5 mg/L KN where high amount of KN present in the medium.
The various concentrations in combination of the growth regulators used in this study induced the proliferation of shoots derived from the nodal segments of S. rebaudiana. Kinetin was less effective at inducing multiple shoots compared to BAP. According to Rafiq et al. (2007), no specific increase in multiple shoot formation occurred when the explants were cultured on KN based media. Similar results were found in Vriesea scalaris, where KN alone had no effect in in vitro multiplication (Silva et al., 2009). Nevertheless, KN can be more effective to induce multiple shoot when combined with BAP (Verma et al., 2011). According to Anbazhagan et al. (2010), the combinations of BAP and KN work well for both shoot proliferation and their elongation from the nodal explants of S. rebaudiana.
According to the present study, 2.0 mg/L IBA used along with 5% sugar showed the highest root induction and elongation. Verma et al. (2011) reported that half strength MS medium supplemented with 2.0 mg/L IBA proved the best with 87.8% rooting and early root initiation of in vitro cultures of S. rebaudiana. Hwang (2006) reported the maximum numbers of roots using a treatment of 1.0 mg/L IBA in the MS medium with up to 100% rooting. Tadhani et al. (2006) also obtained the highest rate of root induction in 1.0 mg/L IBA included medium. Tesfa et al. (2016), reported that increase in sucrose concentration from 0 to 50 g/L (0 - 5%) along with a definite concentration of NAA (5 mg/L), significantly increased the average root length and root number to 4.58 and 18.00 in sugarcane genotype N52, and to 4.54 cm and 21.76 in genotype N53, respectively. Tesfa et al. (2016) also showed that further increase in sucrose concentration to 70 g/L (7%) resulted in reduction in average root length and number of roots in both sugarcane genotypes. Earlier reports also confirmed that higher concentrations of sucrose in the medium have a negative impact on overall rooting due to accumulation of rooting inhibitors, the reduction of rooting promoters in the medium, and the transformation of added sugars into insoluble and storage form (Ahmed et al., 2004).
Root length increased on IBA containing medium and this was accentuated as the IBA concentration increased (Trauttman and Visser, 1990). The IBA seems to be the best auxin in S. rebaudiana to the initiation of the root induction. The potential of IBA in root induction has also been reported in many species (Epstein et al., 1993). IBA has been observed to induce strong rooting response and has been extensively used to promote rooting in a wide range of plant species by Rani and Rana (2010), Steephen et al.(2010) and Yadav and Singh (2011b).
Sucrose is a prime carbon source of stevia micropropagation and developing vigorous plantlets, but the performances of other disaccharides or mono-saccharide are not fully explained as far as optimum stevia tissue culture is concerned. There is a general agreement in the literature that sucrose is required in the medium for stevia tissue culture. The concentration and type of exogenous carbon sources added to medium to serve as energy and also to maintain the osmotic potential (Lipavska and Konradova, 2004). Plant cells and tissues under in vitro conditions are mixotrophic in nature and needs supply of external carbon source for its metabolic activities (Lipavska and Konradova, 2004).
The present study showed that the plants grown in culture medium containing 5% sucrose gave the best response and showed 100% survival rate even after 3 months of acclimatization. The success of using high concentration of sucrose for pre-transplant stage in orchid micropropagation is stated by many researchers. Collins and Dixon (1992), studied different sucrose concentrations in in vitro culturing and observed that for the Australian terrestrial orchid Diuris longifolia, 20 g/L sucrose plus charcoal had a similar rooting effect as 40 g/L sucrose without charcoal. According to Kerbauy (1993), high agar levels, together with the sucrose, promote great longitudinal root growth and lateral aerial growth, while low levels of these components favor the formation of protocorms in in vitro culture of Oncidium varicosum (Orchidaceae). Ishii et al. (1998) observed that in Phalaenopsis the presence of sucrose in the culture medium caused protocorm formation and its absence caused callus proliferation.
This increase in the amount of sucrose in the culture should be taken with caution and should not be progressive, because, according to Capellades et al, (1991) and Hdider and Desjardins (1994), higher sucrose concentrations in in vitro cultures favor carbohydrate accumulation and hinder photosynthesis.
During this study sterilized coir dust in Jiffy™ bags were used as the hardening medium in order to promote water retention since the plantlets were kept in an acclimatization chamber consist of high temperature and high humidity. Meera and Sathyanarayana (2010) reported that with sand as the hardening media, mist house had higher survival rates than greenhouse. Sand did not retain much water in greenhouse to ensure proper growth and survival compared with mist house with frequent misting. In all other cases, the survival was found to be better in greenhouse (28%) compared with mist house (23%). With frequent misting, water logging occurred around the plantlets, and they started to rot which lead to reduction in survival. This was absent in sand due to its high porosity and rate of percolation. They also reported that the survival percentage recorded with cocopeat under greenhouse was around 75.5% that was almost 60 times higher than the lowest survival recorded with 100% sand under greenhouse (1.5%).
This study proved that the CSUP technique can be successfully applied to micropropagate S. rebaudiana as a low cost application where equipment cost can be reduced by 97% (Peiris et al., 2012). According to the Table 3, the total cost reduction percentage was 99.5%.
Attempting to reduce cost of S. rebaudiana micropropagation, Sharma et al. (2013) reported 85% cost reduction for the medium by replacing laboratory reagent grade sucrose by locally available commercial sugar, bacteriological grade agar by Isabgol as the gelling agent and distilled water by tap water. In our study also sugar available at the market was used rather than the analytical grade sucrose which is another cost reduction. All of the aforementioned studies prove evidence for the credibility of the results obtained from the current research.
Stevia rebaudiana micropropagation protocol carried out using seesap (CSUP) low cost media sterilization showed maximum in vitro shoot multiplication of 11.8 shoots/shoot on the MS medium supplemented with 1.0 mg/L BAP, 1.5 mg/L KN in combination and 3% sugar at 5.8 pH. The highest number of roots and maximum root length were observed on the half MS medium supplemented with 2.0 mg/L IBA with 5% sugar combination. The plants grown in pre-transplant culture medium containing 5% sucrose showed 100% survival rate in acclimatization.
The authors have not declared any conflict of interests.
The authors gratefully acknowledge Horizon Campus, Malabe, Sri Lanka for providing financial assistance to carry out this study. They are also grateful to Ms. Hansini Jayanetti, Technician, Horizon Campus for her technical assistance.
REFERENCES
|
Ahmed S, Sharma A, Bhushan B, Singh AK, Wali VK (2004). Effect of carbohydrate source, pH supporting media on in vitro rooting of banana (musa spp.) cv. Gr naine plantlets. African Journal of Agricultural Research 9:1135-1140.
Crossref
|
|
|
|
Anbazhagan M, Kalpana M, Rajendran R, Natarajan V, Dhanavel D (2010). In vitro production of Stevia rebaudiana Bertoni. Emirates Journal of Food and Agriculture 22(3):216-222.
Crossref
|
|
|
|
|
Capellades M, Lemeur R, Debergh P (1991). Effects of sucrose on starch accumulation and rate of photosynthesis in Rosa cultured in vitro. Plant Cell Tissue and Organ Culture 25:21-26.
Crossref
|
|
|
|
|
Collins MT, Dixon KW (1992). Micropropagation of an Australian terrestrial orchid Diuris longifolia R Br. Australian Journal of Experimental Agriculture 32:131-135.
Crossref
|
|
|
|
|
Das A, Gantait S, Mandal N (2011). Micropropagation of an elite medicinal plant: Stevia rebaudiana Bert. International Journal of Agricultural Research 6:40-48.
Crossref
|
|
|
|
|
Epstein E, Sagee O, Zahir A (1993). Uptake and metabolism of Indole-3 acetic acid and Indole-3 butyric acid by Petunia cell suspension culture. Plant Growth Regulation 13(1):31-40.
Crossref
|
|
|
|
|
George S, Sherrington SL (1984). Tissue culture in forest trees: Clonal propagation of Tectona grandis L. (teak) by tissue culture. Plant Science Letters 17(3):259-268.
Crossref
|
|
|
|
|
Hdider C, Desjardins Y (1994). Effects of sucrose on photosynthesis and phosphoenolpyruvate carboxylase activity of in vitro cultured strawberry plantlets. Plant Cell, Tissue and Organ Culture 36:27-33.
Crossref
|
|
|
|
|
Hwang SJ (2006). Rapid in vitro propagation and enhanced stevioside accumulation in Stevia rebaudiana Bert. The Journal of Plant Biology 49(4):267-270.
Crossref
|
|
|
|
|
Ishii Y, Takamura T, Goi M, Tanaka M (1998). Callus induction and somatic embriogenesis of Phalaenopsis. Plant Cell Reports 17:446-450.
Crossref
|
|
|
|
|
Kerbauy GB (1993). The effects of sucrose and agar on the formation of protocorm-like bodies in recalcitrant root tip meristems of Oncidium varicosum (Orchidaceae). Lindleyana 8:149-154.
|
|
|
|
|
Kinghorn AD (1992). Food Ingredient Safety Review: Stevia rebaudiana Leaves. Herbal Research Foundation, Boulder, CO USA.
|
|
|
|
|
Lipavska H, Konradova H (2004). Somatic embryogenesis in conifers: The role of carbohydrate metabolism. In Vitro Cellular and Developmental Biology 40:23-30.
Crossref
|
|
|
|
|
Meera Manjusha AV, Sathyanarayana BN (2010). Acclimatization studies in Stevia (Stevia rebaudiana Bert.). Acta Horticulturae 865:129-133.
Crossref
|
|
|
|
|
Miyazaki Y, Wantenabe H (1974). Studies on the cultivation of Stevia rebaudiana Bertoni on the propagation of plant. The Journal of Tropical Agriculture 17:154-157.
|
|
|
|
|
Nakamura S, Tamura Y (1985). Variation in the main glycosides of Stevia (Stevia rebaudiana). Japanese Journal for Tropical Agriculture 29:109-116.
|
|
|
|
|
Peiris SE, De Silva E, Edussuriya M, Attanayake AMUK, Peiris BCN (2012). CSUP technique: a low cost sterilization method using sodium hypochlorite to replace the use of expensive equipment in micropropagation. Journal of the National Science Foundation of Sri Lanka 40(1):49-54.
Crossref
|
|
|
|
|
Rafiq M, Dahot MU, Mangrio SM, Naqri HA, Qarshi IA (2007). In vitro clonal propagation and biochemical analysis of field established Stevia rebaudiana Bertoni. Pakistan Journal of Botany 39(7):2467-2474.
|
|
|
|
|
Ramírez-Mosqueda MA, Iglesias-Andreu LG, Ramírez-Madero G, Hernández-Rincón EU (2016). Micropropagation of Stevia rebaudiana Bert. in temporary immersion systems and evaluation of genetic fidelity. South African Journal of Botany 106:238-243.
Crossref
|
|
|
|
|
Rani S, Rana JS (2010). In vitro Propagation of Tylophora indica-Influence of Explanting Season, Growth Regulator Synergy, Culture Passage and Planting Substrate. Journal of American Science 6(12):385-392.
|
|
|
|
|
Richman AS, Gijzen M, Starralt AN, Yang Z, Brandle JE (1999). Diterpene synthesis in Stevia rebaudiana Bert. recruitment and up-regulation of key enzymes from the gibberellin biosynthetic pathway. The Plant Journal 19:411-421.
Crossref
|
|
|
|
|
Sharma V, Singh I, Sharma S (2013). Formulation of medium with low cost options for in vitro caulogenesis in ethnomedicinal herb Stevia rebaudiana. Trends in Biotechnology Research 2(1):36-40.
|
|
|
|
|
Silva ALL, Franco ETH, Dornelles EB, Reichert Bortoli CL, Quoirin M (2009). In vitro multiplication of Vriesia scalaris E. Morrem (Bromeliaceae). Iheringia Série Botânica 64:151-156.
|
|
|
|
|
Sivaram L, Mukundan U (2003). In vitro culture studies on Stevia rebaudiana. In Vitro Cellular and Developmental Biology 39:520-523.
Crossref
|
|
|
|
|
Starratt AN, Kirbyb CW, Pocsa R, Brandle JE (2002). Rebaudioside F, A diter pene glycoside from Stevia rebaudiana Bert. Phytochemistry 59:367-370.
Crossref
|
|
|
|
|
Steephen M, Nagarajan S, Ganesh D (2010). Phloroglucinol and silver nitrate enhances axillary shoot proliferation in nodal explants of Vitex negundo L. - an aromatic medicinal plant. Iranian Journal of Biotechnology 8(2):82-89.
|
|
|
|
|
Tadhani MB, Jadeja RP, Rena S (2006). Micropopagation of Stevia rebaudiana Bertoni using multiple shoot culture. Journal of Cell and Tissue Culture Research 6:545-548.
|
|
|
|
|
Tesfa M, Admassu B, Bantte K (2016). In Vitro Rooting and Acclimatization of Micropropagated Elite Sugarcane (Saccharum officinarum L.) Genotypes - N52 and N53. Journal of Tissue Science and Engineering 7:164.
Crossref
|
|
|
|
|
Trauttman IA, Visser JH (1990). An in vitro study of organogenesis in guayule (P. argentatum). Plant Science 72:275-281.
Crossref
|
|
|
|
|
Uddin MS, Chowdhury MSH, Khan MMMH, Uddin MB, Ahmed R, Baten MA (2006). In vitro propagation of Stevia rebaudiana Bert. in Bangladesh. African Journal of Biotechnology 5(13):1238-1240.
|
|
|
|
|
Verma S, Yadav K, Singh N (2011). Optimization of the Protocols for Surface Sterilization, Regeneration and Acclimatization of Stevia rebaudiana Bertoni. American-Eurasian Journal of Agricultural and Environmental Sciences 11(2):221-227.
|
|
|
|
|
Yadav K, Singh N (2011b). In vitro flowering of shoots regenerated from cultured nodal explants of Spilanthes acmella Murr. - an ornamental cum medicinal herb. Analele Universitãþii din Oradea Fascicula Biologie 18(1):66-70.
|
|
