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Plant Growth Regulator Effects on in Vitro Propagation and Stevioside

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Plant Growth Regulator Effects on in Vitro Propagation and Stevioside PROPAGATION AND TISSUE CULTURE HORTSCIENCE 51(12):1573–1580. 2016. doi: 10.21273/HORTSCI11093-16 production of Stevia via seeds is not efficient (Brandle et al., 1998; Gantait et al., 2015). Besides this, the seeds show a wide variation Plant Growth Regulator Effects with regard to the SGs content as well as the morphological features (the shape and color on In Vitro Propagation and of leaves) (Tamura et al., 1984). Clonal propagation via vegetative cuttings is practical Stevioside Production in Stevia for small-scale production, but it is not suit- able due to the small number of individuals rebaudiana and economically viable because of high labor Bertoni costs for large-scale production (Brandle et al., Barbel€ Rock-Okuyucu€ 1998; Gantait et al., 2015). A genetically School of Tobacco Expertise, Celal Bayar University, Akhisar/Manisa, homogeneous population, which produces high yields of the desired SGs, can be Turkey achieved by in vitro propagation of a selected Meltem Bayraktar1 plant (Gantait et al., 2015; Lemus-Mondaca et al., 2012; Tamura et al., 1984). The use of Biotechnology Department, Graduate School of Nature and Applied apical and axillary meristems, shoot tips, or Sciences, Ege University, Bornova/Izmir 35100, Turkey; and Genetic and nodal explants for in vitro production, en- Bioengineering Department, Faculty of Engineering and Architecture, Ahi ables the conservation of the genotype during Evran University, Kirsehir 40100, Turkey clonal propagation. In addition, in vitro plant propagation using axillary buds was reported Ismail Hakki Akgun and Aynur Gurel to be a simple and economic method to obtain Bioengineering Department, Faculty of Engineering, Ege University, many genetically uniform true-to-type plants Bornova/Izmir 35100, Turkey within a short period (Faisal et al., 2007). The presence of diterpene glycosides was Additional index words. dulcoside A, HPLC, in vitro, rubusoside, steviolbioside, stevioside investigated in callus culture, suspension cul- ture (Bondarev et al., 2001; Mathur and Abstract Stevia rebaudiana . is of great importance due to its steviol glycosides (SGs) which Shekhawat, 2013), etiolated in vitro regener- are natural sweeteners used by the food industry as well as having medicinal purposes. In ants, callus culture grown in the light, etio- the present study, the effect of plant growth regulators (PGRs) and explant types on in vitro lated heterotrophic callus (Ladygin et al., S. rebaudiana propagation and shoot growth of were studied, the effect of PGRs on SGs 2008), hairy root culture (Pandey et al., production was determined. For this purpose, nodal explants and shoot tip explants were 2016; Yamazaki and Flores, 1991), in vitro cultured on woody plant medium (WPM) supplemented with cytokinins [6-benzyladenine root culture (Reis et al., 2011), in leaf-derived + + (BA), kinetin (Kn), or thidiazuron (TDZ)] or cytokinins auxins combinations [BA callus cultures (Sivaram and Mukundan, + + + indoleacetic acid (IAA); BA naphthaleneacetic acid (NAA); Kn IAA; Kn NAA]. 2003), salts (NaCl and Na CO ), proline or + 2 3 Although, the best shoot proliferation was obtained on WPM supplemented with BA NAA polyethylene glycol–treated callus, and suspen- combinations, shoots grown on PGR-containing media produced callus at the base of the sion culture (Gupta et al., 2014, 2015). How- shoots and showed chlorosis and necrosis. Additionally, shoots showed at all concentrations ever, there is little information about the effect of TDZ, and at higher concentrations of BA, morphological changes such as malformed of PGRs on SGs of in vitro-propagated Stevia leaves and poor shoot growth. In contrast to PGR-containing media, on the PGR-free shoots. The synthesis of SGs is initiated in the control medium, the development of shoots and roots occurred simultaneously and healthy chloroplasts and takes place mainly in the and well-developed plantlets were obtained. Thus, we developed an economical viable leaves (Brandle and Telmer, 2007; Ladygin means of in vitro propagation by minimizing the micropropagation steps and removing the et al., 2008). The SGs production varies from requirement of PGRs. According to the high-performance liquid chromatography (HPLC) clone to clone as well as depending on the results, PGR-free control medium (WPM) led to considerably higher stevioside content in physical conditions under which Stevia plants the leaves compared with the PGR(s)-containing media and the highest stevioside [34 are regenerated. It is of particular interest to L1 mg·g dry weight (DW)] and rebaudioside A content was only detected on the control develop new S. rebaudiana varieties with a high medium without PGRs. Steviolbioside, rubusoside, and dulcoside A were detected SGs content and to obtain sufficient propagat- m qualitatively in the leaves of shoots grown on WPM supplemented with 2.27 M TDZ, ing and planting material which possesses this m m + m m + m m + 4.54 M TDZ, 2.22 M BA 2.69 M NAA, 2.22 M BA 5.37 M NAA, 2.32 M Kn quality characteristic. The extracts with homo- m m + m 5.71 M IAA, or 2.32 M Kn 2.69 M NAA. geneous and high secondary metabolite con- tents can be achieved in vitro under controlled physical conditions, using a suitable growth Stevia rebaudiana Bertoni belongs to the stevioside and rebaudioside A (Kinghorn, media. Furthermore, it is possible to achieve family Asteraceae and is a perennial that is 2002). The sweetening properties of Stevia within a considerably short time a large number primarily native to eastern Paraguay (Brandle leaves have already been used since the of, with regard to development and growth, et al., 1998). S. rebaudiana accumulates sixteenth century by the native Paraguayan uniform Stevia plantlets by in vitro propaga- mainly in its leaves 34 noncaloric sweet- populations in the Amambay region (Soejarto, tion. Therefore, the purpose of the present tasting SGs (Ceunen and Geuns, 2013). The 2002). In the early 1970s, Japan started the study was to develop an efficient and econom- two main SGs produced in the leaves are commercial cultivation of S. rebaudiana,and ical in vitro propagation protocol for a selected sometime later the first Stevia products have clone line of S. rebaudiana. Furthermore, to been offered on the Japanese market. Since determine the effect of some PGRs on the shoot Received for publication 27 June 2016. Accepted then, Stevia has been introduced in several growth and SGs production. for publication 15 Sept. 2016. countries and can be used as food additive and This research was supported by Celal Bayar Uni- for therapeutic purposes (Brandle et al., 1998; Materials and Methods versity Scientific Research Projects Commission Lemus-Mondaca et al., 2012). (ALS 2008-069). Stevia rebaudiana We are so grateful to Erdal Bedir and Norm Kimya In general, Stevia plants are propagated by shoot cultures San.Tic.Ltd.Sxti. seeds or vegetative cuttings. Since the Stevia In vitro clonally propagated plantlets, 1Corresponding author. E-mail: meltembayraktar5@ seeds are very small and infertile, they have which were obtained from single seed descent gmail.com. poor germination rate. Therefore, large-scale seedlings of S. rebaudiana,wereusedasthe HORTSCIENCE VOL. 51(12) DECEMBER 2016 1573 plant material. To obtain a sufficient number 10 min by removing the bags from the plants 0.8 mL·min–1. A sample of 10 mLwasinjected of plants, nodal segments and shoot tips (with once a day for a period of 2 weeks. The (partial loop injection) each run. The spray one axillary bud) of 4-week-old plantlets were acclimation bags were completely removed at chamber temperature was set to 40 °C, the transferred to glass tubes each containing 10 mL the end of the 2-week period. The plants were drift tube temperature to 105 °C, the gas of WPM (Lloyd and McCown, 1980) supple- then transferred from the climate chamber to pressure to 50 psi, and the filter of the ELSD mented with 3% (w/v) sucrose, and solidified the greenhouse conditions 3 weeks after the to 6. The retention time was 4.22 min for with 0.65% (w/v) plant agar (Duchefa Bio- beginning of acclimatization and were main- stevioside and 6.12 min for rebaudioside A. chemie B.V., Haarlem, The Netherlands). The tained there for a period of 5 weeks. Com- Liquid chromatography–mass spectrometry pH of all media was adjusted to 5.8 before the pletely acclimatized plants were transferred to analysis. Liquid chromatography–mass spec- addition of the gelling agent, and they were field conditions at the end of 8 weeks. trometry (LC-MS) experiments for the qualita- autoclaved at 121 °C at 1.04 kg·cm–2 for tive identification of steviol derivatives were 15 min. For plant multiplication, they were SGs analysis performed on a Thermo Accela UPLC in- transferred every 4 weeks to fresh solidified Chemicals. HPLC grade acetonitrile and strument, equipped with a TSQ Quantum medium. methanol were purchased from Merck (Darm- Access MAX Triple Quadrople Mass Spec- stadt, Germany). The ultrapure water, used for trometer (Thermo Scientific, Bremen, Ger- PGR application and culture conditions the analysis, was obtained from an in-house many). Thermo Accela UPLC system consists To investigate the effect of different ultrapure water system (Sartorius Arium 611; of a high-pressure quaternary pump, an auto- PGRs on shoot proliferation and growth as Sartorius Stedim Biotech, Gottingen,€ Germany). sampler, and a column oven. Separations were well as on SGs production, two different Stevioside and rebaudioside A were isolated carried out by in-house generated method with explant types as specified above, were used. from the leaves of S. rebaudiana by our research the following conditions. For all separations, The explants were cultured in glass tubes group with a purity of + 90%. a Teknokroma RP C18 250 mm · 4.6 mm · each containing 10 mL of WPM supple- HPLC sample preparation. Appoximately 5 mm column (Teknochroma, Barcelona, mented with various concentrations of dif- 75 mg of leaf samples were sonicated three Spain) was used.
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