Stevia rebaudiana Bertoni is used for medicinal and food purposes. The effectiveness of different culture systems in Stevia micropropagation was evaluated. Node explants were cultured in semisolid or RITA® bioreactor containing woody plant medium (WPM) without plant growth regulators (PGRs). The effect of three medium volumes (100, 200, and 300 mL) and four immersion frequencies (10 seconds every 1, 4, 6, or 8 hours) were assessed. The shoots and roots developed simultaneously in the RITA® bioreactor treatment containing 100 mL of WPM at 10 seconds/4 hours immersion frequency and the healthiest plantlets were obtained from this RITA® treatment using 5 mL of medium per explant. The root induction rate was 100% in nonhyperhydrated shoots obtained from both semisolid and RITA® bioreactor, and all of them were successfully acclimatized. Thus, an economical and viable protocol was developed by performing the micropropagation in one step, eliminating the use of agar and PGRs, and decreasing the amount of medium used per explant.
Bärbel Röck-Okuyucu, Meltem Bayraktar, Ismail Hakki Akgun and Aynur Gurel
Stevia rebaudiana is of great importance due to its steviol glycosides (SGs) which are natural sweeteners used by the food industry as well as having medicinal purposes. In the present study, the effect of plant growth regulators (PGRs) and explant types on in vitro propagation and shoot growth of S. rebaudiana were studied, the effect of PGRs on SGs production was determined. For this purpose, nodal explants and shoot tip explants were cultured on woody plant medium (WPM) supplemented with cytokinins [6-benzyladenine (BA), kinetin (Kn), or thidiazuron (TDZ)] or cytokinins + auxins combinations [BA + indoleacetic acid (IAA); BA + naphthaleneacetic acid (NAA); Kn + IAA; Kn + NAA]. Although, the best shoot proliferation was obtained on WPM supplemented with BA + NAA combinations, shoots grown on PGR-containing media produced callus at the base of the shoots and showed chlorosis and necrosis. Additionally, shoots showed at all concentrations of TDZ, and at higher concentrations of BA, morphological changes such as malformed leaves and poor shoot growth. In contrast to PGR-containing media, on the PGR-free control medium, the development of shoots and roots occurred simultaneously and healthy and well-developed plantlets were obtained. Thus, we developed an economical viable means of in vitro propagation by minimizing the micropropagation steps and removing the requirement of PGRs. According to the high-performance liquid chromatography (HPLC) results, PGR-free control medium (WPM) led to considerably higher stevioside content in the leaves compared with the PGR(s)-containing media and the highest stevioside [34 mg·g−1 dry weight (DW)] and rebaudioside A content was only detected on the control medium without PGRs. Steviolbioside, rubusoside, and dulcoside A were detected qualitatively in the leaves of shoots grown on WPM supplemented with 2.27 μm TDZ, 4.54 μm TDZ, 2.22 μm BA + 2.69 μm NAA, 2.22 μm BA + 5.37 μm NAA, 2.32 μm Kn + 5.71 μm IAA, or 2.32 μm Kn + 2.69 μm NAA.