Steviol glycosides (SvGls) are plant secondary metabolites belonging to a class of chemical compounds known as diterpenes. SvGls have been discovered only in a few plant species, including in the leaves of Stevia rebaudiana Bertoni. Over the last few decades, SvGls have been extensively researched for their extraordinary sweetness. As a result, the nutritional and pharmacological benefits of these secondary metabolites have grown increasingly apparent. In the near future, SvGls may become a basic, low-calorie, and potent sweetener in the growing natural foods market, and a natural anti-diabetic remedy, a highly competitive alternative to commercially available synthetic drugs. Commercial cultivation of stevia plants and the technologies of SvGls extraction and purification from plant material have already been introduced in many countries. However, new conventional and biotechnological solutions are still being sought to increase the level of SvGls in plants. Since many aspects related to the biochemistry and metabolism of SvGls in vivo, as well as their relationship to the overall physiology of S. rebaudiana are not yet understood, there is also a great need for in-depth scientific research on this topic. Such research may have positive impact on optimization of the profile and SvGls concentration in plants and thus lead to obtaining desired yield. This research summarizes the latest approaches and developments in SvGls production. Key points • Steviol glycosides (SvGls) are found in nature in S. rebaudiana plants. • They exhibit nutraceutical properties. • This review provides an insight on different approaches to produce SvGls. • The areas of research that still need to be explored have been identified.

This study developed a plant regeneration protocol for Trifolium nigrescens (Viv.) via somatic embryogenesis (SE). Immature zygotic embryos at torpedo (TsE) and cotyledonary (CsE) stage were cultured on media with different auxins and cytokinins at different concentrations. The cultural requirements for SE differed between the explants used: the addition of 6-furfurylaminopurine (kinetin) or N 6 -[2-isopentenyl]-adenine (2iP) along with 2,4-dihydrophenoxyacetic acid (2,4-D) or 1-naphthaleneacetic acid (NAA) was needed to elicit the embryogenic response of CsE, but an exogenous cytokinin totally inhibited 2,4-D-induced SE from TsE. When applied alone, neither the cytokinin nor NAA induced SE in TsE or CsE. In all effective cultures the first somatic embryos appeared directly from the upper part of the hypocotyl (TsE and CsE) and from the margin of cotyledons (TsE) on day 7. Embryogenic callus occurred on CsE after 10 days. At comparable concentrations 2,4-D was a more potent SE inducer than NAA, but most of the embryoids induced on media with 2,4-D displayed morphological abnormalities, whereas those produced in the presence of NAA generally resembled zygotic embryos.Plant regeneration was achieved after transfer of somatic embryos or embryo-derived first shoots to medium without plant growth regulators (PGRs). The frequency of plant recovery was about 30% for embryoids obtained on media containing 2,4-D, and for material from media with NAA the recovery rates were 44-68% (somatic embryos) and 72-100% (embryoid-derived shoots). Regenerants appeared identical to each other and to wild plants; they produced flowers and had the chromosome complement typical for the species, 2n = 16, in root tip cells.

Microscopy and flow cytometry (FCM) were used to study somatic embryogenesis (SE) from zygotic embryos of Trifolium nigrescens Viv. to determine if there were any relationships between characteristics of somatic embryos (morphology, anatomy, genome size stability) and their regenerability. Embryoids were induced on Murashige and Skoog (MS) medium containing 4 mg l -1 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg l -1 N 6 -[2-isopentenyl]-adenine (2iP) either directly from hypocotyls or via an intervening callus, depending on the duration of culture. The morphology of somatic embryos varied from zygoticlike structures to abnormal structures including hornshaped, polycotyledonary, and fused embryoids. The incidence of abnormalities was higher in callus cultures than in direct regeneration. Horn-shaped embryoids were the most frequent type of abnormal embryos. Only embryoids having zygotic-like morphology regenerated into plantlets. Histological observations revealed that the absence of shoot and root apical meristems along with parenchymatization of embryos were major obstacles to conversion of horn-shaped embryoids. The estimated 2C value for T. nigrescens was 0.9 pg. FCM analysis revealed differences in DNA content between embryoids induced via an intervening callus and those produced directly from explants. Individuals with species-specific as well as increased DNA content were detected among those zygotic-like embryos derived from callus, but all horn-shaped embryoids had increased genome sizes. The observed lack of differences in DNA content between zygotic-like and horn-shaped embryoids, from direct SE, indicated that these phenotypic abnormalities were of physiological origin. The mean DNA content of regenerants was species-specific, suggesting that only diploid embryoids were capable for regeneration into plantlets. Keywords Auxin Á Clover Á Flow cytometry Á Histology Á Nuclear DNA content Á Somatic embryo morphology Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid 2iP N 6 -[2-isopentenyl]-adenine FCM Flow cytometry MS Murashige and Skoog (1962)

The changes in the activity of some antioxidant enzymes and endogenous H 2 O 2 level in zygotic sunflower embryos during organogenesis and somatic embryogenesis were monitored. Pathways of regeneration were induced on media differing with sucrose concentration 87 mmol dm À3 for shoot [shoot induction medium (SIM) medium] and 350 mmol dm À3 [embryo induction medium (EIM) medium] for somatic embryo induction. Water potential of the explants cultured on SIM increased, while the embryos maintained on EIM showed middle water deficit stress. The pattern of superoxide dismutase (SOD) isoforms was similar in organogenic and embryogenic culture; however, the intensity of MnSOD bands was higher on SIM than on EIM. Differences in catalase activity were observed: high activity on SIM predominated, whereas on EIM it was reduced. The activity of guaiacol peroxidase in the explants producing shoots and somatic embryos differed at the beginning of culture, but became comparable at the time of shoot and somatic embryo formation (day 5). H 2 O 2 content was unchanged in organogenic culture, but on EIM it increased on day 1 followed by significant decrease. The results indicate that sugar concentration per se, or via induction of different developmental pathways influences the activity of antioxidant enzymes and also H 2 O 2 level in cultured sunflower embryos.

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