В представленной работе проведен критический анализ наиболее интересных гипотез предназначениядольменов Западного Кавказа. Приведен ряд аргументов, свидетельствующих против основнойнаучной гипотезы их назначения, существующей в настоящее время – сооружения для погребений,главным из которых является полное отсутствие в подавляющем большинстве дольменов остатковпогребального содержимого. На основе анализа личных исследований и данных литературныхисточников предложена и обоснована новая и наиболее вероятная гипотеза первоначальногоназначения дольменов Западного Кавказа – укрытия для детей от крупных хищников, случайныходиночных охотников из других племен и некоторых стихийных явлений.

The growth and the biosynthesis of steviol glycosides (SGs) in Stevia rebaudiana cell cultures subjected to the impact of different cultivation factors were investigated under in vitro conditions. The changes in composition and concentration of constituted components of the nutrient medium was found to exert a significant influence on the of cell culture growth and the biosynthesis of SGs. It was found that combination of growth regulators in the nutrient medium is more important factor for proliferation of isolated cells of S. rebaudiana while the level of SGs accumulation depends to greater extent on genotypical features of cell strain. Light was shown to activate the biosynthesis of SGs, providing evidence for the suggestion about chloroplasts participation in this activation. In morphogenic callus cultures, the content of SGs was about ten times higher, indicating that the level of the accumulation of the compounds in question depends on cell differentiation and specialization.

The short-day plant stevia ( Stevia rebaudiana ) was earlier reported to produce electron-dense thylakoids in the chloroplasts under long-day conditions. In the present work, such thylakoids were analyzed by transmission electron microscopy and gas chromatography–mass spectrometry. It was found that they contain glyceryl tricaprate, which belongs to triacylglycerols. A hypothesis is advanced that glyceryl tricaprate acts as a photoprotector for short-day plants against redundant solar radiation.

Using transmission electronic microscopy and mass spectrometry electron-dense thylakoids of chloroplasts of Stevia rebaudiana leaves during active vegetable growth of this plant were studied in relation to the biosynthesis of diterpenoid glycosides (DGs). It was found that these compounds are absent in these thylakoids, but they contain a water-insoluble weakly polar ent-kauren, a known biosynthetic precursor of DGs as well as gibberellins. This finding provides a base for the suggestion that similar, electron-dense, thylakoids were observed earlier by other authors in other plant species. These data allowed us to conclude that an intensive biosynthesis of ent-kauren is likely related to adaptation of the short-day plants including Stevia rebaudiana to vegetable growth under the long day conditions.

The ultrastructure of trichomes of intact and in vitro plants of stevia were investigated under scanning and transmission electronic microscopy. Three types of trichomes were found on both the upper and lower surfaces of the leaf: viz. rounded glands and large and small trichome. The density of trichomes on the surface of upper leaves is higher than on lower leaves, while in vitro plants are characterized with lower density of trichomes than intact ones. The positive correlation between number of trichomes on leaves and content of steviol glycosides (SGs) in them was found. Three basic stages of ontogeny of gland cells were revealed which reflect the sequence of secrete accumulation.

The accumulation of steviol glycosides (SGs) in cells of Stevia rebaudiana Bertoni both in vivo and in vitro was related to the extent of the development of the membrane system of chloroplasts and the content of photosynthetic pigments. Chloroplasts of the in vitro plants, unlike those of the intact plants, had poorly developed membrane system. The callus cells grown in the light contained proplastids of almost round shape and their thylakoid system was represented by short thylakoids sometimes forming a little number of grana consisting of 2–3 thylakoids. In cells of the etiolated in vitro regenerants and the callus culture grown in the dark, only proplastids practically lacking the membrane system were observed. All the chloroplasts having developed thylakoids and forming at least a little number of grana were equipped with photochemically active reaction centers of photosystems 1 and 2. Leaves of in vivo plants accumulated greater amount of the pigments than leaves of the in vitro plants. In both the callus culture grown in the light and the etiolated in vitro regenerants, the content of the pigments was one order of magnitude lower than that in leaves of the intact plants. The callus tissue grown in the dark contained merely trace amounts of the pigments. Leaves of the intact and the in vitro plants did not exhibit any significant differences in photosynthetic O2 evolution rate. However, photosynthetic O2 evolution rate in the callus cells was much lower than that in the differentiated plant cells. The in vitro cell cultures containing merely proplastids did not practically produce SGs. However, after transferring these cultures in the light, both the formation of chloroplasts and the production of SGs in them were detected.

The contents of three major steviol glycosides (SGs) (stevioside and rebaudiosides A and C) in vegetative and generative organs during ontogeny of Stevia rebaudiana Bertoni were analysed with HPLC. Plant organs contained different amounts of the SGs, which declined in the following order: leaves, flowers, stems, seeds, roots. The highest content of the SGs was found in upper young actively growing shoot sections, whereas lower senescent shoot sections exhibited the lowest amount of such compounds. During ontogeny a gradual increase in the SG content was observed in both mature stevia leaves and stems, and this process lasted up to the budding phase and the onset of flowering.

Growth and development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycosides (SGs) were investigated. It was found that, owing to the highly favorable conditions of shoot cultivation created in such an apparatus, the intensity of shoot growth and SG production appeared to be 1.5 - 2.0 times higher than those of the shoots grown in tubes. These results indicate the existence of a positive correlation between these two processes. The data obtained suggest that the enhanced SG production is due to the differentiation of chlorenchyma cells and formation of specific subcellular structures for the glycoside to be accumulated.

The composition and content of steviol-glycosides (SGs)(1) in in vitro cultures of stevia (Stevia rebaudiana Bertoni) were investigated. A comparative analysis of production of these compounds in intact plants, in vitro plants, dedifferentiated callus and suspension cultures, morphogenic callus and in vitro regenerated shoots were conducted. Qualitative composition of the SGs in in vitro plants was found to be identical to that of intact plants, but their content in the former plants appeared to be about five or six times lower. A significant decrease in this value was not observed upon long-term cultivation (for about 5 years) of the plants. Non-differentiated cell cultures, such as callus and cell suspension, were shown to synthesize only minor amounts of the SGs, and their content varied greatly during the growth cycle of the culture. Qualitative composition of the SGs in the cell cultures appeared to be highly scant as compared with that of the donor plants. No correlation between the SG content in organs of the donor plants and that in the cell cultures obtained was found. Factors determining plant cultivation conditions and influencing the accumulation of both, fresh and dry cell biomass were not able to completely induce the SG synthesis in non-differentiated cell cultures. This process was found to be restored only after the appearance of morphogenic structures and shoot formation. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

The effects of growth regulators such as NAA, 2,4-D, kinetin, and benzyladenine (BA) on calluso-genesis and growth of stevia (Stevia rebaudiana Bertoni) cultured cells were investigated. It was found that, on leaf blades, callus two-three times as large as on stem fragments was formed. NAA appeared to be more effective for cell culture growth than 2,4-D, while BA exhibited a stronger effect than kinetin. A significant retardation of callus growth and complete inhibition of organogenesis took place at 2,4-D concentrations ranging from 0.2 to 1.0 mg/l, whereas NAA, at similar concentrations, markedly stimulated callus growth. The rate of primary callus growth on leaf and stem explants was the highest at NAA to BA ratios of 2.0 mg/l/2.0 mg/l and 4.0 mg/l/1.0 mg/l, respectively. Analogous concentrations of these growth regulators were also found to be effective for further stevia cell subculturing. The combination of NAA and BA was also most suitable for induction of shoot formation by stevia explants, but, in this case, the optimal concentration of these compounds was tenfold lower (0.1-0.2 mg/l). The addition of gibberellic acid to callus and suspension cultures resulted in a significant increase in their fresh weight because of cell expansion and hydration.

The ultrastructure of trichomes of intact and in vitro plants of stevia were investigated under scan ning and transmission electronic microscopy. Three types of trichomes were found on both the upper and lower surfaces of the leaf: viz. rounded glands and large and small trichome. The density of trichomes on the surface of upper leaves is higher than on lower leaves, while in vitro plants are characterized with lower density of trichomes than intact ones. The positive correlation between number of trichomes on leaves and content of steviol glycosides (SGs) in them was found. Three basic stages of ontogeny of gland cells were revealed which reflect the sequence of secrete accumulation.