Barbara Monacelli

Metabolites in cell suspension cultures, calli, and in vitro regenerated organs of Hypericum perforatum cv. Topas

Abstract Methanolic extracts from cell suspension cultures, calli, and in vitro regenerated shoots and roots of Hypericum perforatum cv. Topas have been evaluated for their ability to produce active metabolites (hypericins, hyperforins and flavonoids). Biosynthesis of hypericins is connected with the formation of secretory structures (black globules) in regenerated vegetative buds. A further degree of leaf development is necessary to stimulate the production of hyperforins and flavonoids. Xanthones are the main metabolic products in suspension cells, undifferentiated calli and roots regenerated from plantlets or formed by callus. No xanthones are detected in the aerial parts of regenerated plantlets accumulating hypericins, hyperforins and flavonoids.

Two isoflavones and a flavone from the fruits of Maclura pomifera

Abstract Stabilized and optimized cell suspension cultures of Maclura pomifera showed a flavonoid accumulation qualitatively different from that of the intact plant. For a better comparison between in vivo and in vitro production, a re-examination of the fruit extract was undertaken. The study, involving super fluid chromatography, led to the isolation and structure determination of flavonoids not previously reported in the plant. Three of these compounds are new. The presence of flavonoids in stems and leaves was also investigated.

Nuclear DNA changes during plant development and the morphogenetic response in vitro of Nicotiana tabacum tissues

Abstract Cytophotometric and biochemical analyses indicate changes with plant development in the amount and organization of the nuclear DNA in epidermal and subepidermal tissues excised from different portions of tobacco stems: DNA is progressively lost with tissue ageing, while amplification of repeated sequences occurs in tissues developed after phase change. These genome variations are related to the morphogenetic response of the tissues cultured in vitro, which differs both quantitatively and qualitatively: loss of DNA sequences progressively impairs morphogenetic capability; DNA amplification may be a factor of the explants ability to regenerate flowers. These results point to the possible role of the nuclear DNA condition of differentiated tissues in determining their morphogenetic expression in vitro.

Lipolytic isoenzymes from Euphorbia latex

Abstract The activity and substrate specificity of latex lipases from Euphorbia species (E. characias, E. wulfenii, E. pinea, E. myrsinites and E. dendroides) were investigated. High lipolytic activity was found only in E. characias and for the first time in E. wulfenii latex. For both species the lipolytic activity on various triglycerides, and under different temperature and pH conditions, in both crude latex and in partially purified enzymes was quantified. Optimised extraction and purification methods permitted the recovery of the enzymatic fraction with high lipolytic activity. This fraction is probably constituted by a pool of different lipolytic enzymes. Finally, lipolytic activity was also measured for E. characias and E. wulfenii during vegetative and reproductive stages.

Cellular localisation of the anti-cancer drug camptothecin in Camptotheca acuminata Decne (Nyssaceae).

In Camptotheca acuminata, we studied the cellular sites of accumulation of the alkaloid camptothecin (CPT), in both plants grown in the field and those grown in a greenhouse, subjecting the latter to stress (i.e., draught, nutritional deficit, and pruning). Fresh sections of the leaf, stem, and root were analysed for the presence of CPT by examining the autofluorescence that the CPT molecule emits when exposed to UV radiation. In the plants grown in the field, CPT was observed only rarely. In the greenhouse plants, CPT had accumulated in crystalline form in the vacuole of specialised cells (i.e., segregator idioblasts), which were not morphologically distinguishable from the cells of the surrounding tissues. In the organs examined, the segregator idioblasts were localised in parenchymatic and epidermal tissues. CPT crystals were also detected in the glandular trichomes on both the stem and leaf.

The role of isoprenoid accumulation and oxidation in sealing wounded needles of Mediterranean pines

This study was carried out to investigate how the emission of isoprenoids that follows wounding of pine needles is restrained and how this can be associated to wound sealing processes and to defense mechanisms against pathogenic attacks. Needles of two Mediterranean pines (Pinus pinea and Pinus halepensis) emitted a high amount of monoterpenes immediately after wounding but the emission became undetectable within 24 h. Histochemical analysis revealed that the purple stain coloring the isoprenoids in fresh needle sections turned black 24 h after wounding the needles. This suggests that isoprenoids accumulated at the wounding sites. Sections below the wounds showed that the ducts were still full of isoprenoids stained in purple. This excluded the possibility that the emission from wounded needles ceases because the ducts were empty. Chromatographic analysis of the resin covering the wounds indicated a high amount of sesquiterpenes 1 h after wounding and a considerable increase in oxygenated isoprenoids 24 h after wounding. Our results indicate that the emission from wounded needles may be rapidly limited and eventually stopped by the accumulation of oxidation products of the same emitted isoprenoids. This mechanism is similar to that used to seal trunk wounding and may constitute a physical and chemical barrier against pest attacks.

Laticifers in Camptotheca acuminata Decne: Distribution and structure

Summary.In this paper, a system of laticifers in Camptotheca acuminata Decne (Nyssaceae) is described. Laticifers were already present in the leaf primordia of the shoot apex. In the mature leaves, laticifers were found in the midrib and in the larger veins, both in the parenchymatic region delimited by vascular bundles and in the cortex just external to the phloem. In the stem, laticifers were present in both the primary and secondary body, running parallel to the longitudinal axis. They were located in the pith and in the cortex proximal to the phloem. No laticifers were found in the roots. The histochemical analyses indicated that the main compounds accumulated in laticifers were phenols. Neutral lipids and fatty acids were also present. Ultrastructural observations showed osmiophilic globules both in the vacuoles and in the peripheral regions of the cytoplasm of the laticifer cells. Plastids were present, although altered, with some parallel membranes and lacking starch grains. The discovery in C. acuminata of a laticifer system, which had never been described for the order Cornales, could be of taxonomic value, also considering that this order has traditionally represented one of the most problematic groups of flowering plants.

Cell Suspension Cultures of Cassia didymobotrya: Optimization of Growth and Secondary Metabolite Production by Application of the Orthogonal Design Method

Summary The optimization of both the cell growth and the accumulation of secondary metabolites in cell suspension cultures of Cassia didymobotrya Fres. was achieved for the first time by applying an orthogonal design method. The influence of the concentration of 2,4-D, kinetin, and sucrose as well as of the amount of the inoculum was studied.

Xanthones from calli of Hypericum Perforatum subsp. Perforatum

Two new xanthone derivatives, 1-hydroxy-5,6,7-trimethoxyxanthone and 3-O-methylpaxanthone were isolated from callus of Hypericum perforatum subsp. perforatum together with the known paxanthone, cadensin G, 1-hydroxy-6,7-dimethoxyxanthone, 1,3,6,7-tetrahydroxyxanthone, and 1,3,5,6-tetrahydroxyxanthone. The structures of the new compounds were established by spectroscopic methods.

Synthesis and/or accumulation of bioactive molecules in the in vivo and in vitro root

Abstract Roots of many species are studied because of the presence of high-value bioactive molecules, yet few studies have attempted to determine the biosynthetic pathways of these compounds or the way in which synthesis is regulated. The presence of secondary metabolites in the root does not necessarily mean that this organ is also the site of synthesis. Thus the identification of organ-specific intermediate precursors and key enzymes is important for understanding the biosynthetic pathway and the regulation of bioactive molecules. This knowledge could allow researchers to predict the suitability of in vitro systems, such as regenerated roots and hairy roots, for producing the molecules of interest. In the present review, the production of bioactive molecules in in vivo roots is compared to that in in vitro untransformed and transformed roots, concentrating on recent developments in the study of the biosynthesis of the anti-cancer alkaloid camptothecin in Camptotheca acuminata Decne. The results of a recent study performed in our laboratory on the production of camptothecin and other secondary metabolites in in vivo and in vitro C. acuminata roots are also presented.