In vitro regeneration of wild pear (<Emphasis Type="Italic">Pyrus pyraster</Emphasis> Burgsd) clones tolerant to Fe-chlorosis and somaclonal variation analysis by RAPD markers

An in vitro adventitious regeneration system under selective pressure was established in Pyrus pyraster Burgsd to obtain somaclones with higher adaptability to calcareous soils. P. pyraster is important species, both for its relative closeness to cultivated pear and for reforestation of marginal farmland and for the production of timber. Shoot regeneration was induced from leaves and vegetative apices of in vitro-grown shoots on a modified LP medium supplemented with naphtaleneacetic acid (1.07 μM) and benziladenine (BA, 8.9 μM). After 30 days, explants were transferred to an expression medium consisting of the same basal medium with only BA present. Selective treatments utilized MS medium with Fe-EDTA replaced by equimolar amount of FeSO4 with either KHCO₃ or NaHCO₃. Through the selection process 11 putatively tolerant lines were obtained from vegetative shoot apices. RAPD analysis was performed on these lines to allow comparison to the mother clone. A total of seven 10-mer primers were used to amplify all the genotypes and 74 scorable fragments were produced. These were analysed using the Dice similarity index, showing genetic variability among the 11 regenerated clones and between them and the mother clone.     

Methyl jasmonate promotes anthocyanins’ production in Prunus salicina × Prunus persica in vitro shoot cultures

In vitro shoot cultures of Prunus salicina × Prunus persica, “Citation®” rootstock, were treated with 50-μM methyl jasmonate (MJ) or 100-μM abscisic acid (ABA); in MJ-treated shoots, total anthocyanins increased significantly (1.88 mg/g fresh weight) relative to controls (0.43 mg/g fresh weight). Cyanidin 3-glucoside was the most abundant anthocyanin in both MJ-treated and control explants. The addition of ABA to the culture medium did not elicit anthocyanins’ accumulation.     

Factors affecting in vitro shoot formation from vegetative shoot apices of apple and relationship between organogenic response and cytokinin localisation

ABSTRACT

The effects of macro- and micro-elements, benzyladenine (BA) concentration, and the period of auxin application on adventitious shoot formation from callus originating from vegetative shoot apices were tested on apple (Malus domestica Borkh) rootstock Jork 9. The putative relationship between organogenic response and cytokinin localisation was also studied by an immunolocalisation technique for in situ determination of free cytokinins. The use of MS (Murashige & Skoog, 1962) salts in the medium instead of those of LP (Quoirin & Lepoivre, 1977) had a strong positive effect both on shoot formation rate and on the number of shoots produced. The highest organogenic response from callus was induced using 17.8 μM BA in the presence of 2.7 μM NAA and by maintaining the explants for 20 days in darkness, then transferring them to fresh auxin-free medium and to the light. The in situ localisation studies, performed using antibodies with a marked specificity against zeatin and isopentenyladenine, revealed changes in the localisation of free zeatin in the tissues during the shoot-forming process, in particular during the active cell division phase leading to callus formation, and in the initial phase of bud formation. Changes in zeatin distribution in the tissues of the vegetative shoot apex during shoot formation may indicate a role for this cytokinin free base in cell differentiation and organogenesis.     

Effect of CPPU on in vitro axillary shoot proliferation and adventitious shoot regeneration in kiwifruit

Abstract

The effect of CPPU, N‐(2‐chloro‐4‐pyridyl)‐N‐phenylurea, on the development of axillary buds and on adventitious shoot regeneration was investigated in mature leaves of in vitro‐cultured shoots of Actinidia chinensis Planch (Sel. K190) and Actinidia deliciosa A. Chev. cultivars Hayward and Tomuri. In the multiplication phase, 2.2 mM CPPU induced proliferation rates comparable to 4.4 mM benzyadenine (BA) both in Hayward and in Tomuri, while a higher CPPU concentration reduced proliferation. In A. chinensis, significant differences in multiplication rates were not detected between BA and CPPU. However, shoots developed on CPPU appeared hyperhydric, and had very short internodes, reduced leaf laminas, higher water, carotenoid and phenol contents and considerably lower chlorophyll level in comparison with the BA‐grown shoots. On the other hand, in adventitious shoot regeneration CPPU was more effective than zeatin (ZEA) and BA in A. deliciousa cultivars and the best morphogenic response was obtained with the lowest concentration (10 mM) in cv Hayward, while 16 mM was the most efficient treatment in cv Tomuri. In A. chinensis, CPPU was as efficient as ZEA when the highest concentration was used.     

Metabolomics Analysis and Modeling Suggest a Lysophosphocholines-PAF Receptor Interaction in Fibromyalgia

Fibromyalgia Syndrome (FMS) is a chronic disease characterized by widespread pain, and difficult to diagnose and treat. We analyzed the plasma metabolic profile of patients with FMS by using a metabolomics approach combining Liquid Chromatography-Quadrupole-Time Of Flight/Mass Spectrometry (LC-Q-TOF/MS) with multivariate statistical analysis, aiming to discriminate patients and controls. LC-Q-TOF/MS analysis of plasma (FMS patients: n = 22 and controls: n = 21) identified many lipid compounds, mainly lysophosphocholines (lysoPCs), phosphocholines and ceramides. Multivariate statistical analysis was performed to identify the discriminating metabolites. A protein docking and molecular dynamic (MD) study was then performed, using the most discriminating lysoPCs, to validate the binding to Platelet Activating Factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) Receptor (PAFr). Discriminating metabolites between FMS patients and controls were identified as 1-tetradecanoyl-sn-glycero-3-phosphocholine [PC(14∶0/0∶0)] and 1-hexadecanoyl-sn-glycero-3-phosphocholine [PC(16∶0/0∶0)]. MD and docking indicate that the ligands investigated have similar potentialities to activate the PAFr receptor. The application of a metabolomic approach discriminated FMS patients from controls, with an over-representation of PC(14∶0/0∶0) and PC(16∶0/0∶0) compounds in the metabolic profiles. These results and the modeling of metabolite-PAFr interaction, allowed us to hypothesize that lipids oxidative fragmentation might generate lysoPCs in abundance, that in turn will act as PAF-like bioactivators. Overall results suggest disease biomarkers and potential therapeutical targets for FMS.     

Assessment of the antiproliferative activity on murine melanoma cells of extracts from elicited cell suspensions of strawberry,strawberry tree,blackberry and red raspberry

The antiproliferative activity of extracts from cell suspensions of strawberry (Fragaria x ananassa Duch.), blackberry (Rubus fruticosus L.), red raspberry (Rubus idaeus L.) and strawberry-tree (Arbutus unedo L.) on murine B16-F10 melanoma cells was investigated. To enhance the synthesis of the bioactive compounds, various elicitors, i.e. temperature (cold and cold/warm cycle), light (blue and red) and increased nitrogen concentration, were also applied to the cell cultures to evaluate the effects on their antiproliferative activity. The extracts from all the species reduced murine melanoma cell proliferation (between 30% and 38% relative to the control) and an improvement of antiproliferative activity on murine melanoma cells, relative to the un-elicited berry suspensions, was shown by the extracts from strawberry and strawberry-tree cells treated with red and blue light.     

In vitro plant regeneration from callus of shoot apices in apple shoot culture

A new reliable protocol for the induction of adventitious shoot formation and plant regeneration from apple callus has been developed. High regeneration frequency was obtained with this method in four different genotypes (Jork9, M26, Gala and McIntosh) and callus maintained regeneration ability for several months. The procedure consists of inducing vegetative shoot apices, excised from in vitro shoots, for 20 days in darkness on an MS medium without glycine, supplied with 17.8 μM BA, 2.7 μM NAA and 250 mg l⁻¹ cefotaxime. The explants are then transferred to a fresh auxin-free medium and given light. Histological studies revealed that all the regenerated shoots originated from callus. Regenerated shoots were multiplied, rooted and successfully established in soil. Received: 2 April 1999 / Revision received: 10 November 1999 / Accepted: 15 November 1999     

N-Alkyl dien- and trienamides from the roots of Otanthus maritimus with binding affinity for opioid and cannabinoid receptors

Two new thienylheptatrienamides (1, 5) and one new neo-lignan (12), together with thirteen known compounds (2, 3, 4, 6–11, 13–16) were isolated from the roots of Otanthus maritimus. The structures of the new compounds were elucidated on the basis of extensive 1D and 2D NMR experiments as well as high resolution mass spectrometry. All the isolated amides (1–10), the known pontica epoxide (11) and the new neo-lignan (12) were evaluated for their binding affinity to the CB1 and CB2 as well as to the μ and δ opioid receptors. Some alkylamides showed moderately high binding affinity for CB2 receptors and 1-[(2E,4E,8Z)-tetradecatrienoyl]piperidine (10) resulted the most active one with a K_i value of 160 nM. As far as we know, this is the first example of a tertiary alkylamide that binds CB2 receptors with significant potency. Compounds that showed the highest affinity for cannabinoid receptors (6–8, 10) were much less potent against opioid receptors. Primary structure–activity relationship is discussed. Docking experiments were carried out with the aim to understand the key interactions of the most active compounds with CB2 receptor.     

Limonoids from Melia azedarach Fruits as Inhibitors of Flaviviruses and Mycobacterium tubercolosis

The biological diversity of nature is the source of a wide range of bioactive molecules. The natural products, either as pure compounds or as standardized plant extracts, have been a successful source of inspiration for the development of new drugs. The present work was carried out to investigate the cytotoxicity, antiviral and antimycobacterial activity of the methanol extract and of four identified limonoids from the fruits of Melia azedarach (Meliaceae). The extract and purified limonoids were tested in cell-based assays for antiviral activity against representatives of ssRNA, dsRNA and dsDNA viruses and against Mycobacterium tuberculosis. Very interestingly, 3-α-tigloyl-melianol and melianone showed a potent antiviral activity (EC₅₀ in the range of 3–11μM) against three important human pathogens, belonging to Flaviviridae family, West Nile virus, Dengue virus and Yellow Fever virus. Mode of action studies demonstrated that title compounds were inhibitors of West Nile virus only when added during the infection, acting as inhibitors of the entry or of a very early event of life cycle. Furthermore, 3-α-tigloyl-melianol and methyl kulonate showed interesting antimycobacterial activity (with MIC values of 29 and 70 μM respectively). The limonoids are typically lipophilic compounds present in the fruits of Melia azeradach. They are known as cytotoxic compounds against different cancer cell lines, while their potential as antiviral and antibacterial was poorly investigated. Our studies show that they may serve as a good starting point for the development of novel drugs for the treatment of infections by Flaviviruses and Mycobacterium tuberculosis, for which there is a continued need.