Claudia Simões

Anthocyanin production in callus cultures of Cleome rosea: modulation by culture conditions and characterization of pigments by means of HPLC-DAD/ESIMS.

Leaf and stem explants of Cleome rosea formed calluses when cultured on MS medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (PIC). The highest biomass accumulation was obtained in the callus cultures initiated from stem explants on medium supplemented with 0.90 microM 2,4-D. Reddish-pink regions were observed on callus surface after 6-7 months in culture and these pigments were identified as anthocyanins. Anthocyanins production was enhanced by reducing temperature and increasing light irradiation. Pigmented calluses transferred to MS1/2 with a 1:4 ratio NH(4)(+)/NO(3)(-), 70 g L(-1) sucrose and supplementation with 0.90 microM 2,4-D maintained a high biomass accumulation and showed an increase of 150% on anthocyanin production as compared with the initial culture conditions. Qualitative analysis of calluses was performed by high performance liquid chromatography coupled to diode array detector and electrospray ionization mass spectrometry (HPLC-DAD/ESIMS). Eleven anthocyanins were characterized and the majority of them were identified as acylated cyanidins, although two peonidins were also detected. The major peak was composed by two anthocyanins, whose proposed identity were cyanidin 3-(p-coumaroyl) diglucoside-5-glucoside and cyanidin 3-(feruloyl) diglucoside-5-glucoside.

ROLLINIA MUCOSA CELL SUSPENSION CULTURES: ESTABLISHMENT AND GROWTH CONDITIONS

Different types and concentrations of plant growth regulators were tested in order to obtain the best callus and cell suspension culture growth conditions of Rollinia mucosa (Jacq.) Baill. (Annonaceae). Picloram was shown to be the most efficient for induction and production of friable calluses, independent of the concentration used. Cellular morphology and viability, fresh and dry weights, pH and medium sugar concentration were determined for cell suspension cultures. Dissimilation curves were used for the characterization of the growth of cell suspension cultures. Picloram provided the most rapid growth and produced the highest biomass, with little variation in morphology (differentiated cells). It also provided the highest dissimilation, when compared with cell suspension cultures maintained in media with 2,4-D or NAA + BA + GA3. Stable cell suspension cultures can be established in MS medium supplemented with 20.8 μM picloram.

Lignans from leaves, seedlings and micropropagated plants of Rollinia mucosa (Jacq.) Baill. – Annonaceae

Rollinia mucosa produces furofuranic lignans (magnolin, epiyangambin, yangambin) that are antagonists of platelet-activating factor (PAF). The biosynthetic capacity and the potential for the accumulation of furofuranic lignans, including epieudesmin, of the plants cultured both in vivo and in vitro conditions were evaluated. The production and the pattern of lignans accumulated were dependent on the origin of the plant material and the plant organ. The major accumulation of lignans was observed in leaves. In the mature leaves of in vivo grown seedlings magnolin and yangambin predominated, in contrast to leaves from in vitro propagated plants that presented epiyangambin as the major lignan.

In vitro propagation of Cleome spinosa (Capparaceae) using explants from nursery-grown seedlings and axenic plants

SummaryTwo independent experiments were performed to establish micropropagation of Cleome spinosa from stem segments. In the first experiment, direct shoot organogenesis on hypocotyl explants from 2-mo.-old nursery-grown seedlings was obtained on Murashige and Skoog medium with different combinations of benzyladenine (BA) and 6-furfurylaminopurine, added either individually or in combination. Best proliferation rates occurred in the presence of 2.2 and 4.4 μM BA and the highest mean number of shoots was produced in response to 4.4 μM BA. In the second experiment, regeneration via direct organogenesis was also obtained from nodal and internodal segments of axenic plants cultured in the presence of BA (4.4 and 8.8 μM) in association with indole-3-acetic acid (IAA) (0.57 and 1.14 μM). Internodal explants were the most responsive on all media tested. The best mean number of shoots per explant was achieved on medium with 4.4 μM BA in association with 0.57 μM IAA. Histological studies of the globular structures formed at the apical portion of the explants revealed direct shoot regeneration and adventitious shoot differentiation from meristematic centers around the vascular bundles of the primary regenerants. All shoots elongated and rooted on MS0 medium. The acclimatization rates ranged between 70 and 84%. Plants reached to maturity and flowered 4 mo. after transfer to ex vitro conditions.

Biotechnological Production of Plant Secondary Metabolites

Description: Modern techniques have been developed to overcome problems associated with the extraction of natural products from plants. These techniques include production of secondary metabolites by biotechnological methods such as plant tissue culture and microbial biotransformation of natural substances. Such methods have led to an increased yield of secondary metabolite amount, the discovery of new biochemical derivatives and agricultural development. For instance, use of these techniques in agricultural area have led to some beneficial traits such as formation of new varieties of known plant species, better crop quality, higher yield, better nutritive properties, more resistant species to insects and pests. Microbial biotransformation is beneficial in plant secondary metabolite production and derivatization (chemical modification of any compound made by a microorganism) and is chiefly applicable to several varieties of terpenes and steroids. This E-book demonstrates recent developments in this field. It will be of particular interest to the professionals in pharmaceutical and biotechnological industries, as well as natural product chemists, medicinal chemists, plant biochemists, and molecular biologists.

Somatic embryogenesis and plant regeneration from callus cultures of Cleome rosea Vahl

This paper describes a protocol for the efficient vegetative propagation of Cleome rosea by somatic embryogenesis. Leaf and stem explants from nursery-grown seedlings of C. rosea were cultivated on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA), a -naphthaleneacetic acid (NAA), 4-amino-3,5,6-trichloropicolinic acid (picloram) or 2,4-dichlorophenoxyacetic acid (2,4-D). Nodular calli were produced from both explant types in the presence of 4.5 and 9.0 µM 2,4-D. Embryo development and maturation were achieved when calli from stem explants were transferred to media containing a ten-fold reduction of 2,4-D concentration initially used (0.45 and 0.90 µM). Leaf-derived calli did not form embryos with the same treatments. The highest frequency of embryogenic callus formation (85%) and number of embryo per callus (13.45 ± 2.8) were achieved during the first subculture on medium supplemented with 0.90 µM 2,4-D. Embryo conversion into plantlets was achieved following transfer to growth regulator-free MS medium solidified with 2 g.L-1 phytagel. An acclimatization rate of 53% was found three months after transfer to ex vitro conditions and the recovered plants presented a normal phenotypic aspect.

Effects of cromakalim and glibenclamide on arteriolar and venular diameters and macromolecular leakage in the microcirculation during ischemia/reperfusion.

We studied changes in arteriolar and venular diameters and macromolecular leakage altered by ischemia/reperfusion (I/R) and topically applied histamine after I/R and how these changes were modulated by cromakalim (KATP-channel opener) and glibenclamide (KATP-channel blocker). Golden hamsters were prepared for intravital microscopy of the cheek pouch. Ischemia was induced by an inflatable silicon rubber cuff mounted around the neck of the cheek pouch prepared for intravital microscopy. Saline, histamine, cromakalim, and glibenclamide were applied in the superfusion solution. FITC-dextran was injected i.v. 30 min before initiation of ischemia as a marker of macromolecular leakage. Cromakalim 10−6M, but not 10−8M, caused arteriolar dilation in ischemic and normal (nonischemic) preparations, and glibenclamide, 10−10M and 10−8M, had no effects on vessel diameters. Application of cromakalim 10−6M increased arteriolar diameter (+54%) and macromolecular leakage in normal and nonischemic cheek pouches and had an additive effect on macromolecular leakage in ischemic (I/R) preparations but had no effect on histamine-induced increase in macromolecular leakage. Glibenclamide, 10−10M and 10−8M, inhibited I/R-induced but not histamine-induced increases in macromolecular leakage. We concluded that cromakalim may increase macromolecular leakage. This effect is additive to I/R-induced leakage suggesting that stimulation of KATP-channels could take part in the regulation of macromolecular leakage in postcapillary venules. The KATP-blocker glibenclamide inhibited I/R-induced but not histamine-induced macromolecular leakage at concentrations that had no constricting effect on arterioles, and therefore, it cannot be excluded that glibenclamide reduced plasma leakage by some unknown mechanism.