Photosynthetic and transpiration responses of <Emphasis Type=Italic>in vitro</Emphasis>-regenerated <Emphasis Type=Italic>Solanum nigrum</Emphasis> L. plants to <Emphasis Type=Italic>ex vitro</Emphasis> adaptation

In vitro regeneration of black nightshade (Solanum nigrum L.) plants was achieved through callus-mediated shoot organogenesis followed by 30 d indoor ex vitro adaptation to nutritional stress under environmental ambience and thereafter 6-d outdoor acclimatization in pots prior to field establishment. Relevant physiological parameters including pigment content, chlorophyll a fluorescence, net photosynthetic rate (P _N), transpiration rate (E), and stomatal conductance (g _s) of in vitro-regenerated plants were investigated during the course of ex vitro adaptation. During the first 4 d of indoor transplantation to potting substrate, there was a marginal reduction in the leaf chlorophyll and carotenoid contents but P _N and E were strongly reduced. The stomatal conductance and E/P _N ratio were significantly higher in plants up to 20 d of indoor adaptation than those of comparable age grown naturally from seeds. The shape of the OJIP fluorescence transient varied significantly with acclimatization, and the maximum change was observed at 2.0 ms. The 2.0 ms variable fluorescence (V _j), 30 ms relative fluorescence (M ₀), photon trapping probability (TR₀/Abs), and photosystem II (PSII) trapping rate (TR₀/RC) showed initial disturbance and subsequent stabilization during 30 d of indoor acclimatization. Energy dissipation (DI₀/RC) and electron transport probability (ET₀/TR₀) showed an initial phase of increase during the 4 d after plants were transplanted outdoors. During the 6-d outdoor acclimatization after transfer of plants to soil, no significant change in total chlorophylls and carotenoids, E, and g _s were observed, but P _N improved after reduction on the first d. The OJIP-derived parameters experienced change on the first d but were stabilized quickly thereafter. There was no significant difference between outdoor acclimatized plants and those of the seed-grown plants of comparable age with respect to photosynthetic and fluorescence parameters. Direct transfer of plants without indoor acclimatization, however, showed a completely different trend with respect to P _N, E, and OJIP fluorescence transients. The bearing of this study on optimizing micropropagation is discussed.     

Attenuation of fibrosis <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> with <Emphasis Type=Italic>SPARC</Emphasis> siRNA

Introduction  

SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs.     

Presence of <Emphasis Type=Italic>C. albidus</Emphasis>, <Emphasis Type=Italic>C. laurentii</Emphasis> and <Emphasis Type=Italic>C. uniguttulatus</Emphasis> in Crop and Droppings of Pigeon Lofts (<Emphasis Type=Italic>Columba livia</Emphasis>)

Columba livia is an important reservoir and carrier of Cryptococcus neoformans, Cryptococcus uniguttulatus, Cryptococcus laurentii and Cryptococcus albidus. Upper digestive tract of this species is also known as a habitat for Cryptococcus neoformans. Given the increasing clinical interest of this microorganism, 331 swabs from crop and 174 dropping samples from pigeon lofts in Grand Canary Island have been studied. The obtained results show an extensive presence samples 81 positive (24.47%) of Cryptococcus spp. in analysed crops: 32 (9.66%) for C. neoformans, 24 (7.2%) for C. uniguttulatus, 23 (6.9%) for C. albidus and 2 (0.6%) for C. laurentii. In the same way, Cryptococcus spp was also isolated in 82 (47.13%), dropping samples: C. neoformans in 59 (33.9%), C. uniguttulatus, in 9 (5.17%), C. laurentii in 8 (4.59%) and C. albidus in 6 (3.44%) of the investigated samples, respectively. The cryptococcosis produced by species of cryptococci other than C. neoformans has become more important during the last decade, supporting the study on the role of pigeon in the epidemiology of this disease.     

Arbutin production in <Emphasis Type=Italic>Ruta graveolens</Emphasis> L. and <Emphasis Type=Italic>Hypericum perforatum</Emphasis> L. in vitro cultures

Optimization of conditions for hydroquinone biotransformation into its β-d-glucoside, arbutin, in agitated shoot cultures of Ruta graveolens L. and Hypericum perforatum L. allowed us to obtain a maximum content of this important therapeutic and cosmetic product of 7.8 and 7.2% (dry weight), respectively. These contents are higher than respective values required for standardization of known arbutin-containing plant raw materials according to the European Pharmacopoeia and national pharmacopoeias of European countries.     

Over-expression of <Emphasis Type=Italic>ThpI</Emphasis> from <Emphasis Type=Italic>Choristoneura fumiferana</Emphasis> enhances tolerance to cold in Arabidopsis

Thermal hysteresis proteins (Thps) known as antifreeze proteins for their antifreeze activity, depress the freezing point of water below the melting point in many polar marine fishes, terrestrial arthropods and plants. For the purpose of breeding cold-resistant plants, we designed to introduce the Thp gene into the plants. The physiological and biochemical effect of high-lever expression of the modified Choristoneura fumiferana Thp (ThpI) in Arabidopsis thaliana plants was analyzed. Under low temperature stress, the ThpI transgenic plants exhibited stronger growth than wild-type plants. The elevated cold tolerance of the ThpI over-expressing plants was confirmed by the changes of electrolyte leakage activity, malonyldialdehyde and proline contents. These results preliminarily showed that the Thp possibly be used to enhance the low temperature-tolerant ability of plants.     

Efficient separation and purification of allophycocyanin from <Emphasis Type=Italic>Spirulina</Emphasis> (<Emphasis Type=Italic>Arthrospira</Emphasis>) <Emphasis Type=Italic>platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Interaction of <Emphasis Type=Italic>Bdellovibrio bacteriovorus</Emphasis> with bacteria <Emphasis Type=Italic>Campylobacter jejuni</Emphasis> and <Emphasis Type=Italic>Helicobacter pylori</Emphasis>

Interaction of Bdellovibrio bacteriovorus 100NCJB with bacteria Campylobacter jejuni (strains 1, 2, 3, 4, and 5) and Helicobacter pylori, strain TX30a, was confirmed. The results indicate that lytic activity of bdellovibrios both in liquid media and cells attached to a surface was observed. The potential use of the antimicrobial activity of predatory bacteria for environmental bioprotection and public health is discussed.     

Manifestation of salt tolerance of <Emphasis Type=Italic>Spirulina platensis</Emphasis> and <Emphasis Type=Italic>Spirulina maxima</Emphasis> cyanobacteria of the genus <Emphasis Type=Italic>Arthrospira (Spirulina)</Emphasis>

The salt tolerance of two representatives of genus Spirulina (Arthrospira) Spirulina platensis and Spirulina maxima has been investigated. They both are the wide-spread objects of photobiotechnology and it has been shown that the content of 5–15 % sea-water in medium has not caused the decreasing of biomass yield more than 15–20% as compared with control. The further decreasing of biomass was proportionate to sea-water content in medium. The investigation of reactivity of native (intravital) exometabolites secreted into cultural medium has showed that the sea-water content influence the oxidative activity (OA) of exometabolites and hour’s rhythmics.     

New allele of the <Emphasis Type=Italic>COCHLEATA</Emphasis> gene in pea <Emphasis Type=Italic>Pisum sativum</Emphasis> L.

Analysis with the polymerase chain reaction showed that the Khlorofill-4 pea Pisum sativum chlorophyll-deficient mutant with reduced stipules has an altered structure of the COCHLEATA (COCH) gene, carrying a new mutant COCH allele. The phenotype of the mutant was described in comparison with another form having reduced stipules (stipules reduced) and the control. Leaves of the coch mutant are smaller and have other proportions than in the control; stipules are absent from leaves of the first nodes and are narrow, bandlike, or spoonlike at later ontogenetic stages. It was concluded that the cell number in the stipule epidermis is reduced in the st and coch mutants compared to the wild type.     

Introduction of <Emphasis Type=Italic>OsglyII</Emphasis> gene into <Emphasis Type=Italic>Oryza sativa</Emphasis> for increasing salinity tolerance

Mature seed-derived embryogenic calli of indica rice (Oryza sativa L. cv. PAU201) were induced on semisolid Murashige and Skoog medium supplemented with 2.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 0.5 mg dm⁻³ kinetin + 560 mg dm⁻³ proline + 30 g dm⁻³ sucrose + 8 g dm⁻³ agar. Using OsglyII gene, out of 3180 calli bombarded, 32 plants were regenerated on medium containing hygromycin (30 mg dm⁻³). Histochemical GUS assay of the hygromycin selected calli revealed GUS expression in 50 % calli. Among the regenerants, 46.87 % were GUS positive. PCR analysis confirmed the presence of the transgene of 1 kb in 60 % of independent plants. Further, these plants have been grown to maturity in glasshouse. In vitro screening for salt tolerance showed increase in fresh mass of OsglyII putative transgenic calli (185.4 mg) as compared to control calli (84.2 mg) on 90 mM NaCl after 15 d. When exposed to 150 mM NaCl, OsglyII putative transgenic plantlets showed normal growth while the non-transgenic control plantlets turned yellow and finally did not survive.     

<Emphasis Type=Italic>In vitro</Emphasis> organogenesis of <Emphasis Type=Italic>Passiflora alata</Emphasis>

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO₃ incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA₃ and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.     

The order of the <Emphasis Type=Italic>bs,Skdh</Emphasis>, and <Emphasis Type=Italic>Aadh1</Emphasis> genes in chromosome 5R of rye <Emphasis Type=Italic>Secale cereale</Emphasis> L.

Segregation analysis was performed in the progenies obtained in analyzing crosses of hybrids of spring and winter accessions of rye Secale cereale L. and wild S. montanum subsp. anatolicum (Grossh.) Tzvel. (syn. S. strictum (J. Presl) J. Presl). The test genes controlled the brittle stem (bs), the allelic variants of aromatic alcohol dehydrogenase (Aadh1) and shikimate dehydrogenase (Skdh), and the growth habit (Vrn1). A linkage was observed in the inheritance of the brittle stem and the aromatic alcohol dehydrogenase and shikimate dehydrogenase alloenzymes. The order of genes was established to be bs-Skdh-Aadh1, and the genetic distances were estimated to be bs-(9.0%)-Skdh, bs-(10.8%)-Aadh1, and Skdh-(5.3%)-Aadh1. The recombination coefficient between the Skdh and Aadh1 genes varied from 2.2 to 18.2%, averaging 5.3%. The growth habit was inherited independently of the bs-Skdh-Aadh1 linkage group.     

Remobilization of <Emphasis Type=Italic>Tol2</Emphasis> transposons in <Emphasis Type=Italic>Xenopus tropicalis</Emphasis>

Background  

The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency.