A bacterial artificial chromosome (BAC) library of Malus floribunda 821 and contig construction for positional cloning of the apple scab resistance gene Vf.

The apple scab resistance gene Vf, originating from the wild species Malus floribunda 821, has been incorporated into a wide variety of apple cultivars through a classical breeding program. With the aim of isolating the Vf gene, a bacterial artificial chromosome (BAC) library consisting of 31 584 clones has been constructed from M. floribunda 821. From the analysis of 88 randomly selected BAC clones, the average insert size is estimated at 125 kb. If it is assumed that the genome size of M. floribunda 821 is 769 Mb/haploid, the library represents about 5x haploid genome equivalents. This provides a 99% probability of finding any specific sequence from this library. PCR-based screening of the library has been carried out using eight random genomic sequence-characterized amplified regions (SCARs), chloroplast- and mitochondria-specific SCARs, and 13 high-density Vf-linked SCAR markers. An average of five positive BAC clones per random SCAR has been obtained, whereas less than 1% of BAC clones are derived from the chloroplast or mitochondrial genomes. Most BAC clones identified with Vf-linked SCAR markers are physically linked. Three BAC contigs along the Vf region have been obtained by assembling physically linked BAC clones based on their fingerprints. The overlapping relatedness of BAC clones has been further confirmed by cytogenetic mapping using fiber fluorescence in situ hybridization (fiber-FISH). The M. floribunda 821 BAC library provides a valuable genetic resource not only for map-based cloning of the Vf gene, but also for finding many other important genes for improving the cultivated apple.     

DNA methylation profiles differ between field- andin vitro-grown leaves of apple

A reliable method has been previously developed to detect cytosine methylation at the 5′-CCGG-3′ sequence using isoschizomers (Msp I and Hpa II) and a modified amplified fragment length polymorphism (AFLP) technique. With this method, DNA methylation profiles were investigated in leaf tissues of apple (Malus × domestica cv. Gala) grown under two different growth conditions, field and tissue culture. A total of 1,622 AFLP bands were detected using 32 pairs of primers, and these banding patterns were assembled into three groups. Type I AFLP bands were present in both EcoR I/Hpa II and EcoR I/Msp I lanes. Type II bands were present in the EcoR I/Msp I lanes, but not in EcoR I/Hpa II lanes. Type III bands were present in EcoR I/Hpa II lanes, but not in the EcoR I/Msp I lanes. For leaf tissues of field- and in vitro-grown apples, the ratios of types I, II, and III to the total number of amplified fragments were 70 %, 24 %, and 6 %, and 71 %, 23 %, and 6 %, respectively. Although the ratios of the three types of banding patterns were similar in both leaf tissues, a few bands specific to either field-grown trees or in vitro-grown shoots were observed. This study provided evidence that changes in methylation occurred in apple leaf tissues subjected to tissue culture growth conditions.     

Chronic-arginine supplementation improves endothelial cell vasoactive functions in hypercholesterolemic and atherosclerotic monkeys

Chronic exposure to l-arginine results in regression of atherosclerotic lesions and reversal of endothelial dysfunction. We investigated whether chronic l-arginine supplementation induces regression of atherosclerotic lesions and reversal of endothelial dysfunction in atherogenic rhesus monkeys and the mechanism which leads to these effects. About 12 male rhesus monkeys were fed 1% cholesterol and 18 g butter for 6 months to create an experimental model of hypercholesterolaemia and atherosclerosis (Group I) and 12 monkeys were fed standard stock diet for 6 months (Group II). After, 6 months these two groups were further divided into 2 sub-groups which in addition to their respective diets were fed 2.5% l-arginine in drinking water for additional 6 months (Group III and Group IV). Systemic nitric oxide (NO) formation was assessed as plasma nitrite and cGMP formation every 3 months. Oxygen free radical (OFR) generation and malondialdehyde production as an index of lipid peroxidation were determined. Changes in isometric tension were compared in isolated ring segments of thoracic aorta from normal and hypercholesterolemic animals.Cholesterol feeding progressively reduced plasma nitrite and cGMP generation (p<0.05). Dietary l-arginine partly restored the levels of plasma nitrite and cGMP (p<0.05) but did not change plasma cholesterol levels. l-arginine significantly reduced aortic intimal thickening, blocked the production of carotid and coronary intimal plaques and completely preserved endothelium-dependent vasodilator function. Further, l-arginine significantly inhibited generation of the reactive oxygen species (ROS) generation and lipid peroxidation.Chronic oral supplementation with l-arginine blocks the progression of plaques via restoration of nitric oxide synthase substrate availability and reduction of vascular oxidative stress. (Mol Cell Biochem 269: 1–11, 2005)     

Effects of salt formulations, carbon sources, cytokinins, and auxin on shoot organogenesis from cotyledons of Pinus pinea L.

Adventitious shoots were induced on cotyledons of Pinus pinea. Among seven salt formulations, three carbon sources (sucrose, glucose and fructose), and two cytokinins [6-benzyladenine (BA) and thidiazuron (TDZ)] with and without [-naphthaleneacetic acid (NAA)], cotyledons grown on 1/2 MS medium containing sucrose and 30 M BA produced the highest frequency of shoot organogenesis (>90%) and mean number of shoots/explant (>40% of cotyledons produced over 20 shoots/cotyledon). As for the site of shoot organogenesis along the cotyledonary explant, the highest number of shoots per explant was observed along the basal segment of the cotyledon (distal to the hypocotyl) over all cytokinin and auxin treatments tested. Shoots were elongated on a growth regulator-free medium containing activated charcoal.     

Influence of Media Components and pH on Somatic Embryo Induction in Three Genotypes of Soybean

The influence of media components on the initiation of somatic embryogenesis in three genotypes of soybean was investigated. The following genotypes were used: Iroquois, Macon, and Savoy. Media modifications included sucrose concentration, type and concentration of auxin at two pH levels, and pH level independently. Immature cotyledons were used as the source of explant. Cotyledons were placed on a medium containing MS salts, B5 vitamins, sucrose, and auxin. Gelrite (0.2%) was used as the solidifying agent. Sucrose concentrations of 1, 2, 3, 4.5, or 6% were used. The auxins used included 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthaleneacetic acid (NAA) with each at concentrations of 45.2, 90.4, 135.7, 180.9, and 226.2 M. The pH of each the media was adjusted to either 5.7 or 7.0 with 1 N NaOH. In an additional experiment, the effect of the two pH levels, 5.7 and 7.0, was investigated independently. Overall, the frequency of somatic embryogenesis significantly varied among the different genotypes used in this study, with Iroquois showing the highest response. Frequency of somatic embryogenesis also varied in response to the different treatments used, including sucrose and auxin. The highest initiation (91.7%) and mean number of somatic embryos per responding explant (14.9) of Iroquois was observed in a medium containing 2% sucrose. The highest initiation (97.1%) and mean number of somatic embryos per responding explant (19.5) was observed in Iroquois on 135.7 M 2,4-D and Savoy on 135.7 M 2,4-D, respectively, for the auxin by pH level experiment. No significant differences were observed among the two pH treatments used.     

Characterization and expression of β-1,3-glucanase genes in peach

Beta-1,3-glucanase is one of the pathogenesis-related (PR) proteins involved in plant defense responses. A peach beta-1,3-glucanase gene, designated PpGns1, has been isolated and characterized. The deduced amino acid sequence of the product of PpGns indicates that it is a basic isoform (pI 9.8), and contains a putative signal peptide of 38 amino acids but has no C-terminal extension. Amino acid sequence comparisons revealed that PpGns1 is 69% and 67% identical to citrus and soybean beta-1,3-glucanases, respectively. Southern analysis of total genomic DNA also indicates that at least three genes for beta-1,3-glucanases exist in peach, forming a small gene family. Characterization of four additional clones by PCR has identified a second beta-1,3-glucanase gene, PpGns2. PpGns2 has been partially sequenced, and when compared to PpGns1, it shows high sequence homology, 96% and 99% nucleotide identity in the first and (partial) second exons, respectively. The deduced partial sequence of the PpGns2 product displays only two differences from PpGns1 in the signal peptide and one in the (partial) mature protein (141 amino acids). The 5'-flanking promoter regions of these two genes share 90% identity in nucleotide sequences interrupted by five major gaps (4-109 nt long). The promoter region contains various sequences similar to cis-regulatory elements present in different stress-induced plant genes. In leaves and stems of peach shoot cultures grown in vitro, PpGns1 is induced within 12 h after exposure to a culture filtrate of Xanthomonas campestris pv. pruni or ethephon. However, it is not induced following treatment with mercuric chloride.