Regular Production of Transgenic Wheat Mediated by Agrobacterium tumefaciens

Wheat is the number one crop both in acreage and total yield in the world. Therefore, it is very important to improve wheat by gene engineering techniques even though it belongs to the plants insensitive to gene transformation, especially to Agrobacterium-mediated transformation. Wheat immature embryos of 1.0-1.5mm in size, C58c1 of Agrobacterium strain harboring pPTN249, pPTN270, pPTN254, and pSIS-GFP respectively (all the vectors contain the aphA selectable gene driven by enhanced 35S promoter and a target gene controlled by ubi promoter or E35S promoter), AB medium for Agrobacterium activate culture, WCC medium for co-culture, were used in this study. The embryos with 4 days of pre-culture were transferred onto selection medium with 10mg/L geneticin, 50mg/L ticarcillin, 50mg/L vancomycin, and 50mg/L cefotaxine after 30 minutes of infection and 2 days of co-cultivation with Agrobacterium. Followed callus production,shoot regeneration on selection medium, 114 resistant plantlets were obtained from 10 transformation experiments of four genotypes. By nptⅡ ELISA (nptⅡ enzyme assay), PCR, Southern blot and leaf bleach,29 positive plants were identified from two genotypes of Bobwhite and Yanglmai 10, with an average transformation efficiency of 0.82 %. The result tested by Southern blot also showed that the transgenic plants with single- copy integration of target gene took 65.52% among total positive plants. The ELISA value of transgenic plant was also related to the copies of alien DNA integrating into wheat chromosomes, the transgenic plants with single copy integration giving higher ELISA value than the ones with 2 or 3 copies integration.     

Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.     

Phylogeny and nodulation signal molecule of rhizobial populations able to nodulate common beans—other than the predominant species Rhizobium etli—present in soils from the northwest of Argentina

We examined the bean rhizobia community other than the predominant species Rhizobium etli present in soils of a region that is part of the range occupied by the host in Northwest Argentina, which showed Rep and 16S rDNA RFLP polymorphism. Two populations represented by isolates T29N3L and T44N22P were found to be distinct chromosomal genotypes and closely related to species Rhizobium tropici and Agrobacterium rhizogenes. Their symbiotic genes were analyzed and found to cluster with those from R. tropici as well as with rhizobia isolated from leguminous trees. Three nodulation metabolites produced by T44N22P were detected which are tetra- and pentameric chitocompounds, N-methylated, O-carbamoylated, and N-substituted either by a C_18:0 or C_18:1 acyl chain at their non-reducing end, and all them sulphated at the reducing end. Isolates T29N3L and T44N22P exhibited broad host range but unlike T29N3L, only T44N22P was able to efficiently nodulate Medicago truncatula.     

Production of extensive chickpea (Cicer arietinum L.) protein hydrolysates with reduced antigenic activity.

Chickpea protein isolate was used as starting material for the production of hypoallergenic protein hydrolysates. Western blotting of the protein isolate showed that IgE in sensitized patient sera strongly bound to the basic polypeptidic chains and recognized the acidic ones of 11S globulin. During the hydrolysis process by the individual and/or sequential action of endo- and exoproteases, a high reduction of antigenic activity was observed. Results suggest that the presence of intact or partially hydrolyzed basic polypeptide chains of 11S globulin are responsible for the formation of IgE complexes in protein hydrolysates obtained by exoprotease treatment; however, the digestion of these polypeptide chains by individual action of endoprotease caused a high loss of antigenic activity. The most effective reduction of antigenicity, >90%, was observed in extensive hydrolyzed chickpea proteins obtained by sequential treatment with endo- and exopeptidases. This chickpea protein hydrolysate could be useful for the elaboration of specialized hypoallergenic food products.     

Cloning of a novel phytase from an anaerobic rumen bacterium,Mitsuokella jalaludinii,and its expression in Escherichia coli

The full length phytase gene of Mitsuokella jalaludinii was successfully cloned and was found to be 1 047 bp in length, with 348 amino acids, and was designated as PHY7 phytase gene. A comparison of the sequence of PHY7 phytase gene of M. jalaludinii with various microbial phytase gene sequences showed that it was not similar to those from other bacteria except Selenomonas ruminatium, thus suggesting that they may both express a new class of phytase. The PHY7 phytase gene was subsequently subcloned into bacterial expression vector, p ET32 a, for expression in Escherichia coli strain Rosetta-gami. Expression of the recombinant phytase gene was optimised and characterised. The recombinant phytase was estimated to be approximately 55 k Da by SDS-PAGE analysis. The recombinant phytase exhibited optimum activity at 55°C, p H 4.5 and showed good p H stability from p H 3.5 to 5.5(78% relative activity). Metal ions such as Ca2+, Mg2+, and K+ were found to exert significant stimulatory effect on the recombinant phytase activity while Cu2+, Fe3+, and Zn2+ greatly inhibited the enzyme activity. The recombinant phytase showed moderate resistance to trypsin proteolysis, but susceptible to pepsin proteolysis. The results of the study showed that several characteristics of recombinant phytase were slightly different from the native enzyme. Unfavourable characteristics such as reduced p H stability and metal ion effects should be taken into consideration during feed enzyme formulation.     

Effects of vertical hedge interval of vetiver grass on erosion on steep agricultural lands

Vetiver grass is widely used to reduce soil erosion and has been applied in many areas of the world. However, studies of the effect of vertical hedge intervals on runoff, soil loss and outflow sediment size distribution under a steep slope area are rare. The vetiver grass system (VGS) with three vertical hedge intervals (0·75, 1·5 and 3 m) and no hedgerow were tested at three land slopes (30, 40 and 50 per cent) under three simulated rainfall intensities (60, 85 and 110 mm h⁻¹). It has been observed that vetiver grass (Vetiveria nemoralis) has great potential for reducing runoff and soil loss by about 38·7–68·6 and 56·2–87·9 per cent, respectively. The vetiver strips delayed incipient runoff and reduced peak runoff rate and steady erosion rate. The land slope affected soil loss but did not have a significant effect on runoff. A narrow vetiver hedge interval slightly reduced runoff and soil loss more than a wider one. The soil loss equation obtained in this study revealed that runoff has a higher effect on soil loss. The median sediment size that passed through the vetiver strip increased with rainfall intensity and was mostly dominated by very fine sand, silt and clay. Copyright © 2009 John Wiley & Sons, Ltd.     

Biomass burning in Brazil's Amazonian “arc of deforestation”: Burning efficiency and charcoal formation in a fire after mechanized clearing at Feliz Natal, Mato Grosso

Estimates of greenhouse-gas emissions from deforestation are highly uncertain because of high variability in key parameters and because of the limited number of studies providing field measurements of these parameters. One such parameter is burning efficiency, which determines how much of the original forest's aboveground carbon stock will be released in the burn, as well as how much will later be released by decay and how much will remain as charcoal. In this paper we examined the fate of biomass from a semideciduous tropical forest in the “arc of deforestation,” where clearing activity is concentrated along the southern edge of the Amazon forest. We estimated carbon content, charcoal formation and burning efficiency by direct measurements (cutting and weighing) and by line-intersect sampling (LIS) done along the axis of each plot before and after burning of felled vegetation. The total aboveground dry biomass found here (219.3 Mg ha⁻¹) is lower than the values found in studies that have been done in other parts of the Amazon region. Values for burning efficiency (65%) and charcoal formation (6.0%, or 5.98 Mg C ha⁻¹) were much higher than those found in past studies in tropical areas. The percentage of trunk biomass lost in burning (49%) was substantially higher than has been found in previous studies. This difference may be explained by the concentration of more stems in the smaller diameter classes and the low humidity of the fuel (the dry season was unusually long in 2007, the year of the burn). This study provides the first measurements of forest burning parameters for a group of forest types that is now undergoing rapid deforestation. The burning parameters estimated here indicate substantially higher burning efficiency than has been found in other Amazonian forest types. Quantification of burning efficiency is critical to estimates of trace-gas emissions from deforestation.     

Senescent leaf decomposition in a Mediterranean pear orchard

In a pear orchard, when leaf senescence occurs, nitrogen (N) is added to the soil by the fallen leaves and can be re-used by the tree after undergoing decomposition and mineralization processes. Studies on leaf decomposition and N mineralization in orchards are scarce but essential to understand the N balance in the tree–soil ecosystem in a sustainable or precision agriculture. This study aimed to quantify the contribution of pear tree senescent leaves to N cycling in the orchard and its re-cycling by the crop. ‘Rocha’ pear unlabelled leaves were incubated in situ using the litter-bag technique and ¹⁵N-enriched leaves were placed at the soil surface in undisturbed confined cores.One- to six-year-old pear trees returned to the soil between 1 kg N ha^?1 year^?1 and 6 kg N ha^?1 year^?1 from senescent leaves that decomposed at rates varying from 0.0025 day^?1 (d^?1) to 0.0047 d^?1 (estimated by both techniques, respectively). In the litter-bags, after 506–641 days, only 18–35% of initial DW was recovered in the soil, whereas in the soil cores the weight loss was higher, resulting in only 30–6% of initial DW after 398–406 d. After this period, between 36% and 110% of the initial N of the senescent leaves was recovered as organic ¹⁵N in the surface soil layer (0–7.5 cm), depending on climatic conditions, and being more prone to be absorbed by weeds.