Agrobacterium-mediated transformation of the CrDAT gene and selection of transgenic periwinkle lines with a high vincristine accumulation

Catharanthus roseus contains vincristine and vinblastine, which are outstanding drugs for cancer. In the biosynthetic pathways of terpenoid indole alkaloids (TIAs) in C. roseus, deacetylvindoline 4-O-acetyltransferase (DAT) is a key enzyme that catalyses the last reaction of vindoline biosynthesis to form vinblastine and vincristine. In this study, the CrDAT transgene was transferred into the periwinkle by Agrobacterium-mediated transformation and generated transgenic periwinkle lines with an increase in vincristine accumulation. The C. roseus DAT gene was introduced into C. roseus plants and it was confirmed that CrDAT was successfully transferred into the genome of periwinkle plants and efficiently translated to synthesise recombinant DAT protein. Four transgenic periwinkle lines in T1 generation, T1-1, T1-3, T1-6, and T1-7, expressed recombinant DAT protein with the total protein content in the range of 2.86 μg.mg^?1 to 5.12 μg.mg^?1. Moreover, the vincristine contents of four transgenic lines increased by 1.63?2.48-fold compared to non-transgenic plants, ranging from 6.91 µg.g^?1 (fresh weight) to 10.53 µg.g^?1 (fresh weight). The T1-1 line had the highest vincristine content. Hence, the overexpression of the recombinant DAT protein can improve the vincristine accumulation of transgenic C. roseus plants.

Abbreviation: CrDAT - Catharanthus roseus Deacetylvindoline-4-O-Acetyl Transferase; D4H - Deacetoxyvindoline 4-hydroxylase; ELISA - Enzyme-Linked Immunosorbent Assay Monoterpene indole alkaloid; T0, T1 - Generations of transgenic plants; TIAs - Terpenoid indole alkaloids; WT- The wild-type tobacco plants (non transgenic plant); 35S - Cauliflower mosaic virus 35S promoter     

High vegetative compatibility diversity of Cryphonectria parasitica infecting sweet chestnut (Castanea sativa) in Britain indicates multiple pathogen introductions

Chestnut blight, caused by Cryphonectria parasitica, was identified in Devon, UK, in December 2016. Intensive surveys detected the disease at further sites in Devon (seven), Berkshire (one), Dorset (one), Derbyshire (four) and a cluster of eight sites in southeast London. Over 570 survey samples were tested, and 227 were positive for C. parasitica by isolation and real-time PCR. A total of 227 isolates were tested for mating type, and 197 screened for vegetative compatibility group (VCG) and compared with VCGs known from mainland Europe. The same isolates were also screened for the presence of Cryphonectria hypovirus 1 (CHV-1). Eleven VCGs were identified within the UK population. Five corresponded to already known European VCGs but six were unique. The European VCGs mainly came from the Devon, Dorset, Berkshire and Derbyshire disease outbreaks, whilst unique VCGs were almost exclusively from the southeast London cluster. Both mating types were detected, but only one mating type was present at each site, with the exception of a single Devon site. Perithecia of C. parasitica were never observed at any site. CHV-1 was found in seven isolates from three different locations and was always subtype-I, which has limited hypovirulence. Therefore, although CHV-1 is associated with C. parasitica at some outbreaks, it probably has limited impact on virulence. The diversity of VCGs and their distribution at outbreak sites, together with findings of CHV-1, suggests C. parasitica has been introduced to the UK multiple times over at least two decades through international plant trade.     

Genetic variation in root development responses to salt stresses of quinoa

Soil salinity has become a serious environmental abiotic stress limiting crop productivity and quality. The root system is the first organ sensing the changes in salinity. Root development under elevated salinity is therefore an important indicator for saline tolerance in plants. Previous studies focused on varietal differences in morphological traits of quinoa under saline stresses; however, variation in root development responses to salinity remains largely unknown. To understand the genetic variation in root development responses to salt stress of quinoa, we conducted a preliminary screening for salinity response at two salinity levels of a diverse set of 52 quinoa genotypes and microsatellite markers were used to link molecular variation to that in root development responses to salt stresses of represented genotypes. The frequency distribution of saline tolerance index showed continuous variation in the quinoa collection. Cluster analysis of salinity responses divided the 52 quinoa genotypes into six major groups. Based on these results, six genotypes representative of groups I to VI including Black quinoa, 2-Want, Atlas, Riobamba, NL-6 and Sayaña, respectively, were selected to evaluate root development under four saline stress levels: 0, 100, 200 and 300 mM NaCl. Contrasts in root development responses to saline stress levels were observed in the six genotypes. At 100 mM NaCl, significant differences were not observed in root length development (RLD) and root surface development (RSAD) of most genotypes except Black quinoa; a significant reduction was observed in this genotype as compared to controls. At 200 mM NaCl, significant reduction was detected in RLD and RSAD in all genotypes showing this as the best concentration to discriminate among genotypes. The strongest inhibition of root development was found for all genotypes at 300 mM NaCl as compared to lower saline levels. Among genotypes, Atlas of group III shows as a saline-tolerant genotype confirming previous reports. Variation in root responses to salinity stresses is also discussed in relation to climate conditions of origins of the genotypes and reveal interesting guidelines for further studies exploring the mechanisms behind this aspect of saline adaptation.     

Phosphorus fractions and dynamics as affected by land-use changes in the Central Mexican highlands

Land-use change from forest to agriculture in the volcanic ash-derived soils of Mexico has increased over recent decades. It is likely that land uses and management practices, particularly fertilizer use have affected phosphorus (P) distribution and availability. The objective of this study was to evaluate the effects of land-use types (native forest and maize mono-cropping), and the related P addition, on the forms and distribution of soil P and their isotopic exchangeability. An Andisol, sampled from a cropping site, along with the contiguous area under native forest was treated with ³²P-labelled potassium phosphate (KH₂³²PO₄). The soil samples were extracted after incubation times of 7, 21, 35 and 49 days. Phosphorus content and ³²P recovery in fractions sequentially extracted were assessed for each incubation time. Total soil P was dominated by inorganic fractions (79 to 86%) in both land-use types. Resin-Pi, bicarbonate extractable inorganic P (Bic-Pi) and sodium hydroxide extractable inorganic P (NaOH_0.1-Pi) were all raised with P addition. However, the proportion of organic P fraction was reduced under cropped soil. The recovery of ³²P in soils with P addition indicates that resin-Pi, Bic-Pi and NaOH_0.1-Pi comprised nearly all the exchangeable P. In native soils with no P addition, more than 19% of the ³²P was recovered in Bic-Po and NaOH_0.1-Po forms. This finding indicates that organic P cycling is crucial when soil Pi reserves are presented in an inadequate amount. Ecologically based management has to be designed for replenishment and succeeding maintenance of soil organic P compounds to increase sustainable agricultural production.     

Prediction of Genetic Gain in Sweet Corn using Selection Indexes

Journal of Crop Science and Biotechnology - The cultivation of sweet corn is expanding in Brazil, but there are serious constraints about the availability of commercial cultivars. The selection of...     

Movement behaviour of large female yellow European eel (Anguilla anguilla L.) in a freshwater polder area

Due to a recruitment decline of more than 90% in 30 years, the European eel (Anguilla anguilla L.) has been classified by IUCN as critically endangered. Although the species has been studied intensively to obtain knowledge to improve management, studies about the resident yellow stage are relatively scarce. In this study, 52 large female yellow eels were tagged with acoustic transmitters in a Belgian polder system and tracked by a network of 23 automatic listening stations. We studied both circadian and seasonal movement patterns and the effect of environmental variables on these patterns. Large female yellow eels were most active at night in late summer and early autumn. A generalised linear mixed model showed that their movement is only slightly influenced by environmental variables. Moreover, as yellow eels show high site fidelity (i.e., the majority was detected only in the habitat type of their catch‐release location), they do not encounter many human‐induced connectivity problems in polder systems, which makes these systems highly suitable as eel growth habitat. These results can contribute to an effective eel management regarding habitat protection and restoration.     

Cylindrospermopsin effects on cell viability and redox milieu of Neotropical fish <Emphasis Type="Italic">Hoplias malabaricus</Emphasis> hepatocytes

Cylindrospermopsin (CYN) is a cyanotoxin that is cytotoxic to a wide variety of cells, particularly to the hepatocytes. In this study, the toxic effects of purified CYN were investigated in primary cultured hepatocytes of Neotropical fish Hoplias malabaricus. After isolation, attachment, and recovery for 72 h, the cells were exposed for 72 h to 0, 0.1, 1.0, 10, and 100 μg l^?1 of CYN. Then, cell viability and a set of oxidative stress biomarker responses were determined. Catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione S-transferase activities were not affected by exposure to CYN. Concentration-dependent decrease of glutathione reductase activity occurred for most CYN-exposed groups, whereas non-protein thiol content increased only for the highest CYN concentration. Lipid peroxidation, protein carbonylation, and DNA damage levels were not altered, but reactive oxygen species levels increased in the cells exposed to the highest concentration of CYN. Cell viability decreased in all the groups exposed to CYN. Thus, CYN may cause a slight change in redox balance, but it is not the main cause of cell death in H. malabaricus hepatocytes.     

Effect of an intensive mechanical removal effort on a population of non‐native rainbow trout Oncorhynchus mykiss in a South African headwater stream

1. Invasions by non‐native species can compromise the conservation value of otherwise pristine headwater streams. While both developed and developing countries recognize this threat, few of the latter have suitable budgets to implement control programmes.
2. This study assessed the effectiveness of a mechanical project to remove non‐native rainbow trout Oncorhynchus mykiss from a 6 km section of the upper Krom River, a small headwater stream in the Cederberg Mountains in South Africa's Cape Floristic Region (CFR).
3. From October 2013 to February 2014, 354 O. mykiss were removed by angling (58%), fyke netting (28%) and gill netting (14%). This resulted in a marked reduction, but not eradication, of the O. mykiss population (fish relative abundance decreased from 0.53 ± 0.09 fish per net per night in October 2013 to 0.21 ± 0.09 fish per net per night in February 2014). Following the cessation of manual removals, the relative abundance of O. mykiss had increased to 0.56 ± 0.18 fish per net per night by March 2016, suggesting that without sustained removal effort, the population will rapidly return to its pre‐removal abundance level.
4. Further work is needed to refine the methodology and test the effectiveness of mechanical removal of non‐native freshwater fish in a variety of ecological settings in the CFR. This approach holds potential for meeting the dual goals of reducing the ecological impacts of non‐native fishes and generating employment opportunities in line with the policy objectives of developing nations.

     

The release and uptake of metals from potential biofilm inhibition products during spiny lobster (Sagmariasus verreauxi,H. Milne Edwards 1851) culture

Zinc (Zn) and copper (Cu) are strong inhibitors of bacterial biofilms in aqueous solutions, but are known toxins of crustaceans. A new metal application method; cold‐sprayed metal embedment, known to modulate metal release, was tested for its applications in crustacean larval culture systems. Cold‐spray technology allows metal particles to bond to plastics, while modulating metal ion release and biocide activity to the substrate boundary. In this study, Eastern spiny lobster (Sagmariasus verreauxi) larvae (phyllosoma) were cultured in the presence of cold‐sprayed Zn and Cu metal surfaces. Metal loss was monitored gravimetrically on embedded surfaces, assessment of water ion concentrations and analysis of phyllosoma body content were undertaken. Phyllosoma moulting, deformity and mortality patterns were monitored. Cold‐sprayed Zn‐ and Cu‐embedded surfaces were depleted with losses of 0.69% and 31.2% noted respectively. Culture water concentrations of these metals were elevated and accumulation by phyllosoma occurred. Water Zn concentrations of 18.5 μg L^?1 were associated with chronic eyestalk moult deformities; the first report of Zn causing a non‐lethal moult deformity in crustacean larvae. The Cu surface lost a third of its metal mass with a water concentration of 40 μg L^?1 causing acute toxicity and localization of composite granules in the midgut gland. Cu associated mortality was noted by Day 2 of culture with a LD 50 experienced by Day 9. Future work on the use of bioactive metals in aquaculture systems will focus on a range of different metal alloys, and improved modulation of ion release mechanisms through increased particle embedment depth and separation.     

Irrigation of paddy soil with industrial landfill leachate: impacts in rice productivity,plant nutrition,and chemical characteristics of soil

Treated industrial effluents have high levels of nutrients and dissolved organic matter. The irrigation of rice by flooding can increase nutrient uptake and grain yield. Therefore, this study evaluated the nutrient contents in the shoots and grain of the rice crop and also the chemical of the soil after irrigation of the crop with leachate of the treated industrial effluent. A greenhouse experiment was conducted using pots filled with 20 kg of soil in a randomized block design with three replications. The treatments consisted of control (irrigation with distilled water) and four concentrations of the leachate (25, 50, 75, and 100 %) for irrigation. At the end of the experiment, the nutrient contents in tissues of rice plants, sterility of spikelets, and grain mass were evaluated. Results showed that irrigation with the leachate at 25 % content increased the macro- and micronutrients’ concentrations in the shoot biomass and grain, except for potassium and iron. Irrigation with the industrial leachate decreased tillering and grain yield; however, it increased chlorophyll content, sterility of spikelets, and sodium intake at this leachate concentration. The potassium and sodium levels and the electrical conductivity values of soils irrigated with treated industrial leachate were increased. The use of the treated leachate from industrial effluents is an alternative that reuses the nutritional load, but the volume of leachate should be limited and monitored to prevent the sodicity in the soil and problemsdue to eutrophication.