Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration

Genetic transformation of mature trees is difficult because adult tissues are recalcitrant to Agrobacterium tumefaciens infection and transformation and because transgenic mature events are less competent for regeneration. We have shown that reinvigoration allows manipulation of the vegetative phase to increase the potential for transformation and regeneration without loss of competence for flowering and fruiting. To produce transgenic plants from clementine mandarin (Citrus clementina hort. ex Tanaka), we optimized the conditions of the source material both ex vitro and in vitro. Grafting of mature buds on juvenile rootstocks in the spring and preventing multiple bud sprouting by removing all but one bud permitted selection of vigorous first flushes for in vitro culture. Use of additional virulence genes from A. tumefaciens to increase transformation frequency and optimization of culture media and conditions to enhance explant cell competence for T-DNA integration and organogenesis resulted in efficient and reliable transgenic plant production. Transformed regenerants from explants, cultured in media without antibiotics, were identified by a screenable marker (either beta-glucuronidase or green fluorescent protein (GFP)), creating the possibility of generating transgenic clementine plants without antibiotic resistance marker genes. Stable integration of foreign genes was demonstrated by Southern blot analysis, and expression of these foreign genes was confirmed by detection of GFP fluorescence in leaves, floral organs and fruits of the transgenic plants.     

Effect of an entomopathogenic nematode,Steinernema carpocapsae Weiser (Nematoda,Steinernematidae), on carabid beetles in field trials with annual legumes

Two species of carabid beetle:Bembidion properans Steph. andPterostichus cupreus L. were the most often captured into pitfall traps in the spring period on plots with various cultivars of annual legumes. The greatest density ofP. cupreus population in the field with annual legumes was observed in May, in the feeding and eggs laying period by pea weevilSitona lineatus L. (Col. Curculionidae) — the main legumes pest. In June the most frequent carabid wasB. properans, the small beetles destroying the eggs or young larvae ofSitona lineatus. No effect of usingS. carpocapsae nematodes to infect nontarget carabid species was found in case different doses of invasive larvae on the soil surface into traps, although the mortality of captured ground beetles was high. The length of life of Carabidae collected from traps with nematodes were not significantly decreased in comparison with the control group.

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Zusammenfassung Zwei Carabiden-Arten,Bembidion properans Steph. undPterostichus cupreus L. waren die beiden häufigsten Arten, die im Frühjahr in Bodenfallen in verschiedenen Kulturen einjähriger Leguminosen gefangen wurden. Die größte Dichte vonP. cupreus wurde im Mai festgestellt während der Fraß- und Eilege-Periode des Haupt-Leguminosenschädlings, des ErbsenrüßlersSitona lineatus L. (Col., Curculionidae). Im Juni warB. properans der häufigste Laufkäfer, der die Eier und Junglarven vonSitona lineatus zur Beute nahm. Die Hinzugabe invasiver Larven des NematodenSteinernema carpocapsae in mit gefangenen Laufkäfern besetzten Bodenfallen erbrachte keine gesicherte Infektion, obgleich die Carbadiden-Dichte in den Gefäßen hoch war. Die Lebensdauer von Carabidae in mit Nematoden besetzten Gefäßen unterschied sich nicht von jener in Kontrollgefäßen.

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With 2 figures and 2 tables     

Über den Befall einjähriger Leguminosen durch den Erbsenrüßler,Sitona lineatus L. (Col., Curculionidae)

In early spring period early field pea and other cultivars of pea were most attractive plants forS. lineatus adults in comparison with bean cv. In May, in egg laying period, bean cv appeared to be the most preferable. These field studies are the first step on further experiments on determining the effect of host plant on susceptibility of the weevils to infection by entomopathogens. *** DIRECT SUPPORT *** A0135136 00002     

14-3-3 protein down-regulates key enzyme activities of nitrate and carbohydrate metabolism in potato plants

The 14-3-3 protein is one of the best candidates for coordinating all plant metabolic pathways. To verify this suggestion transgenic potato plants with repression of one (J4 and J5 plants), two (G1 plants), and six (G3 plants) constitutive 14-3-3 protein isoforms as well as plants overexpressing the 14-3-3 protein were studied. Reduction in the 14-3-3 protein level in the J4 and J5 transformants, the G1 transformants, and the G3 transformants was close to 29, 41.5, 38, and 55%, respectively. In the case of the 14-3-3 overexpressing plants (J2), a 30% increase in protein content was detected. Changes in nitrate reductase (NR), sucrose phosphate synthase (SPS), and starch synthase (SS) activities in the transgenic plants perfectly reflect the overall 14-3-3 protein level. The highest increase in enzyme activities was observed for the G3 plants and the lowest for the J4 transformants. The same was detected for the measured metabolites. The highest increase in the protein, starch, and sucrose levels was detected in the tubers from the G3 transgenic plants. Because there was almost no change in the isoform ratio in the transgenic plants when compared to the control, it is suggested that it is the overall content of the 14-3-3 protein, rather than the content of particular isoforms, which plays a crucial role in the regulation of enzyme activities and thus in metabolite synthesis. The properties of the 14-3-3 overexpressing plants are very similar to those of the control ones, suggesting that the protein is in excess in the nontransformants and a further increase in its content is not recognized by cell metabolism. A considerable influence of the 14-3-3 protein level on potato plant metabolism was demonstrated. This effect was observed in key metabolic enzyme activities and metabolite content as well. A high variability between mean values, representing individual transgenes, with respect to nitrate reductase, sucrose phosphate synthase, and starch synthase activities in the examined genotypes was noted. These changes were closely correlated with metabolite levels, among them protein, starch, reducing sugars, and sucrose. The results obtained for the five types of transgenic potato plants in comparison with the control were statistically assessed using discriminate function and cluster analyses.     

ADP ribosylation factor regulates metabolism and antioxidant capacity of transgenic potato tubers

In our recent studies we have evidenced that repression of ADP-ribosylation factor (ARF) in potato plants results in 14-3-3 gene activation. The significant alteration in plant phenotype and in carbohydrate content clearly indicates that there may also be changes in other metabolite syntheses. In this paper we present the data on contents of compounds, occurring in transgenic potato tubers from field trial, known to be important for the human diet. We also determine which of the ARF-antisense plant features resulted from ARF repression. This determination was accomplished by the analysis of ARF-antisense plants transformed with cDNA encoding 14-3-3 protein in reverse orientation. The sucrose accumulation and the decrease in glycoalkaloids level were found to be characteristic features of all transgenic plants. The increase in antioxidant capacity of transgenic potato tubers should also be pointed out. The analysis of fat from modified potato tubers revealed a nutritionally valuable composition of fatty acids, including the significant increase of linoleic acid level.     

Organophosphorus pesticide residues in Mexican commercial pasteurized milk

A study was conducted to measure residues of 13 organophosphorus (OP) pesticides, widely used as dairy cattle ectoparasiticides or in crops used for animal feed, in homogenized and pasteurized Mexican milk samples. Four different milk brands with high distribution were collected biweekly during a 12 month period (n = 96) in supermarkets. OP pesticide residues were measured by gas chromatography with a flame photometric detector. Approximately 39.6% of the samples contained detectable levels of OP pesticide residues. Eight samples contained residues exceeding established maximum residue limits (MRL), and the OP pesticides present in these samples were dichlorvos (five samples), phorate, chlorpyrifos, and chlorfenvinphos (one sample, respectively). Average residues of 13 OP pesticides measured were below established MRLs ranging between 0.0051 and 0.0203 ppm.     

Combined use of gliadins and SSRs to analyse the genetic variability of the Spanish collection of cultivated diploid wheat (Triticum monococcum L. ssp. monococcum)

This work studied the combined use of gliadins and SSRs to analyse inter- and intra-accession variability of the Spanish collection of cultivated einkorn (Triticum monococcum L. ssp. monococcum) maintained at the CRF-INIA. In general, gliadin loci presented higher discrimination power than SSRs, reflecting the high variability of the gliadins. The loci on chromosome 6A were the most polymorphic with similar PIC values for both marker systems, showing that these markers are very useful for genetic variability studies in wheat. The gliadin results indicated that the Spanish einkorn collection possessed high genetic diversity, being the differentiation large between varieties and small within them. Some associations between gliadin alleles and geographical and agro-morphological data were found. Agro-morphological relations were also observed in the clusters of the SSRs dendrogram. A high concordance was found between gliadins and SSRs for genotype identification. In addition, both systems provide complementary information to resolve the different cases of intra-accession variability not detected at the agro-morphological level, and to identify separately all the genotypes analysed. The combined use of both genetic markers is an excellent tool for genetic resource evaluation in addition to agro-morphological evaluation.     

Characterization of field‐evolved resistance to Bacillus thuringiensis‐derived Cry1F δ‐endotoxin in Spodoptera frugiperda populations from Argentina

BACKGROUND

Transgenic maize (Zea mays L.) event TC1507 (Herculex^® I insect protection), expressing Cry1F δ‐endotoxin derived from Bacillus thuringiensis var. aizawai, was commercialized in 2003 in the Americas. Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) susceptibility to Cry1F was monitored annually across several regions in Argentina using diagnostic concentration bioassays. Reduced performance of TC1507 maize against S. frugiperda was reported in 2013. A resistant population was established in the laboratory and the dominance of Cry1F resistance was characterized.

RESULTS

During 2012–2015, high‐survivorship of several populations was observed in the resistance monitoring program. Reciprocal crosses of a Cry1F‐resistant population with a Cry1F‐susceptible population were evaluated to calculate effective dominance (D_ML) based on mortality levels observed at 100 µg/ml Cry1F. Two additional dominance levels (D_LC and D_EC) were calculated using lethal (LC₅₀) or effective concentration (EC₅₀) derived from concentration–response bioassays. Estimates indicated that Cry1F resistance in S. frugiperda in Argentina was either highly recessive (D_ML = 0.005) or incompletely recessive (D_LC < 0.26 and D_EC < 0.19).

CONCLUSION

This study is the first documented confirmation and characterization of S. frugiperda Cry1F field‐evolved resistance in Argentina. The resistance to Cry1F in S. frugiperda populations collected in Argentina, is autosomal and incompletely recessive similar to the resistance reported in Brazil. © 2017 The Authors. Pest Management Science published by John Wiley © Sons Ltd on behalf of Society of Chemical Industry.     

Changes in Gut Microbiota Linked to a Reduction in Systolic Blood Pressure in Spontaneously Hypertensive Rats Fed an Extra Virgin Olive Oil-Enriched Diet

Fat type in diet is responsible for specific changes in gut microbiota (GM). Extra virgin olive oil (EVOO) has been shown to be beneficial for blood pressure and to produce effects on GM. To analyze the cause-effect relationship between intestinal microbial changes and blood pressure, we studied the effect of EVOO on fecal microbiota and systolic blood pressure (SBP) levels in spontaneously hypertensive rats (SHR). SHR were fed either an enriched EVOO diet or a standard diet for a period of 12 weeks. At the end of the experimental period, the microbial profiles in the feces were studied in both groups by using PCR-denaturing gradient gel electrophoresis. Real-time PCR was used to quantify the selected bacterial groups. The results demonstrated significant differences when using Lactobacillus (p<0.05), clostridia XIV (p<0.01) and universal (p<0.05) primers. A significant (r=?0.475; p=0.04) inverse correlation between the abundance of clostridia XIV and SBP, which depends on the type of diet, was also observed. Finally, the results suggested an increase in the microbial diversity of the feces of the animals fed the EVOO diet. These results strongly connect the pattern of GM in SHR fed a diet enriched with EVOO to the lower levels of SBP observed in these animals at the end of the feeding period.