Corrado Fogher

Field assessments of gene flow from transgenic to cultivated rice (Oryza sativa L.) using a herbicide resistance gene as tracer marker

Abstract Development of plant genetic engineering has led to the deployment of transgenic crops and, simultaneously, to the need for a thorough assessment of the risks associated with their environmental release. This study investigated the occurrence of gene flow from transgenic rice to non-transgenic rice plants under agronomic conditions using a herbicide resistance gene as a tracer marker. Two field experiments were established in the paddy fields of two main Mediterranean rice-growing areas of Spain and Italy. In both locations analyses of phenotypic, molecular and segregation data showed that pollination of recipient plants with pollen of the transgenic source occurred at a significant frequency. A gene flow slightly lower than 0.1% was detected in a normal side-by-side plot design. Similar results were found in a circular plot when the plants were placed at 1-m distance from the transgenic central nucleus. A strong asymmetric distribution of the gene flow was detected among this circle and highest values (0.53%) were recorded following the direction of the dominant wind. A significant lowest value (0.01%) was found in the other circle (5 m from the transgenic plants) as was expected according to the characteristics of rice pollen. Such circular-field trial designs could also prove to be very useful in studying the gene flow to other commercial cultivars of rice with the aim of establishing strategies to prevent pollen dispersal from commercial transgenic fields to the neighbouring conventional fields.

DNA extraction from olive oil and its use in the identification of the production cultivar

Abstract DNA recovery from food samples might be of great importance when the raw material used in the production process has to be traced. We were interested in verifying the presence of nucleic acids in extra virgin olive oil in order to determine the cultivar of origin of the olives used for the production. A reliable DNA extraction method for extra virgin olive oil has been defined, as far as both quantity and quality are concerned, and the possibility of using this DNA for fingerprinting the original cultivar has been demonstrated. DNA extraction was tested on four monovariety oils, plus four commercial extra virgin olive oils. The DNA in the extracted solution was of chloroplast and nuclear origin since we were able to amplify cloned cultivar RAPD and AFLP fragments homologous to nuclear DNA of other species. It has also been shown that DNA purified from oil can be used for AFLP analysis and that the profile of the DNA purified from a monovariety oil corresponds to the profile of the DNA purified from the leaves of the same cultivar.

Recombinant human acid β-glucosidase stored in tobacco seed is stable, active and taken up by human fibroblasts

Gaucher disease, the most common genetic lysosomal disorder, is caused by the lack of functional acid β-glucosidase (GCase) and is currently treated at a very high cost by enzyme replacement therapy. In an attempt to provide a safe and cost-effective production system, human placental GCase was produced and purified from transgenic tobacco seeds. Plant-derived recombinant GCase was found to be enzymatically active, uptaken by human fibroblasts and free of immunogenic xylose and fucose residues. This report demonstrates the potential of plant bioreactors in the large-scale production of injectable proteins required for lifelong therapy.

Soybean Kunitz, C-II and PI-IV inhibitor genes confer different levels of insect resistance to tobacco and potato transgenic plants

Abstract In modern, highly intensive agriculture, the control of insect pests is basically achieved with the application of chemical pesticides. Heavy reliance on this sole strategy is associated with several drawbacks, and the development of alternative or complementary methods to chemical control is desirable. In this work, three soybean genes (KTi3, C-II and PI-IV)coding for serine proteinase inhibitors were isolated by PCR and transferred to Agrobacterium tumefaciens EHA 105, which in turn was used for transforming tobacco leaf and potato tuber discs. Biochemical assays confirmed that transgenic plants synthesized serine proteinase inhibitors; rates of expression varied among plants. The level of insect resistance (tested with Spodoptera littoralis Boisduval) was particularly high in tobacco, where many plants caused the death of all larvae. In potatoes, larval mortality was much less frequently achieved, but the results were still encouraging in that larval weight gain was reduced by 50% in the presence of adequate amounts of inhibitor. When 8-day-old larvae were fed different KTi3-expressing tobacco plants, a highly significant (P<0.01) correlation was observed between inhibitor content and larval live weight. Larval weight gain was found to be dependent on midgut proteolytic activity. On the basis of the evidence collected, it is suggested that further work is required to identify more specific inhibitors for the main proteinases of the target insect.

Regeneration of Populus nigra transgenic plants expressing a Kunitz proteinase inhibitor (KTi3) gene

Transgenic poplar (Populus nigra, cv. Jean Pourtet) plants were recovered as a result of Agrobacterium tumefaciens-mediated transformation performed with EHA105 pBI-KUN strain. Plasmid pBI-KUN contains a 650 bp insert derived from the soybean (Glycine max L.) KTi3, gene, coding for a Kunitz trypsin proteinase inhibitor. A total of 58 independent transgenic lines were obtained from 200 co-cultivated leaf explants. Southern blot hybridization analysis demonstrated the presence of KTi3 gene in the poplar genome. Northern blot analysis of different kanamycin-resistant plantlets confirmed the accumulation of KTi3 mRNA and revealed different levels of expression. The trypsin inhibitory activity was determined in poplar transgenic tissues by means of specific assay. Moreover, the trypsin-like digestive proteinases of the polyphagous moth Lymantria dispar (Lepidoptera, Lymantriidae) and Clostera anastomosis (Lepidoptera, Notodontidae) were detected and inhibited in vitro by Kunitz proteinase inhibitor from selected transgenic plants. Two insect bioassays were performed on P. nigra transgenic plant lines, using larvae of the above mentioned insects. In both cases larval mortality and growth as well as pupal weight were not significantly affected when the insects were fed on transgenic leaves and control leaves, respectively.

Spread of herbicide-resistant weedy rice (red rice, Oryza sativa L.) after 5 years of Clearfield rice cultivation in Italy.

The weedy relative of cultivated rice, red rice, can invade and severely infest rice fields, as reported by rice farmers throughout the world. Because of its close genetic relationship to commercial rice, red rice has proven difficult to control. Clearfield (Cl) varieties, which are resistant to the inhibiting herbicides in the chemical group AHAS (acetohydroxyacid synthase), provide a highly efficient opportunity to control red rice infestations. In order to reduce the risk of herbicide resistance spreading from cultivated rice to red rice, stewardship guidelines are regularly released. In Italy, the cultivation of Cl cultivars started in 2006. In 2010, surveillance of the possible escape of herbicide resistance was carried out; 168 red rice plants were sampled in 16 fields from six locations containing Cl and traditional cultivars. A first subsample of 119 plants was analysed after herbicide treatment and the resistance was found in 62 plants. Of these 119 plants, 78 plants were randomly selected and analysed at the level of the AHAS gene to search for the Cl mutation determining the resistant genotype: the Cl mutation was present in all the resistant plants. Nuclear and chloroplast microsatellite markers revealed a high correlation between genetic similarity and herbicide resistance. The results clearly show that Cl herbicide-resistant red rice plants are present in the field, having genetic relationships with the Cl variety. Finding plants homozygous for the mutation suggests that the crossing event occurred relatively recently and that these plants are in the F2 or later generations. These observations raise the possibility that Cl red rice is already within the cultivated rice seed supply.

Expression of the Vitreoscilla Hemoglobin (VHb)-Encoding Gene in Transgenic White Poplar: Plant Growth and Biomass Production, Biochemical Characterization and Cell Survival under Submergence, Oxidative and Nitrosative Stress Conditions

Expression of the vhb gene, encoding the hemoglobin protein from Vitreoscilla spp. (VHb), has been shown to increase cell growth and protein synthesis, modify the oxygen-dependent product biosynthesis and the susceptibility to oxidative and nitrosative stresses in several host microrganisms, and to improve plant tolerance to flooding-submergence. A chimeric construct consisting of the CaMV35S promoter fused to the vhb gene and nopaline synthase terminator was transferred into white poplar (Populus alba L.) via Agrobacterium tumefaciens in order to test the generality of these phenomena. The presence of the vhb gene was demonstrated by Southern blot analysis. Accumulation of the vhb transcript and protein was detected in all the selected transgenic poplar lines. In vitro growth bioassays revealed that the vhb gene expression in transgenic poplar plants did not significantly affect their growth pattern. One out of the six selected transgenic lines showed significantly higher values for plant height and stem biomass in greenhouse conditions and exhibited enhancement of root biomass production and stem diameter when compared to the wild-type plants. However, no significant differences in chlorophyll a, b, total carotenoid and protein contents were observed. Two selected transgenic lines were characterized in more detail for tolerance to submergence, oxidative and nitrosative stresses. Under in vitro and in vivo submergence conditions, growth parameters and total protein content of transgenic VHb poplars were similar to those observed in the wild-type plants. In addition, leaf discs from the transgenic plants maintained in standard growth conditions did not reveal increased tolerance to oxidative stress by hydrogen peroxide compared to wild-type plants. In a parallel study, cell suspension cultures obtained from both wild-type and VHb transgenic lines were evaluated for growth and survival in the presence of oxidative and nitrosative stresses. No significant differences were observed between the tested VHb and wild-type poplar lines. Our results show that VHb expression in plants can have erratic effects since the enhancement of plant growth and biomass production and the tolerance to submergence, oxidative and nitrosative stresses are not consistently dependent on the presence of this specific function. Consequently, the genetic manipulation of plant oxygen metabolism must be carefully evaluated and extensive biochemical, molecular and cellular investigations are required to assess the real value of the final products.

Development of SCAR Markers for Germplasm Characterisation in Olive Tree (Olea europea L.)

A set of 14 SCAR markers were developed starting from RAPD, AFLP and SAMPL analysis of several olive germplasm accessions. Eight RAPD, two AFLP and four SAMPL fragments were converted into dominant and codominant SCARs by cloning and sequencing the selected fragments. The markers obtained were evaluated on forty different olive cultivars from different Italian production areas (mainly from Liguria). The combined use of these SCARs made possible to univocally identify 26 cultivars while the remaining 14 will require the development of further markers since most of them are placed in a main group containing six genetically similar cultivars (among which Frantoio and Taggiasca) and four minor groups containing two cultivars each. A total of 31 different haplotypes were identified and the analysis of several individual plants indicated no intra-cultivar variability. Considering the SCAR polymorphism two alleles were scored for each markers with the only exception of markers IGPS3 and IGPS4 showing 4 alleles with 7 recognised groups and 5 alleles with 4 groups, respectively. Though less polymorphic in comparison with other markers like SSRs, the developed SCARs proved useful in genotype identification. In addition, they could potentially be used for breeding applications and forensic analysis.

Evaluation of biodiversity of lactic acid bacteria microbiota in the calf intestinal tracts

Amplified fragment length polymorphism (AFLPs) were used to analyse the naturally occurring flora of lactic acid bacteria (LAB) in gastrointestinal tracts of two healthy 65-day-old calves. More than 1,000 of presumptive LAB were collected and cultured from the gastrointestinal tracts and, among the isolated colonies, a total of 311 strains were analysed and separated into eight clusters based on AFLP banding patterns. To precisely determine the species inside the clusters, partial sequences of fragments of the 16S ribosomal DNA gene were determined, and sequence homology searches were conducted through GenBank on few strains per cluster. The most representative genera of LAB were Lactobacillus (169 isolates, 54% of total) and Streptococcus (99 isolates, 32% of total), while the most frequent species was identified as L. mucosae with 86 different isolates (51% of the Lactobacillus spp. and 28% of the total). This report gives a first characterization of LAB strain biodiversity recovered directly from calf intestine and is the first account of the presence of the L. mucosae species in calves. Moreover it demonstrates that the AFLP is a robust and useful technique for characterizing the strain level of LAB microflora.

Endonuclease genes up-regulated in tissues undergoing programmed cell death are expressed during male gametogenesis in barley

In the process of programmed cell death (PCD), a key role has been attributed to endonucleases capable to cleave nuclear DNA at internucleosomal sites. In barley (Hordeum vulgare L.), two such nucleases (Bnuc1 and BEN1) were individually identified in unrelated tissues. In the present work, we demonstrate that their genes are also expressed in immature anthers at different stages of pollen development. Further experiments carried out on RNA extracted from immature barley anthers led to discover a novel endonuclease gene, namely Bnuc2 (AJ311603 in the EMBL/GenBank/DDBJ databases), eventually found up-regulated at the tetrad stage. The protein encoded was found to conserve large sequence portions of Bnuc1 and BEN1 endonucleases, including the domain regions involved in secretion and DNA/RNA binding. A survey conducted on barley EST libraries showed that Bnuc2 and BEN1 mRNAs are jointly present also in the transcriptome of 20 DAP spike and that other endonuclease ESTs are co-expressed with Bnuc1 or BEN1 in tissues where PCD has been recorded. Therefore, it can be concluded that during the PCD process, a set of S1-type endonucleases is synthesised regardless of the tissue considered.