Giuseppe Mennella

Production of transgenic eggplant (Solanum melongena L.) resistant to Colorado Potato Beetle (Leptinotarsa decemlineata Say)

Abstract A modified gene of Bacillus thuringiensis var. Tolworthi (Bt), encoding a coleopteran insect-specific CryIIIB toxin, was transferred via Agrobacterium tumefaciens to the female parent of the eggplant commercial F1 hybrid ‘Rimina’. One-hundred and fifty eight transgenic plants were regenerated and tested by PCR and NPTII expression assays. The presence of the CryIIIB toxin in leaf extracts was demonstrated in 57 out of 93 transgenic plants tested by DAS-ELISA assay. High Bt-expressing plants contained a 74-kDa protein cross-reacting with serum anti-CryIIIB toxin. Twenty three out of 44 S. melongena plants tested by insect bioassay showed significant insecticidial activity on neonate larvae of Colorado Potato Beetle (CPB). The Bt transgene and the toxic effect on CPB larvae were transmitted to progenies derived by selfing. Thus, transgenic Bt eggplants represent a very effective means of CPB pest control.

Open field trial of genetically modified parthenocarpic tomato: seedlessness and fruit quality

BackgroundParthenocarpic tomato lines transgenic for the DefH9-RI-iaaM gene have been cultivated under open field conditions to address some aspects of the equivalence of genetically modified (GM) fruit in comparison to controls (non-GM).ResultsUnder open field cultivation conditions, two tomato lines (UC 82) transgenic for the DefH9-RI-iaaM gene produced parthenocarpic fruits. DefH9-RI-iaaM fruits were either seedless or contained very few seeds. GM fruit quality, with the exception of a higher β-carotene level, did not show any difference, neither technological (colour, firmness, dry matter, °Brix, pH) nor chemical (titratable acidity, organic acids, lycopene, tomatine, total polyphenols and antioxidant capacity – TEAC), when compared to that of fruits from control line. Highly significant differences in quality traits exist between the tomato F1 commercial hybrid Allflesh and the three UC 82 genotypes tested, regardless of whether or not they are GM. Total yield per plant did not differ between GM and parental line UC 82. Fruit number was increased in GM lines, and GM fruit weight was decreased.ConclusionThe use in the diet of fruits from a new line or variety introduces much greater changes than the consumption of GM fruits in comparison to its genetic background. Parthenocarpic fruits, produced under open field conditions, contained 10-fold less seeds than control fruits. Thus parthenocarpy caused by DefH9-RI-iaaM gene represents also a tool for mitigating GM seeds dispersal in the environment.

An association mapping approach to identify favourable alleles for tomato fruit quality breeding

BackgroundGenome Wide Association Studies (GWAS) have been recently used to dissect complex quantitative traits and identify candidate genes affecting phenotype variation of polygenic traits. In order to map loci controlling variation in tomato marketable and nutritional fruit traits, we used a collection of 96 cultivated genotypes, including Italian, Latin American, and other worldwide-spread landraces and varieties. Phenotyping was carried out by measuring ten quality traits and metabolites in red ripe fruits. In parallel, genotyping was carried out by using the Illumina Infinium SolCAP array, which allows data to be collected from 7,720 single nucleotide polymorphism (SNP) markers.ResultsThe Mixed Linear Model used to detect associations between markers and traits allowed population structure and relatedness to be evidenced within our collection, which have been taken into consideration for association analysis. GWAS identified 20 SNPs that were significantly associated with seven out of ten traits considered. In particular, our analysis revealed two markers associated with phenolic compounds, three with ascorbic acid, β-carotene and trans-lycopene, six with titratable acidity, and only one with pH and fresh weight. Co-localization of a group of associated loci with candidate genes/QTLs previously reported in other studies validated the approach. Moreover, 19 putative genes in linkage disequilibrium with markers were found. These genes might be involved in the biosynthetic pathways of the traits analyzed or might be implied in their transcriptional regulation. Finally, favourable allelic combinations between associated loci were identified that could be pyramided to obtain new improved genotypes.ConclusionsOur results led to the identification of promising candidate loci controlling fruit quality that, in the future, might be transferred into tomato genotypes by Marker Assisted Selection or genetic engineering, and highlighted that intraspecific variability might be still exploited for enhancing tomato fruit quality.

Thermal Treatment of Eggplant (Solanum melongena L.) Increases the Antioxidant Content and the Inhibitory Effect on Human Neutrophil Burst

The aim of this study was to compare the amount and activity of phytonutrients in raw, grilled, and boiled eggplant fruit using chemical measures and a biological assay of oxidative bursts in human neutrophils. The thermally treated samples showed various changes in their chemical composition (dry matter, soluble solids, acidity, and the amount of alcohol insoluble substances) due to the cooking processes and were much richer in the main phenolic compounds such as chlorogenic and caffeic acids, which are known to be antioxidants. Consequently, their free radical scavenging activity was significantly higher, especially that of superoxide anion. The biological assay of oxidative bursts from human neutrophils in the presence of N-formyl-methionyl-leucyl-phenylalanine confirmed the greater activity of extracts of the cooked eggplants with respect to raw eggplants. Successive extract dilutions showed a significant activity up to 1.25 microg/mL after cooking, while raw fruits resulted in an activity up to 10.00 microg/mL. These results showed that the thermal treatment commonly used before consumption can increase the content and biological activity of antioxidant compounds of eggplants.

Characterization of Health-Related Compounds in Eggplant (Solanum melongena L.) Lines Derived from Introgression of Allied Species

The purpose of the present study was to investigate the levels of either the nutraceutical and health-promoting compounds or the antioxidant properties of innovative eggplant (Solanum melongena L.) genotypes tolerant and/or resistant to fungi, derived from conventional and non-conventional breeding methodologies (i.e., sexual interspecific hybridization, interspecific protoplast electrofusion, androgenesis, and backcross cycles) in comparison with their allied and cultivated parents. Chemical measures of soluble refractometric residue (SRR), glycoalkaloids (solamargine and solasonine), chlorogenic acid (CA), delphinidin 3-rutinoside (D3R), total phenols (TP), polyphenoloxidase (PPO) activity, antiradical activity on superoxide anion and hydroxyl radical were carried out in raw fruit and peel of 57 eggplant advanced introgression lines (ILs), of three eggplant recurrent genotypes and of three allied species during 2005 and 2006. The majority of the ILs, obtained after several backcross cycles, showed positive characteristics with respect to the allied parents such as good levels of SRR, CA, D3R, TP, PPO activity, the scavenging activity against superoxide anion and hydroxyl radical and, in particular, significantly (p <or= 0.05) reduced concentrations of the toxic steroidal glycoalkaloids, solasonine and solamargine. These results showed the possibility to obtain new eggplant genotypes bearing useful traits derived from the allied parents (i.e., resistance/tolerance to plant pathogen fungi) together with nutraceutical and antioxidant properties typical of the cultivated species.

Current status in production and utilization of dihaploids from somatic hybrids between eggplant (Solanum melongena L.) and its wild relatives

The major constrains for practical exploitation of the somatic hybrids between eggplant and its wild relatives have been their sterility and tetraploidy which prevented their incorporation into breeding programs. Here we demonstrate that anther culture was successfully utilized to bring back the ploidy level to the diploid status in tetraploid interspecific hybrids between eggplant and the allied species S. integrifolium and S. aethiopicum gr. gilo. Both the relative species are resistant to Fusarium oxysporum f. sp. melongenae and to some strains of bacterial wilt (Ralstonia solanacearum) which are very destructive diseases of eggplant. Dihaploid androgenetic plants were obtained from the somatic hybrids, from the “double somatic hybrid” obtained by sexual cross of the two somatic hybrids [(eggplant + S. aethiopicum) × (eggplant + S. integrifolium)], and from tetraploid backcrossed plants between the somatic hybrid with S. aethiopicum and eggplant. Phenotypical, molecular, biological and biochemical characterization, and also artificial inoculation with Fusarium oxysporum are consistent with a recombination between the genomes of the species involved in the hybridizations. Dihaploids resistant to Fusarium were successfully backcrossed with eggplant. Besides their utility as potential valuable breeding materials, the introgressed lines obtained may be utilized in genetic and molecular studies about the resistance to Fusarium from S. integrifolium and S. aethiopicum gr. gilo.

ISSR and Isozyme Characterization of Androgenetic Dihaploids Reveals Tetrasomic Inheritance in Tetraploid Somatic Hybrids between Solanum melongena and Solanum aethiopicum Group Gilo

Gene exchanges between Solanum melongena and its allied relative Solanum aethiopicum are a crucial prerequisite for introgression of useful traits from the allied species into the cultivated eggplant. In order to evaluate the extent of genetic recombination between the 2 species, biochemical and molecular markers were employed. A dihaploid population obtained through anther culture of the corresponding tetraploid somatic hybrids was genetically analyzed. The extent of disomic/tetrasomic inheritance and segregation ratios of 3 isozyme systems and intersimple sequence repeat (ISSR) markers were evaluated. The dihaploids, being derived from microspores, allowed for simple, complete, and accurate analyses. The segregation of 280 ISSR markers (110 aethiopicum-specific, 104 melongena-specific, and 66 monomorphic) were evaluated in 71 dihaploids. According to the genetic constitution (simplex/duplex/triplex), almost 64% of the fragments revealed the tetrasomic and/or disomic inheritance. With regard to the assigned species-specific fragments, 68% and 4% were unambiguously the result of tetrasomic and disomic inheritance, respectively. Twenty-four of the 66 monomorphic ISSRs were inherited according to random chromatid segregation. The phenotypes of glucose-6-phosphate dehydrogenase (G-6-PDH), 6-phosphogluconate dehydrogenase (6-PGDH), and shikimate dehydrogenase (SKDH) were studied in 70 dihaploids and inferences were made about the allelic state of their 5 loci. The isozyme markers segregated in the dihaploids in a distorted manner, their segregations did not fit in with any of the expected segregation ratios. However, tetrasomic inheritance might be suggested for G-6-PDH 2 and SKDH 1 loci. Our results demonstrated that gene exchanges occurred readily in the somatic hybrids between S. melongena and S. aethiopicum gr. Gilo.

Phenylpropanoids Accumulation in Eggplant Fruit: Characterization of Biosynthetic Genes and Regulation by a MYB Transcription Factor

Phenylpropanoids are major secondary metabolites in eggplant (Solanum melongena) fruits. Chlorogenic acid (CGA) accounts for 70–90% of total phenolics in flesh tissues, while anthocyanins are mainly present in the fruit skin. As a contribution to the understanding of the peculiar accumulation of these health-promoting metabolites in eggplant, we report on metabolite abundance, regulation of CGA and anthocyanin biosynthesis, and characterization of candidate CGA biosynthetic genes in S. melongena. Higher contents of CGA, Delphinidin 3-rutinoside, and rutin were found in eggplant fruits compared to other tissues, associated to an elevated transcript abundance of structural genes such as PAL, HQT, DFR, and ANS, suggesting that active in situ biosynthesis contributes to anthocyanin and CGA accumulation in fruit tissues. Putative orthologs of the two CGA biosynthetic genes PAL and HQT, as well as a variant of a MYB1 transcription factor showing identity with group six MYBs, were isolated from an Occidental S. melongena traditional variety and demonstrated to differ from published sequences from Asiatic varieties. In silico analysis of the isolated SmPAL1, SmHQT1, SmANS, and SmMyb1 promoters revealed the presence of several Myb regulatory elements for the biosynthetic genes and unique elements for the TF, suggesting its involvement in other physiological roles beside phenylpropanoid biosynthesis regulation. Transient overexpression in Nicotiana benthamiana leaves of SmMyb1 and of a C-terminal SmMyb1 truncated form (SmMyb1Δ9) resulted in anthocyanin accumulation only of SmMyb1 agro-infiltrated leaves. A yeast two-hybrid assay confirmed the interaction of both SmMyb1 and SmMyb1Δ9 with an anthocyanin-related potato bHLH1 TF. Interestingly, a doubled amount of CGA was detected in both SmMyb1 and SmMyb1Δ9 agro-infiltrated leaves, thus suggesting that the N-terminal region of SmMyb1 is sufficient to activate its synthesis. These data suggest that a deletion of the C-terminal region of SmMyb1 does not limit its capability to regulate CGA accumulation, but impairs anthocyanin biosynthesis. To our knowledge, this is the first study reporting a functional elucidation of the role of the C-term conserved domain in MYB activator proteins.

Mapping Quantitative Trait Loci Affecting Biochemical and Morphological Fruit Properties in Eggplant (Solanum melongena L.)

Eggplant berries are a source of health-promoting metabolites including antioxidant and nutraceutical compounds, mainly anthocyanins and chlorogenic acid; however, they also contain some anti-nutritional compounds such as steroidal glycoalkaloids (SGA) and saponins, which are responsible for the bitter taste of the flesh and with potential toxic effects on humans. Up to now, Quantitative Trait Loci (QTL) for the metabolic content are far from being characterized in eggplant, thus hampering the application of breeding programs aimed at improving its fruit quality. Here we report on the identification of some QTL for the fruit metabolic content in an F2 intraspecific mapping population of 156 individuals, obtained by crossing the eggplant breeding lines “305E40” × “67/3.” The same population was previously employed for the development of a RAD-tag based linkage map and the identification of QTL associated to morphological and physiological traits. The mapping population was biochemically characterized for both fruit basic qualitative data, like dry matter, °Brix, sugars, and organic acids, as well as for health-related compounds such chlorogenic acid, (the main flesh monomeric phenol), the two peel anthocyanins [i.e., delphinidin-3-rutinoside (D3R) and delphinidin-3-(p- coumaroylrutinoside)-5-glucoside (nasunin)] and the two main steroidal glycoalkaloids, solasonine, and solamargine. For most of the traits, one major QTL (PVE ≥10%) was spotted and putative orthologies with other Solanaceae crops are discussed. The present results supply valuable information to eggplant breeders on the inheritance of key fruit quality traits, thus providing potential tools to assist future breeding programs.

Involvement of the Putative N-Acetylornithine Deacetylase from Arabidopsis thaliana in Flowering and Fruit Development

In eukaryotic cells, the non-proteinogenic amino acid ornithine is the precursor of arginine and polyamines (PAs). The final step of ornithine biosynthesis occurs in plants via a cyclic pathway catalyzed by N(2)-acetylornithine:N-acetylglutamate acetyltransferase (NAOGAcT). An alternative route for ornithine formation, the linear pathway, has been reported for enteric bacteria and a few other organisms; the acetyl group of N(2)-acetylornithine is released as acetate by N(2)-acetylornithine deacetylase (NAOD). NAOD activity has never been demonstrated in plants, although many putative NAOD-like genes have been identified. In this investigation, we examined the effect of down-regulation of the putative Arabidopsis thaliana NAOD gene by using AtNAOD-silenced (sil#17) and T-DNA insertional mutant (atnaod) plants. The ornithine content was consistently reduced in sil#17 and atnaod plants compared with wild-type plants, suggesting that in addition to NAOGAcT action, AtNAOD contributes to the regulation of ornithine levels in plant cells. Ornithine depletion was associated with altered levels of putrescine and spermine. Reduced AtNAOD expression resulted in alterations at the reproductive level, causing early flowering and impaired fruit setting. In this regard, the highest level of AtNAOD expression was observed in unfertilized ovules. Our findings suggest that AtNAOD acts as a positive regulator of fruit setting and agree with those obtained in tomato auxin-synthesizing parthenocarpic plants, where induction of SlNAOD was associated with the onset of ovary growth. Thus, here we have uncovered the first hints of the functions of AtNAOD by connecting its role in flower and fruit development with the regulation of ornithine and PA levels.