A. Balestrazzi’s research while affiliated with University of Pavia and other places

Seed longevity is a complex trait that depends on numerous factors. It varies among species and populations, and within different seed morphs produced by the same plant. Little is known about variation in longevity in different seed morphs or the physiological and molecular basis of these differences. We evaluated the longevity and oxidative stress status in heteromorphic seeds aged in two different storage conditions. We compared controlled ageing tests (seed storage at 45°C and 60% relative humidity; a method of accelerated ageing used to estimate longevity in genebank conditions) with storage in a genebank for up to 40 years (−18°C and 8% seed moisture content). We employed as study species two wild wheats characterized by seed heteromorphism: Aegilops tauschii and Triticum monococcum subsp. aegilopoides . We estimated the ROS content and the expression of genes coding for enzymes related to the H 2 O 2 scavenging pathway. Results confirmed that seed longevity varies between different seed morphs. Different storage environments resulted in different longevity and survival curves. ROS levels, even if with variable patterns, were higher in several aged seed lots. We observed consistency in the expression of two genes ( GSR and CAT ) related to ROS scavenging in the late phase of pre‐germinative metabolism. Differences in seed longevity between morphs were observed for the first time under genebank conditions. Our results suggest also that controlled ageing tests should be used with caution to infer ranks of longevity under cold storage.

The link between MtTdp1α (tyrosyl-DNA phosphodiesterase) gene, involved in the repair of DNA topoisomerase I mediated DNA damage, and the plant defense response has been investigated in MtTdp1α-depleted Medicago truncatula transgenic lines obtained by intron-spliced hairpin RNA approach, compared to control line (CTRL, empty vector). Reduction of cuticle permeability highlighted by chlorophyll efflux assays positively correlated with the level of MtTdp1α gene silencing. The increased cuticle thickness was confirmed by transmission electron microscopy which revealed an apparent expansion of the epicuticular waxes deposited on the outer surface. RNA-Seq analysis, carried out in the MtTdp1α-depleted plants, revealed the different expression of resistance (R) genes, PAMP (pathogen-associatedmolecular pattern) triggered immunity (PTI) genes and transcription factors (TFs) involved in the regulation of the plant defense response.

Breeders are always looking for new and novel cultivars to be competitive in the flower market. Low seed germination rate is still a major problem encountered in traditional rose breeding programs. Mutagenic agents, such as ionizing radiation, may be used to enhance seed vigor and increase the productivity, improving sprouting and emergence of buds that are carried out through seed coating. The effects of ionizing radiation on seed vigor are in most cases genotype-dependent, thus irradiation treatments need to be optimised for each different cultivar. In the present study, gamma rays (0, 50, 100 and 200 Gy) were applied on hybrid tea rose seeds in order to set up a radiation protocol for increasing seed germination. Seeds coming from six different croßes of Rosa hybrida commercial cultivars were used in order to gain information on radio-tolerance and germination ability.

The latent presence of bacteria in the plant tissues can cause severe economic damage due to their sudden appearance in the medium, generally related to stress. Chemicals and antibiotics have been used to control contamination. Extracts from the leaves of different Salvia species were found to have a general antibacterial activity towards several human and plant pathogens. The extract of Salvia somalensis containing high levels of carnosic acid was tested as agent against bacterial contamination in micropropagation. Contaminants of in vitro multiplied clones of Zanthedeschia aethiopica, Ranunculus hyb. and Paeonia hyb. were isolated. Purified bacterial lines were identified based on metabolic or molecular tests. Bacterial suspensions were treated with increasing concentrations of the sage extract (CA 50, 100, 200, 400 mg/L) to analyze bacteriostatic and bactericide activity. The effect of compounds on morphogenic parameters of in vitro grown Myrtus communis 'tarentina', Dianthus caryophyllus, Solidago virgaurea and Zanthedeschia aethiopica was tested to determine toxicity levels. The cultures showed increased senescence and root growth inhibition proportional to the increase of carnosic acid content in the medium.

In the present work, Petunia × hybrida leaf discs maintained on regeneration medium for 8 days were used to assess the effects of genotoxic stress induced by in vitro culture. The investigation was carried out by comparing the response of intact leaves excised from Petunia × hybrida plantlets grown in vitro and the regenerating leaf discs. In situ detection by histochemical staining and alkaline-Single Cell Gel Electrophoresis (SCGE) analysis demonstrated that both reactive oxygen species accumulation and DNA damage were enhanced in explants cultured in vitro. Significant up-regulation of the PhOGG1 (8-oxoguanine DNA glycosylase/lyase), PhAPX (ascorbate peroxidase) and PhMT2 (metallothionein) genes involved in DNA repair and antioxidant defence was observed in the explants cultured in vitro, compared to intact leaves. The Petunia × hybrida leaf discs were exposed to increasing (0, 100, 150, 200 and 400 mM) doses of the model genotoxic agent hydrogen peroxide (H2O2) and then analysed. The DNA diffusion assay highlighted the dose- and time-dependent fluctuations of programmed cell death/necrosis events in response to H2O2. Leaf discs treated with increasing H2O2 concentration and untreated controls were analysed by FPG-SCGE to assess the level of oxidative DNA damage at different time points following treatments. The PhOGG1, PhAPX and PhMT2 genes were significantly up-regulated in response to H2O2, reaching the highest transcript levels with the 150 mM dose. Based on the reported data, these genes might be used as molecular indicators of the genotoxic stress response in Petunia × hybrida cells.

Background and AimsThe germination test currently represents the most used method to assess seed viability in germplasm banks, despite the difficulties caused by the occurrence of seed dormancy. Furthermore, seed longevity can vary considerably across species and populations from different environments, and studies related to the eco-physiological processes underlying such variations are still limited in their depth. The aim of the present work was the identification of reliable molecular markers that might help in monitoring seed deterioration.Methods Dry seeds were subjected to artificial ageing and collected at different time points for molecular/biochemical analyses. DNA damage was measured using the RAPD (random amplified polymorphic DNA) approach while the seed antioxidant profile was obtained using both the DPPH (1,1-diphenyl, 2-picrylhydrazyl) assay and the Folin-Ciocalteu reagent method. Electron paramagnetic resonance (EPR) provided profiles of free radicals. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to assess the expression profiles of the antioxidant genes MT2 (type 2 metallothionein) and SOD (superoxide dismutase). A modified QRT-PCR protocol was used to determine telomere length.Key ResultsThe RAPD profiles highlighted different capacities of the two Silene species to overcome DNA damage induced by artificial ageing. The antioxidant profiles of dry and rehydrated seeds revealed that the high-altitude taxon Silene acaulis was characterized by a lower antioxidant specific activity. Significant upregulation of the MT2 and SOD genes was observed only in the rehydrated seeds of the low-altitude species. Rehydration resulted in telomere lengthening in both Silene species.Conclusions Different seed viability markers have been selected for plant species showing inherent variation of seed longevity. RAPD analysis, quantification of redox activity of non-enzymatic antioxidant compounds and gene expression profiling provide deeper insights to study seed viability during storage. Telomere lengthening is a promising tool to discriminate between short- and long-lived species.

Ornamental crops are routinely used in breeding programs aimed at obtaining new gene combinations. The search for novel genotypes, highly tolerant to environmental stresses, is a relevant goal for ornamental breeders. The availability of marker genes, used as indicators of the plant stress response in mutational breeding, might assist in the selection of new valuable genotypes. The Tdp1 gene encoding tyrosyl-DNA phosphodiesterase has been extensively investigated in animal cells, due to the role of this DNA repair enzyme in the protection against oxidative DNA damage. However limited information on its function are currently available in plants. Within this context, the Tdp1 gene and other DNA repair genes might be useful tools in defining novel breeding approaches. The search for the PhTdp1α and PhTdp1β cDNAs, encoding the TDP1α and β isoforms from Petunia hybrida, was carried out by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), using degenerated oligonucleotides, designed from the available plant Tdp1α and Tdp1β amino acid sequences. A detailed bioinformatic investigation of the different Petunia cDNA fragments is currently in progress while the response of Petunia cells, in terms of Reactive Oxygen Species accumulation and antioxidant activity, has been assessed.

An experimental trial was established in greenhouse with transgenic white poplars from an elite genotype (Populus alba L. cv “Villafranca”) expressing the bar gene for tolerance to Basta® herbicide. The steady-state level of bar mRNA was evaluated in different seasons, over a two-year period. Notwithstanding the presence of the 35SCaMV promoter, significant fluctuations in the amount of the bar transcript in relation to season and organ position were observed. The highest expression levels were reached in apical leaves in summer, while a consistent drop in transgene expression occurred at the onset of dormancy and in winter buds. A similar expression pattern was evidenced for the poUBI gene used as endogenous marker. The genetically modified poplars were tolerant to intense and repeated glufosinate ammonium treatments, maintaining an intact shoot sprouting and a good height increment.