Alma Balestrazzi

In vitro culture and genetic engineering of Populus spp.: synergy for forest tree improvement

Populus species and hybrids are intensively cultivated as sources of woody biomass for the forest products industry and for reforestation of lowlands in temperate regions of the world. However, the long generation time of trees, the presence of seasonal dormancy and the prolonged period required for evaluation of mature traits are strong limitations for classical breeding and selection. The development of methods for in vitro culture and genetic engineering has increased the possibility of producing poplar genotypes improved in insect pest resistance, herbicide tolerance, growth rate and wood quality, or reduction in undesirable traits. Poplar has become a model system in forest tree biotechnology due to several useful features: small genome size, short rotation cycle, rapid growth rate and ease of vegetative propagation. The combination of molecular techniques and classical breeding will help create forest trees with positive effects on the environment. However, risks associated with the biotechnological applications (concerning the impact on biodiversity, long-term adaptation, transgene inheritance and stability) should be carefully evaluated and field tests performed with transgenic poplar.

Expression of the Stilbene Synthase (StSy) Gene from Grapevine in Transgenic White Poplar Results in High Accumulation of the Antioxidant Resveratrol Glucosides

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis- isomers of resvera trol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplaris a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 μg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.

Seed priming: state of the art and new perspectives

Priming applied to commercial seed lots is widely used by seed technologists to enhance seed vigour in terms of germination potential and increased stress tolerance. Priming can be also valuable to seed bank operators who need improved protocols of ex situ conservation of germplasm collections (crop and native species). Depending on plant species, seed morphology and physiology, different priming treatments can be applied, all of them triggering the so-called ‘pre-germinative metabolism’. This physiological process takes place during early seed imbibition and includes the seed repair response (activation of DNA repair pathways and antioxidant mechanisms), essential to preserve genome integrity, ensuring proper germination and seedling development. The review provides an overview of priming technology, describing the range of physical–chemical and biological treatments currently available. Optimised priming protocols can be designed using the ‘hydrotime concept’ analysis which provides the theoretical bases for assessing the relationship between water potential and germination rate. Despite the efforts so far reported to further improve seed priming, novel ideas and cutting-edge investigations need to be brought into this technological sector of agri-seed industry. Multidisciplinary translational research combining digital, bioinformatic and molecular tools will significantly contribute to expand the range of priming applications to other relevant commercial sectors, e.g. the native seed market.

Genotoxic stress and DNA repair in plants: emerging functions and tools for improving crop productivity

Crop productivity is strictly related to genome stability, an essential requisite for optimal plant growth/development. Genotoxic agents (e.g., chemical agents, radiations) can cause both chemical and structural damage to DNA. In some cases, they severely affect the integrity of plant genome by inducing base oxidation, which interferes with the basal processes of replication and transcription, eventually leading to cell death. The cell response to oxidative stress includes several DNA repair pathways, which are activated to remove the damaged bases and other lesions. Information concerning DNA repair in plants is still limited, although results from gene profiling and mutant analysis suggest possible differences in repair mechanisms between plants and other eukaryotes. The present review focuses on the base- and nucleotide excision repair (BER, NER) pathways, which operate according to the most common DNA repair rule (excision of damaged bases and replacement by the correct nucleotide), highlighting the most recent findings in plants. An update on DNA repair in organelles, chloroplasts and mitochondria is also provided. Finally, it is generally acknowledged that DNA repair plays a critical role during seed imbibition, preserving seed vigor. Despite this, only a limited number of studies, described here, dedicated to seeds are currently available.

Genetic transformation of Populus nigra by Agrobacterium tumefaciens.

Two clones of Populus nigra L. were tested in vivo and in vitro for their susceptibility to three different Agrobacterium tumefaciens wild-type strains evaluating number and size of resulting calluses. Strain C58 proved to be the most virulent.Various parameters affecting Agrobacterium-mediated transformation of P. nigra clones were further analyzed using ß-glucuronidase gene transient expression. The clone Jean Pourtet proved to be more susceptible than the clone San Giorgio. A. tumefaciens strain A281 pKIWI105 proved to be the most virulent. The optimal procedure involved dipping of leaf discs into a bacterial suspension (7×108 cells/ml) for 20 min, followed by a 48 h co-cultivation period on semi-solid regeneration medium.Leaf explants were co-cultivated with two disarmed A. tumefaciens strains. Plantlets of San Giorgio were regenerated, tested for ß-glucuronidase activity and rooted on selective medium containing kanamycin. Polymerase chain reaction analysis and Southern blot hybridization confirmed the integration of the neomycin phosphotransferase II gene into the poplar genome.

Transformation of elite white poplar (Populus alba L.) cv. ' Villafranca' and evaluation of herbicide resistance

Abstract Transgenic white poplar plants (Populus alba L.) expressing the nptII gene and the bar gene from Streptomyces hygroscopicus have been produced using Agrobacterium tumefaciens-mediated gene transfer. Eleven kanamycin-resistant plant lines were obtained with a transformation frequency of 7%. Successful genetic transformation was confirmed by Southern and northern analyses. The level of resistance to the commercial preparation of phosphinothricin (Basta; Roussel-Hoechst Agrovet) was evaluated by in vitro and in vivo assays. Using in vitro selective conditions for phosphinothricin, only plantlets from four kanamycin-resistant independent lines remained green and continued to grow and root. After transfer to the growth chamber, all selected transgenic lines were shown to be completely resistant to the herbicide Basta with doses equivalent to 6 l ha–1 (normal field dosage) and were tolerant at concentration of 12 l ha–1. This is the first report describing the genetic transformation of a P. alba clonal cultivar of commercial interest with a gene of agronomic value.

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.

Seed imbibition in Medicago truncatula Gaertn.: Expression profiles of DNA repair genes in relation to PEG-mediated stress.

The expression profiles of genes involved in DNA repair, namely MtTdp1 (tyrosyl-DNA phosphodiesterase), top1 (DNA topoisomerase I), MtTFIIS (transcription elongation factor II-S) and MtTFIIS-like, were evaluated in Medicago truncatula Gaertn. during seed imbibition carried out with the osmotic agent polyethylene glycol (PEG6000, 100g/L). The use of PEG6000 resulted in delayed water up-take by seeds, and reduced levels of oxidative DNA damage, measured in terms of 7,8-dihydro-8-oxoguanine (8-oxo-dG) were observed compared to seeds imbibed with water. The prolonged exposure to PEG6000 caused an increase in DNA oxidative damage; after 24h of treatment with the osmotic agent, the estimated amount of 8-oxo-dG was 1.25-fold higher compared to the value detected in seeds imbibed with water. Three days after imbibition, consistent cell damage and reactive oxygen species (ROS) production were also detected in radicles emerging from the PEG-treated seeds. All of the tested genes were known to be up-regulated during seed imbibition, with the highest transcript levels accumulating at approximately 8-12h of rehydration. Exposure to PEG6000 caused a delayed up-regulation of MtTdp1α and MtTdp1β genes, with transcript peaks occurring at 12-24h, when the highest levels of DNA damage were also recorded. For the top1, MtTFIIS and MtTFIIS-like genes, different expression profiles were observed in response to PEG6000. The possible roles of these genes in the repair response activated during seed imbibition are discussed.

The tyrosyl-DNA phosphodiesterase gene family in Medicago truncatula Gaertn.: bioinformatic investigation and expression profiles in response to copper- and PEG-mediated stress

The Tdp1 gene encoding tyrosyl-DNA phosphodiesterase has been extensively investigated in animal cells, due to the role of this enzyme in the repair of topoisomerase I-DNA covalent lesions. In contrast, information in this regard is totally missing in plants. We report for the first time in plants on the Tdp1 gene family from barrel medic (Medicago truncatula Gaertn.), composed of two members, hereby named MtTdp1α and MtTdp1β. The expression profiles of MtTdp1α and MtTdp1β genes were evaluated in plantlets grown in vitro using copper and polyethylene glycol (PEG 6000) as stress agents. In situ detection of reactive oxygen species (ROS) was carried out by histochemical staining, while the level of oxidative DNA damage, quantified in terms of 7,8-dihydro-8-oxoguanine (8-oxo-dG), increased up to 7.4- and 6.7-fold in response to copper and PEG 6000 treatments, respectively. Quantitative real-time polymerase chain reaction revealed that both Tdp1 genes were significantly up-regulated in response to copper and PEG. The Tdp1 genes were also significantly up-regulated during seed rehydration, an aspect of seed physiology in which DNA repair is a key component. Thus, the Tdp1 genes might be used as novel tools for improving stress tolerance in crops. The expression patterns of the barrel medic top1α and top1β genes, encoding distinct isoforms of DNA topoisomerase I, were also analyzed and discussed to acquire additional information on their specific functions, closely related to that of the Tdp1 gene in animal cells.

Enhanced triterpene saponin biosynthesis and root nodulation in transgenic barrel medic (Medicago truncatula Gaertn.) expressing a novel β‐amyrin synthase (AsOXA1) gene

Triterpene saponins are a group of bioactive compounds abundant in the genus Medicago, and have been studied extensively for their biological and pharmacological properties. In this article, we evaluated the effects of the ectopic expression of AsOXA1 cDNA from Aster sedifolius on the production of triterpene saponins in barrel medic (Medicago truncatula Gaertn.). AsOXA1 cDNA encodes beta-amyrin synthase, a key enzyme involved in triterpene saponin biosynthesis. One of the four transgenic lines expressing AsOXA1 accumulated significantly larger amounts of some triterpenic compounds in leaf and root than did control plants. In particular, the leaf exhibited significantly higher levels of bayogenin, medicagenic acid and zanhic acid. The amounts of medicagenic acid and zanhic acid, which represent the core of the M. truncatula leaf saponins, were 1.7 and 2.1 times higher, respectively, than the amounts extracted from the control line. In root, the production of bayogenin, hederagenin, soyasapogenol E and 2beta-hydroxyoleanolic acid was increased significantly. The increase in the total amounts of triterpenic compounds observed in the leaves of transgenic lines correlated with the AsOXA1 expression level. Interestingly, the plants expressing AsOXA1 showed, under different growth conditions, improved nodulation when compared with the control line. Nodulation enhancement was also accompanied by a significant change in the soyasapogenol B content. Our results indicate that the ectopic expression of AsOXA1 in barrel medic leads to a greater accumulation of triterpene saponins and enhanced root nodulation.