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The intensive application of agro-chemicals, and in particular of copper (Cu)-based compounds, causes increasing pollution of agricultural land, with serious risks for human health. Aromatic plants used for purposes other than food, can be considered for the exploitation and/or remediation of metal-polluted substrates, since contamination of the essential oils by these metals was not significant. Myrtle (Myrtus communis L.) is a Mediterranean evergreen shrub whose essential oil has many commercial applications. In this work, the effect of an excess of Cu in respect to control conditions was assessed on M. communis growth and foliar terpene composition. Metal accumulation in roots and shoots was also evaluated for the possible use of this species in phytoremediation. The amount of Cu applied in our experiments minimally affected the terpene profiles of in vitro grown plants, whereas no variations were detected in in vivo plants. The presence of the metal in the soil did not significantly impair plant growth, thus allowing its cultivation on polluted substrates. On the other hand, the amount of Cu in the plant was not enough to result in a significant reduction of Cu levels in the soil. Therefore, myrtle plants proved to be good candidates for the re-vegetation of Cu-contaminated lands.

ElecTIS is a new single container bioreactor which does not require forced air blowing, instead making the culture material mobile and the liquid medium stationary. The timed up-and-down movement of the basket containing the shoot culture ensures periodic contact with the liquid medium positioned at the base of the container. In this study we tested for the first time its use in the recovery of blackberry shoot cultures (Rubus fruticosus L., cvs Thornfree and Chester), coming from 5 months of slow growth storage (SGS), at 4 °C and in the dark. The shoot recovery at standard culture conditions was performed on two different types of ElecTIS, i.e., one with a smaller basket (ElecTISS, 234 cm2 of culture area), and one with a large basket (ElecTISL, 336 cm2), comparing the culture in TIS (cycle of 8 min every 6 h, equal to 32 min/day) with the traditional one in a gelled medium in glass jars (500 cc). After each one of the three 4-week subcultures, the shoot growth parameters and the relative growth rate highlighted a clear superiority of ElecTIS in promoting the recovery of shoot cultures coming from SGS. The analyses of chlorophyll content and stoma functionality confirmed the superior quality of shoots cultured in the ElecTIS bioreactor, and these shoots were afterwards easily rooted and acclimatized ex vitro.

This study aimed at determining the cadmium phytoextraction potential of three Populus alba L. clones cultivated in the presence of increasing sodium chloride concentrations. Plantlets of a commercial and two autochthonous poplar clones were grown in perlite with nutrient solution enriched in CdSO4 (50 and 100 μM) and NaCl (25 and 50 mM), administered either alone or in combination. The three clones showed significant variation not only in cadmium and salt tolerance, accumulation and content, but also in the effect of the interaction between the two elements on these parameters. The toxic effect of Cd and salt excess on plants was mutually exacerbated by the presence of both. Even though the outcome of the joint treatment was always a decrease in shoot Cd or Na accumulation, the three clones showed variation in the extent of such reduction. Evaluating the total element content per plant shoot, the fast-growing commercial clone displayed the highest phytoextraction potential for Cd and Na, either alone or in mixture. Our results demonstrated for the first time that the Cd response in presence of salt can vary in the different clones.

The environment contamination with different pollutants has become a worldwide problem and the phytoremediation techniques may offer interesting perspectives for counteracting this phenomenon. Many efforts are necessary to improve these techniques aimed to the restoration of contaminated soils and waters, and for this purpose, biotechnologies developed in the last decades are now fully available. Phytoremediation takes advantage of different biological processes involved in the accumulation, complexation, volatilization, and degradation of organic and inorganic pollutants. In recent years, these processes have been studied on a multitude of species and woody plants, in particular, display the most interesting perspectives for their possibility of accumulating high amounts of specific pollutants in their large biomass. in vitro cultures constitute a powerful tool for developing strategies in phytoremediation. Micropropagation and, more in general, cell and tissue culture can be directly exploited in phytoremediation. For research purposes, these techniques offer the advantage of investigating the plant (or plant cells and organs) behaviour under controlled conditions, in comparison with whole plant experiments under field conditions. Under axenic conditions, we can also distinguish the response of plants from those of microorganisms present in the rhizosphere. The attention must be focused also on the rhizosphere and the complex interactions between plants and microbial communities that may play a relevant role in phytoremediation. These subjects have been largely investigated and a huge literature can be found, so our analysis was mainly addressed towards the field of heavy metals contamination and the use of woody plants for remediation. In the second part, the wide subject of genetic engineering is illustrated; the use of genetically modified plants, in fact, is of great usefulness for the understanding of the metabolic processes involved in the mechanisms of pollutants uptake, sequestration and translocation. Special attention is focused on reactive oxygen species, nitric oxide, and phytohormones, which are signaling molecules modulating plant responses to pollutants stress mainly through differentially expressed genes and the antioxidative system activation. In this context, several genes have been functionally characterized and transformed to target plants for enhancing their phytoremediation efficiency. Consequently, whenever possible, and taking into account any possible risk linked to the use of modified organisms, the use of plants overexpressing genes involved in these phenomena is a promising method for improving the efficacy of phytoremediation.

This work investigated if the assessment of tolerance to trace metals can depend on plant density in the experimental design. A non-metallicolous and a metallicolous populations of Silene paradoxa were hydroponically cultivated at increasing density and in both the absence (−Cu conditions) and excess of copper (+Cu conditions). In −Cu conditions, the metallicolous population showed a lower susceptibility to plant density in comparison to the non-metallicolous one, explained by a higher capacity of the metallicolous population to exploit resources. In +Cu conditions, an alleviating effect of increasing density was found in roots. Such effect was present to a greater extent in the non-metallicolous population, thus making the populations equally copper-tolerant at the highest density used. In shoots, an additive effect of increasing plant density to copper toxicity was reported. Its higher intensity in the metallicolous population reverted the copper tolerance rela- tionship at the highest plant densities used. In both populations, a density-induced decrease in root copper accumulation was observed, thus concurring to the reported mitigation in +Cu conditions. Our work revealed the importance of density studies on the optimization of eco-toxicological bio-assays and of metal tolerance assessment and it can be considered the first example of an alleviating effect of increasing plant number on copper stress in a metallophyte.

Slow growth storage has been achieved for Castanea sativa (cv. ' Montemarano ' ) shoot cultures over a duration of 48 mo at a temperature of 8°C, where 82% of explants survived and were able to resume normal growth after transfer to standard culture conditions at 23°C. The evaluation of the chlorophyll content of leaves also showed no differences between material stored for 48 mo and control material subcultured at 23°C. With a storage temperature of 4°C, the survival of shoots was significantly lower at approximately 56% after 12 mo, and no plants recovered after 24-mo storage. The presence of 6-benzyladenine 0.44 ? M in the culture medium proved to be necessary for the recovery of healthy shoots, while pre-treatments with different concentrations of abscisic acid did not significantly influence the survival of shoots following storage conditions. A low level of light during slow growth storage resulted in positive effects on the rate of shoot survival over the longest preservation periods

Embryogenic cultures of horse chestnut (Aesculus hippocastanum L.) can be obtained from different organs and tissues. We describe here the induction from stamen filaments and the procedures applied for the successive phases of somatic embryo development and maturation. Embryogenic tissues are obtained on Murashige and Skoog medium containing 9.0 μM 2,4-dichlorophenoxyacetic acid. Somatic embryos develop after transfer to hormone-free medium enriched with glutamine. Maturation and germination of isolated embryos are achieved by transfer to medium containing polyethylene glycol 4000 and activated charcoal, successive desiccation treatment, and cold storage at 4 °C for 8 weeks.

Embryogenic cultures of horse chestnut (Aesculus hippocastanum L.) can be obtained from different organs and tissues. We describe here the induction from stamen filaments and the procedures applied for the successive phases of somatic embryo development and maturation. Embryogenic tissues are obtained on Murashige and Skoog medium containing 9.0 μM 2,4-dichlorophenoxyacetic acid. Somatic embryos develop after transfer to hormone-free medium enriched with glutamine. Maturation and germination of isolated embryos are achieved by transfer to medium containing polyethylene glycol 4000 and activated charcoal, successive desiccation treatment, and cold storage at 4 °C for 8 weeks.

Embryogenic tissues obtained from stamen filament cultures of horse chestnut (Aesculum hippocastanum L.) were cultured on maturation media supplemented with different combinations of abscisic acid, polyethylene glycol 4000, mannitol or activated charcoal. Somatic embryos were subjected to different desiccation procedures after a culture period on maturation media. After a slow desiccation, obtained by placing the somatic embryos in empty and non-sealed Petri dishes under the laminar air flow for 48 h, an increase in viability, shoot elongation and conversion was observed for the embryos previously cultured on medium enriched with ABA (80 μM) alone or plus PEG (50 g l−1).

Biochar (BC) is a carbon rich product resulting from the biomass pyrolysis process and there have been no reports until now on BC effects in tissue cultures as a suitable substitute for activated charcoal (AC). The results of an experiment on two clones of white poplar (Populus alba L.) grown in culture media with different amounts of BC (0, 0.5 and 1.5 g/dm(3)) showed that its addition did not damage the plants and there were no significant differences comparing the data obtained for the same concentrations of AC. Both BC and AC addition was shown to increase root dry biomass and number of roots per shoot and these effects appeared to be independent of genotype and concentrations of the added products. A greater elongation was also recorded for shoots grown on a substrate containing BC than those grown on media without BC. These effects did not seem to be caused by darkening due to the addition of BC as there are no significant differences between the temperatures of the different culture media, but are probably due to the adsorption of molecules such as ethylene. Indeed, during the experiment, the hormone concentration in the atmosphere was lower in vials containing the media with BC than the BC-free ones after 14 and 21 days: the lower amount of ethylene in the medium with BC could explain the difference in shoot elongation and the abundant root biomass since high ethylene concentration could inhibit organogenesis.

Phytoremediation is a developing strategy for the restoration of contaminated soils and waters. Many efforts are devoted to the development of this technology and biotechnologies are now available for enlarging the possibilities of its exploitation. In vitro cultures constitute, in particular, a powerful tool and in this chapter their role in phytoremediation is extensively explored. The role of cell, tissue culture and micropropagation in the understanding of the processes related to the uptake of pollutants by plants and the direct use for phytoremediation purposes of plants multiplied by in vitro cultures has been examined. In a second paragraph, the attention has been focused on the interactions between plants and microbial communities in the rhizosphere that play a relevant role for applications in phytoremediation and whose investigations are mostly based on in vitro technologies. In the final paragraph, the huge possibilities opened by genetic engineering are illustrated, pointing out how, in genetically modified plants, the overexpression of genes involved in metabolism, uptake, or transport of specific pollutants represents a promising method for enhancing the effectiveness of phytoremediation.

Clonal propagation ofPinus pinea L. was achieved by organogenesis on cotyledon explants and the influence of several factors on adventitious bud production and development was investigated. Gupta and Durzan (DCR) medium with benzyladenine (5 µM) induced higher bud production. Bud development and shoot elongation required subcultures on medium with activated charcoal. Rooting was obtained after 10 d culture on medium containing IBA (10 µM).

Induction of somatic embryogenesis is described in common ash (Fraxinus excelsior L.). Embryogenic tissues are obtained from immature zygotic embryos and cultured on a modified Murashige and Skoog (MS) medium containing 8.8 μM 2,4-dichlorophenoxyacetic acid and 4.4 μM benzyl-adenine. Embryogenic tissue is subcultured and multiplied on medium supplemented with reduced concentration of plant growth hormones. Somatic embryos develop and mature by transfer to hormone-free medium and subsequent culture on medium containing low amount of benzyladenine. Somatic embryo germination and conversion are enhanced by cold storage at 4°C and successive transfer onto Woody Plant Medium (WPM). Fully developed plantlets are then transferred to pots and acclimatized in the greenhouse equipped with a mist system.

Induction of somatic embryogenesis is described in common ash (Fraxinus excelsior L.). Embryogenic tissues are obtained from immature zygotic embryos and cultured on a modified Murashige and Skoog (MS) medium containing 8.8 μM 2,4-dichlorophenoxyacetic acid and 4.4 μM benzyl-adenine. Embryogenic tissue is subcultured and multiplied on medium supplemented with reduced concentration of plant growth hormones. Somatic embryos develop and mature by transfer to hormone-free medium and subsequent culture on medium containing low amount of benzyladenine. Somatic embryo germination and conversion are enhanced by cold storage at 4°C and successive transfer onto Woody Plant Medium (WPM). Fully developed plantlets are then transferred to pots and acclimatized in the greenhouse equipped with a mist system.

This study evaluated the survival and recovery of non-encapsulated and encapsulated shoots of Sequoia sempervirens after storage at 4 °C in the dark for up to 15 months on four different culture media. Survival and regrowth of encapsulated shoots declined within 3 months, regardless of the storage medium composition. By contrast, no significant decrease in survival and regrowth was noted with non-encapsulated shoots after 12 months of storage on Quoirin and Lepoivre medium supplemented, or not, with 1 mg l −1 benzyladenine. Regrowth dropped to 60–61% after 15 months of storage on the same media. Medium-term conservation of S. sempervirens germplasm is therefore possible using in vitro storage of non-encapsulated shoot cultures.

Purpose. This work was planned for providing a useful screening tool for the selection of Populus alba clones suitable for phytoremediation techniques. To this aim, we investigated variation in arsenic, cadmium, copper and zinc tolerance, accumulation and translocation in three poplar clones through an in vitro screening. Poplars have been widely proposed for phytoremediation, as they are adaptable to grow on contaminated areas and able to accumulate metals. The investigation of possible differences among poplar clones in metal tolerance and accumulation deserves to be deeply studied and exploited for the selection of the more suitable tool for phytoremediation purposes. Methods. In vitro multiplied microshoots of a commercial and two autochthonous Populus alba clones were subcultured on hormone-free WPM medium for one month and then transferred for two weeks onto media containing different concentrations of the metals investigated. At the end of the treatments, plantlets were sampled, weighed and mineralised by wet ashing. Metal concentrations were determined by ICP-OES. Results. For the metal concentration used in the experiments, our clones of Populus alba showed variation in metal tolerance, metal accumulation and content. The fast growing commercial clone, even if rarely showing the highest plant metal concentration, displayed the highest metal content, suggesting biomass production as the key factor in evaluating the phytoextraction capacity of Populus alba clones for the metals studied. Conclusions. Data demonstrated that in vitro screening of cuttings represents a valuable way of assessing the ability of different poplar clones, to take up, tolerate and survive metal stress.

An effective procedure for the cryopreservation of horse chestnut (Aesculus hippocastanum L.) embryogenic callus by vitrification/one-step freezing is described here. In particular, the study focused on the possibility of recovering the full proliferation potential of the embryogenic lines after storage in liquid nitrogen. The developmental stage of the embryogenic lines was shown to play an important role. Ninety-min incubation in PVS2 and preservation at -196 degrees C of callus samples, containing a prevalence of embryogenic masses at an advanced stage of somatic embryo maturation (i.e., the torpedo stage), gave optimum regrowth of healthy and proliferating embryogenic callus. Moreover, raising the thawing temperature to 45 degrees C yielded the maximum survival (94%) of torpedo-stage embryogenic samples, recovery of proliferation and, in more than 70% of cases, maturation to the cotyledonary stage. This study opens the way to the possibility of safe, long-term storage in liquid nitrogen of valuable embryogenic lines of horse chestnut, avoiding repeated subculturing.