The transmission of transgenes (uid-A and nptII) was studied in crosses between transgenic plants, with Fragaria x ananassa cultivar Teodora as the donor plant and various Fragaria species as recipient plants. GUS expression and kanamycin resistance were evaluated in embryos and in seedlings after aseptical germination, both in intra- and inter-specific crosses as demonstrated by different ploidy level. This expression in most of the T-1 seedlings shows that one or more functional transgenes were transferred from T-0 plants to T-1 seedlings. These results, obtained in a greenhouse, confirm the possibility of genetic exchange both at the intra- and inter-specific level, though the inter-specific hybrids seem to have reduced germination ability. These observations need to be further investigated.

During the production and assessment of transgenic plants resistant to quarantine viruses, the need to contain genetically modified plants (GMPs) and pathogens severely limits working options. Moreover, in the case of fruit trees, acclimatisation and viral inoculation are very time-consuming, thus a quick and safe method to assess the resistance to quarantine viruses, such as Plum pox virus (PPV), is desirable. This article focuses on the production of transgenic plums together with a contained and rapid evaluation in vitro for PPV resistance. The plum Stanley was transformed by Rhizobium radiobacter (syn. Agrobacterium tumefaciens) using a PPV-M derived hairpin construct (h-UTR/P1) that had previously been shown to confer high and broad-spectrum PPV resistance in model plant. Two transgenic clones, St24 and St28, were obtained. To assess their ability to resist PPV infection, micropropagated shoots of the transgenic clones were micrografted in vitro onto PPV-D infected GF305 rootstock. Following successful grafting, the transgenic scions were analysed by immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) for PPV detection. A total of 97% (47/48) of St24 and 73% (17/23) of St28 tested plants were resistant to the heterologous strain of PPV. In line with an RNA silencing mediated resistance mechanism, the St24 clone was shown to accumulate higher concentration of PPV UTR/P1-specific small interfering RNAs (siRNAs) than St28 one. The results are of practical interest not only developing plum clones that are highly resistant to PPV, but also for setting up quick and contained inoculation test procedure.

Pomegranates contain phenolic compounds with antioxidative and putative antitumoral effects either in fruits and vegetative tissues. The aim of this study was to compare the potential for in vitro multiplication and rooting of 6 genotypes,1 italian cultivar and a wild genotype of pomegranate (Punica granatum L.) and to evaluate content in total phenols and ellagic acid in their in vitro grown shoots. The multiplication rates ranged from 2 to 5 and the rooting percentage from 55% to 85%, in relation to the genotype. The ellagic acid and total phenol content was shown to be genotype dependent as well as. This preliminary characterisation gave information for the choice of the most suitable genotypes to be used for future application in production of bioactive compound from in vitro shoot cultures in this species

In recent years our laboratory has worked on transformation of Prunus species mediated by Agrobacterium tumefaciens in order to obtain plant resistance to sharka disease. In plum, kanamycin selection applied either immediately or with a one-month delay both produced clones with stably integrated transgenes (Damiano et al., 2007a; Ilardi et al., 2007). These clones, derived from hypocotyl sections of seeds of 'Stanley' and rootstock 'St. Julien', infected with strain EHA101 carrying the plasmid pGA482GG/ PPV-CP-33, after three years of in vitro culture, contain the NPTII, GUS and PPV coat protein genes, as confirmed by PCR. Some of the transgenic clones obtained in 2001, derived from 'Stanley', grown in the greenhouse since 2003, express histochemical GUS reaction in the floral organs and pollen grains. In apricot, 'Boccuccia Spinosa' and 'Monaco Bello', antibiotic selection was applied during the multiplication phase due to the high sensitivity of hypocotyl sections and embryo apices to kanamycin (Damiano et al., 2007b). PCR initially confirmed integration of transgenes in some clones, but after six months NPTII, GUS and PPV coat protein genes were no longer detected. Difficulties have been encountered in regeneration of peach: thus far no transformation events have occurred. These results suggest that species and antibiotic selection affect the efficiency of transformation and the stability of the transgenic plants. Regarding the use of the reporter gene, though generally unaccepted for the field release, it could be a useful tool for monitoring pollen spread. Concerning virus resistance strategy, the full coat protein viral gene was integrated in the transgenic lines, but the aim will be to induce resistance by RNA interference, preventing exogenous protein expression.

Shoot apices of in vitro-grown plantlets of white mulberry, Morus alba L. cv Florio, were cryopreserved using either encapsulation-dehydration or vitrification. For encapsulation-dehydration, alginate beads containing apices were dehydrated for 1, 3, 5 or 7 days in a liquid medium containing various sucrose concentrations (0.5, 0.75, 1.0 or 1.25 M). Bead desiccation was performed using silica gel for either 0, 4, 6, 8, 9 or 14 h. For vitrification, apices were directly immersed for either 5, 15, 30 or 60 min in a vitrification solution (PVS2). Following encapsulation-dehydration, treatment of alginate beads with 0.75 M sucrose was more effective in promoting re-growth of explants after immersion in liquid nitrogen than in the presence of 0.5 M sucrose for either 1 or 3 days. Re-growth of explants was also observed following vitrification and this reached 47% with increasing duration of the PVS2 treatment from 5 to 30 min. Overall, the highest frequency of explant re-growth was obtained when explants were subjected to encapsulation-dehydration in the presence of 0.75 M along with a 3 day sucrose dehydration pre-treatment and followed by desiccation for 9 h in silica gel.

Berry species are important sources of polyphenols with antioxidant properties and great attention has been focused on those compounds for their pharmacological potential. The main focus of this study was to determine the in vitro conditions affecting anthocyanin concentration in cell suspension cultures of strawberry (Fragaria x ananassa Duch.), blackberry (Rubus fruticosus L.), red raspberry (Rubus idaeus L.) and strawberry-tree (Arbutus unedo L.). We used as elicitors: temperature, light (blue or red spectra) and changes of medium composition, i.e., increased nitrogen concentration. Anthocyanin production was significantly increased only by elicitation with light, either blue or red spectra, in blackberry and strawberry-tree cultures, and particularly red light in strawberry. In red raspberry cultures anthocyanin accumulation was not affected by any elicitor.

Shoot tips of peach cultivars 'Summer Grand', 'San Giorgio' and 'Babygold 6' were cryopreserved by encapsulation dehydration. Tips were pre-cultured for 1, 3, 5 or 7 days in liquid medium with increasing sucrose concentrations (0.5, 0.75, 1.0 or 1.25 M). Desiccation was performed with silica gel for 0, 4, 6, 9, 14, 20 or 24 hours. The highest regrowth, from 33 to 46% depending on the cultivar, was observed with a pre-culture of encapsulated tips for 3 days on 0.5 M sucrose enriched liquid medium and a desiccation of beads in silica gel for 9 hours prior to immersion in liquid nitrogen.

The CRA-Fruit Tree Research Centre (CRA-FRU) of Rome is the repository of the in vivo national collection of the fruit tree germplasm with about 8.000 genotypes. Cryopreservation of in vitro material, by reducing costs and the risks associated with the loss of genotypes in the in vivo collections, could be a valuable complementary tool for germoplasm conservation. Programs for the application of this strategy were developed at the CRA-FRU and this paper provides an overview of our results using encapsulation-dehydration of fruit species.

Changes in metabolism and protein expression were analysed during cryopreservation of the ancient apple variety Annurca. Our experiments concerned transglutaminase activity, polyamine levels and protein expression associated with shoot tip dehydration. Cryopreserved shoot tips displayed 72% regrowth after treatment in liquid medium with 0.75 M sucrose for 1 day followed by dehydration to 19% moisture content (fresh weight basis). After dehydration, the concentration of polyamines putrescine and spermidine decreased compared with untreated controls, while spermine concentration remained unaffected. Transglutaminase activity was slightly reduced in treated samples, while post-thaw regrowth enzyme activity approached control values. We also detected significant changes in protein expression profiles and identified six proteins related with stress response or involved in the slowing down of the cell cycle. The relationship between biochemical parameters, protein synthesis and cryotolerance is discussed.

Temporary immersion techniques for plant in vitro culture, avoiding the time-consuming sub-culturing necessary with the use of solid media have been applied to various species in order to increase the multiplication rate and to automate as much as possible the plant production. In this work in vitro grown shoots of Italian cultivars 'Montebello' and 'Tonda Gentile Romana' of Corylus avellana L., were cultured on a solid medium or temporary immersion system using a basal culture medium, previously defined. Different carbon sources (sucrose or glucose) and durations of immersion (30, 60 or 120 minutes of immersion per day) were applied to determine the best cultural conditions for shoot proliferation. The temporary immersion system enhanced shoot multiplication and gave no hyperhidric shoots. Shoots obtained with this system were induced to root by immersion for one day in 80 mg.L-1 IBA solution and root expression was performed either in gel rite, agar and/or vermiculite. The most effective treatment in terms of rooting (100% and 80% in 'Montebello' and 'Tonda Romana', respectively) and acclimatisation (up to 100% in 'Montebello') was shown to be the combination of vermiculite with agarised medium. Hazelnut showed a good aptitude to be in vitro multiplied with the temporary immersion system and further studies are now in progress to optimise the protocol in view of a possible wider application of temporary immersion systems (TIS) to this species.

The rootstock 'Citation '['Zaipime'] (Prunus salicina x P. persica) was obtained in USA by F. Zaiger. At present, in Italy, it is under evaluation as rootstock of plum and apricot, while it is no longer recommended for peach, because of the numerous cases of graft incompatibility. In the present work different aspects of micropropagation and correspondent expressed protein profiles are presented. The plants were multiplied onto Murashige and Skoog (1962) basal medium with different combinations of BA, GA(3), NAA. Basal salts, IBA, activated charcoal, explants source and different concentrations of zinc sulphate heptahydrate were used to test their influence on rooting. Plants resulting from different culturing conditions showed a variability in multiplication rate and in rooting aptitude, ranging from 100% of the best rooting percentage to almost 0% of the controls (auxins-free). After 40 days the best acclimatization percentage (100%) was obtained with the rooting media MS macro salt half strength, MS micro elements full strength and IBA (0.5 mg/L), without activated charcoal. Plants rooted on the same medium, but with activated charcoal, showed less survival percentages better growth and development. To obtain a better knowledge of biology of the micropropagation steps, a proteomic approach has been undertaken. Total proteins content extracted from in vitro propagated material at different stages was separated through a 2-DE. Protein profiles were analyzed with Image Master software in order to identify differences between samples. The analysis carried out, so far, prove the existence of modification in protein pattern and concentration during micropropagation stages. These results may also represent some groundwork to the comprehension of graft incompatibility with peach.

In vitro propagated shoots, obtained from axillary buds were treated, to induce rooting, with 2 mg L-1 IBA for all the rooting phase, quick immersion of the basal part of microcuttings in 2 g L-1 IBA solution for 1 or 10 min and transferring to a PGR-free agarised medium; an immersion of basal parts in 20 g L-1 sucrose solution containing increasing concentrations (0-100 mg L-1) of IBA for 1 or 5 days and transferring to a PGR-free agarised medium or an immersion of basal parts of the microcuttings in 20 g L-1 sucrose solution containing 80 mg L-1 IBA for 1 day and transferring to a PGR-free medium with or without agar and with or without vermiculite. The best response in terms of rooting and acclimatisation was obtained with an induction in 80 mg L-1 IBA solution applied for 1 day and a transferring to a liquid medium with vermiculite. This protocol allows to obtain 80% of rooted microcuttings and a 98% of survival after acclimatisation.

Shoot tips of Pyrus pyraster were successfully cryopreserved by encapsulation-dehydration. Na--alginate beads each containing one shoot tip, dehydrated for 2 days in 0.75M sucrose and desiccated to 20% moisture content (fresh weight basis), gave 60% recovery after exposure to liquid nitrogen. Regenerated shoots showed no differences in length and leaf shape compared to the mother plant. Multiplication rate and rooting ability of cryopreserved shoots were lower than those of untreated controls after one subculture, but were completely restored following the third subculture. Fifteen cryopreserved lines derived from single buds were used for genetic analyses by RAPDs and SSRs, in comparison with the mother plant. In RAPD analysis, of a total of 24 primers, only 15 showed reproducible and well resolved bands and were further used. These primers produced a total of 66 fragments ranging from about 500 to 2500 base pair size. SSR (microsatellite) marker amplification was performed using 19 primers which produced 57 reproducible fragments. Microsatellites fragments ranged from 60 to 600 base pairs. Both RAPDs and SSRs did not reveal any polymorphism between cryopreserved lines and the original genotype, suggesting that cryopreservation, using encapsulation-dehydration, does not affect genetic stability of wild pear.;

The development of valid micropropagation method for Diospyros kaki ` Kaki Tipo', the most important persimmon cultivar in Italy, would overcome difficulties related to its in vivo propagation performed by graft on rootstocks obtained from seeds. This method gives problems related to high variability of rootstocks and sometimes, lack of graft affinity. Due to this and for rooting difficulty of cuttings, it could be interesting to use in vitro self-rooted plants. This work investigates the changes of in vitro ` Kaki Tipo' shoots rooting ability in the presence of the same basal medium but different auxins (IAA, IBA, NAA) and with or without induction period ( dark incubation for initial 10 days of treatments). After 40 days, the rooting percentage, number of roots per rooted plants and length of roots were measured. After 21 days from the beginning of rooting treatments, another stock of plants were subjected to proteins extraction and separation. The best rooting results were obtained with IBA and dark treatment. Moreover, there is no rooting without auxins, and dark treatment, excepted for NAA, enhances rooting parameters. Protein analysis of plants determined that NAA and dark condition confers the highest biosynthetic activity.