Soil salinization is a global problem limiting growth and economical yield in fruit species including walnut. Knowledge of the response of varieties and cultivars to salinity can represent an important contribution to future walnut plantation in areas affected by soil salinization. Juglans regia 'Sorrento' is a commercially important Italian variety. The present work aimed to explore the morpho-physiological response to NaCl-induced stress in 'Sorrento' plants using clonally propagated ex vitro plantlets to reduce the impact of genetic variation. Ex vitro acclimatized plants were exposed to salinity conditions by irrigating pot soil with half-strength Hoagland solution containing 100 mM NaCl for 20 days. Salt-induced effects on plant growth, chlorophyll and carotenoid content, osmolality, membrane injury and on activities of antioxidant enzymes catalase and guaiacol peroxidase (G-POD) in leaves were evaluated. Morphological and growth parameters were not significantly altered by salt stress with respect to the control. Slight differences in leaf necrosis and absence of injury in the membranes, stable content of photosynthetic pigments, and an increase in G-POD activity were also found in salt-treated plants. These results collectively suggest that 'Sorrento' has potential for the cultivation in salinized environments.

Cryopreservation by droplet vitrification was applied to hazelnut (Corylus avellana L.). axillary buds of the Italian cultivated variety Tonda Gentile Romana, which were collected from in vitro growing shoots, immersed in ice cooled PVS2 or PVS3 for 60 or 90 min, then transferred to a droplet of vitrification solution, placed on a strip of aluminium foil, and plunged into liquid nitrogen (LN). Additionally, the effect on the recovery of the mother plant after cryopreservation was evaluated, following a cold pre-treatment at 4 °C for 3 months. The highest regrowth percentage (56.7%) was obtained after applying PVS3 for 60 min, while the application of PVS2 for the same amount of time reduced regrowth to 41.5%. Increasing the exposure to vitrification solutions to 90 min reduced regrowth to 43.3% when PVS3 was applied, and 35.6% if PVS2 was used. The cold pre-treatment on the mother plant did not significantly improve overall regrowth. The cryopreservation process did not decline the rooting ability of the recovered shoots.

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.

The biotechnological approach is a promising strategy to overcome some serious diseases which affect stone fruits such as Sharka. However, members of the genus Prunus are among the most challenging with respect to in vitro organogenesis: regeneration and transformation are highly dependent on species, genotype, and source of explants. We focused our activity on the development of adventitious shoot regeneration protocols using leaves of micropropagated Prunus varieties, and on the optimisation of regeneration ratios based on the hypocotyl slices strategy from mature peach seeds (`Nemaguard', `Missour', `Montclar', `Romestar', `Percoco di Tursi'), apricot (`Sancastrese', `Hatif Hatif', Vitillo', Boccuccia spinosa, `Palummella'), and plum (`Stanley', `St. Julien'). The infection of hypocotyl slices of `Stanley' and `St. Julien' with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPV-CP33 carrying the Plum Pox Virus (PPV) coat protein, the GUS and NPTII genes, allowed the creation of 8 transgenic clones of `Stanley' and 3 transgenic clones of `St. Julien'. GUS activity and molecular analysis of regenerated shoots confirmed the insertion of PPV-CP and NPTII genes in the plant genome and the absence of bacterial contamination. Challenging of transgenic `St. Julien' clones with sap prepared from leaves of Nicotiana benthamiana infected with PPV produced no symptoms on inoculated and on post-inoculation generated leaves. DAS-ELISA results confirmed the absence of virus infection in transgenic plants.

Sharka, caused by plum pox virus, is the most devastating disease of stone fruit. Breeding programs for sharka resistance are constrained by the lack of resistant cultivars and by the polygenic nature of the resistance trait. The ectopic expression of viral sequences in the plant can induce resistance to the virus and genetic transformation offers a useful tool to this purpose. Transgenic expression of pathogen-derived sequences encoding self-complementary hairpin RNA that undergo an efficient and predictable post-transcriptional silencing is an agriculturally sustainable strategy to obtain virus-resistant plants, stopping the production of transgenic viral proteins. In a previous work by Di Nicola-Negri et al. (2005) four PPV-derived gene constructs based on hairpin RNAi technology were successfully used to induce high resistance to PPV infection in Nicotiana benthamiana plants. In order to induce sharka resistance in stone fruits, zygotic tissue coming from mature seeds of apricot and peach genotypes were transformed through Agrobacterium tumefaciens infections with the above-mentioned RNAi constructs and with a construct containing the full PPV coat protein gene. Regeneration and transformation efficiency are discussed.

Sharka is the most important disease of stone fruits in terms of agronomic impact and economic importance. The disease is caused by Plum pox virus (PPV) a single-stranded RNA virus. In a recent work, we transformed Nicotiana benthamiana plants with four PPV sequences, covering the PI and HC-Pro genes, arranged to express self-complementary hairpin RNA. Two lines for each construct were challenged with PPV. All the transgenic plants were resistant to PPV infection, with the exception of a fraction containing the P1/HcPro construct. The P1/HcPro lines, in addition to the type of gene construct utilised, differ from the other lines in that they are multiple copy lines. In the current study, three additional P1/HcPro N. benthamiana lines were analysed. All thirty plants challenged with the Italian ISPaVe44 PPV isolate were fully resistant to the virus infection. Between one and three transgenic loci were present in the transformed lines suggesting that a single transgenic locus was not an absolute prerequisite for obtaining plants completely resistant to PPV infection. Additionally, in order to introduce PPV resistance in Prunus species, transformation techniques were developed for Prunus domestica (plum) 'Stanley'. Transgenic plums were obtained, as confirmed by PCR and histochemical GUS assays.

To determine the optimum conditions for Agrobacterium-mediated gene transfer, peach explants including cotyledons, embryonic axes and hypocotyl slices from non-germinated seeds and epicotyl internode slices from germinating seeds were exposed to Agrobacterium-mediated transformation treatments. The GUS (uidA) marker gene was tested using two different A. tumefaciens strains, three plasmids and four promoters [CaMV35s, (Aocs)3AmasPmas (“super-promoter”), mas-CaMV35s, and CAB]. GFP was tested with six A.␣tumefaciens strains, one plasmid (pLC101) and the doubleCaMV35s (dCaMV35s) promoter. The CaMV35s promoter produced more GUS expression than the CAB promoter. A. tumefaciens strains EHA105 and LBA4404 harboring the same plasmid (pBIN19) differed in their effects on GUS expression suggesting an interaction between A. tumefaciens strain and plasmid. A combination of A. tumefaciens EHA105, plasmid pBIN19 and the CaMV35s promoter produced the highest rates of transformation in peach epicotyl internodes (56.8%), cotyledons (52.7%), leaves (20%), and embryonic axes (46.7%) as evaluated by the percentage of explants expressing GUS 14 days after co-cultivation. GFP expression under the control of the dCaMV35s promoter was highest for internode explants but only reached levels of 18–19%. When GFP-containing plasmid pCL101 was combined with each of five A. tumefaciens strains the highest levels of transformation were 20–21% (internode and cotyledons, respectively). When nine peach genotypes were co-cultivated with A. tumefaciens strain EHA105 and GFP-containing plasmid pCL101 the highest levels of transformation were 26–28% (cotyledons and internodes, respectively). While GFP represents a potentially useful transformation marker that allows the non-destructive evaluation of transformation, rates of GFP transformation under the conditions of this study were low. It will be necessary to optimize expression of this marker gene in peach.