Trans-grafting plum pox virus resistance from transgenic plum rootstocks to apricot scions.

Introduction: Trans-grafting could be a strategy to transfer virus resistance from a transgenic rootstock to a wild type scion. However contradictory results have been obtained in herbaceous and woody plants. This work was intended to determine if the resistance to sharka could be transferred from transgenic plum rootstocks to wild-type apricot scions grafted onto them. Methods: To this end, we conducted grafting experiments of wild- type apricots onto plum plants transformed with a construction codifying a hairpin RNA designed to silence the PPV virus and studied if the resistance was transmitted from the rootstock to the scion. Results: Our data support that the RNA-silencing-based PPV resistance can be transmitted from PPV-resistant plum rootstocks to non-transgenic apricot scions and that its efficiency is augmented after successive growth cycles. PPV resistance conferred by the rootstocks was robust, already occurring within the same growing cycle and maintained in successive evaluation cycles. The RNA silencing mechanism reduces the relative accumulation of the virus progressively eliminating the virus from the wild type scions grafted on the transgenic resistant PPV plants. There was a preferential accumulation of the 24nt siRNAs in the scions grafted onto resistant rootstocks that was not found in the scions grafted on the susceptible rootstock. This matched with a significantly lower relative accumulation of hpRNA in the resistant rootstocks compared with the susceptible or the tolerant ones. Discussion: Using transgenic rootstocks should mitigate public concerns about transgenes dispersion and eating transgenic food and allow conferring virus resistance to recalcitrant to transformation cultivars or species.

The Effect of Silver Nanoparticle Addition on Micropropagation of Apricot Cultivars (Prunus armeniaca L.) in Semisolid and Liquid Media.

Silver nanoparticles (AgNPs) are novel compounds used as antimicrobial and antiviral agents. In addition, AgNPs have been used to improve the growth of different plants, as well as the in vitro multiplication of plant material. In this work the effect of AgNPs on in vitro growth of 'Canino' and 'Mirlo Rojo' cultivars, as well as the leaf ion composition, are studied. Different concentrations of AgNPs (0, 25, 50, 75 and 100 mg L-1) were added to two culture systems: semisolid medium with agar (SSM) in jars and liquid medium in temporary immersion system (TIS). Proliferation (number of shoots), shoot length, productivity (number of shoot × average length), leaf surface, fresh and dry weight were measured. Additionally, the silver and other ion accumulation in the leaves were evaluated by inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis. The productivity of 'Canino' and 'Mirlo Rojo' decreased when increasing the concentration of AgNPs in the semisolid medium. However, the use of AgNPs in the TIS improved the proliferation and productivity of 'Canino' and Mirlo Rojo', increasing biomass production, and the concentration of nutrients in the plants, although these effects are genotype-dependent. TISs are the best system for introducing silver into shoots, the optimum concentration being 50 mg L-1 for 'Canino' and 75 mg L-1 for 'Mirlo Rojo'. Principal component analysis, considering all the analyzed ions along the treatments, separates samples in two clear groups related to the culture system used. The use of bioreactors with a liquid medium has improved the productivity of 'Canino' and 'Mirlo Rojo' in the proliferation stage, avoiding hyperhydration and other disorders. The amount of metallic silver that penetrates apricot plant tissues depends on the culture system, cultivar and concentration of AgNPs added to the culture medium. Silver ion accumulation measured in the shoots grown in the TIS was higher than in shoots micropropagated in a semisolid medium, where it is barely detectable. Furthermore, AgNPs had a beneficial effect on plants grown in TIS. However, AgNPs had a detrimental effect when added to a semisolid medium.

Application of Ascophyllum nodosum-Based Soluble Extract on Micropropagation and Regeneration of Nicotiana benthamiana and Prunus domestica.

In the present study, the effect of a commercial extract of the seaweed Ascophyllum nodosum on in vitro micropropagation, shoot regeneration, and rhizoghenesis were studied in Nicotiana benthamiana and Prunus domestica. Results showed that the MS medium supplemented with various concentrations of the Ascophyllum extract (5, 10, 50, and 100 mg L-1) significantly enhanced the number of regenerated buds from N. benthamiana leaf discs to the conventional MS regenerating medium. Increases ranged from 3.5 to 6.5 times higher than the control. The effect of the Ascophyllum extract on N. benthamiana micropropagation was assessed through the measurement of some plant growth parameters. Results showed that the extract alone could not replace the micropropagation medium since shoot length, shoot diameter, root length, and leaf area were significantly reduced. However, its combination with a half-strength MS medium enhanced these parameters. Its effect was also evaluated on regeneration from plum hypocotyl slices. When added to the shoot regeneration medium without any plant growth regulators, the Ascophyllum extract alone could induce shoot regeneration. However, the percentage of bud regeneration and number of regenerated buds were lower than with the conventional shoot regeneration medium containing complete growth regulators. In contrast, the Ascophyllum extract drastically promoted rhizogenesis from plum hypocotyl slices. These results pave the way for the possible use of A. nodosum extracts in in vitro mass propagation of higher plants.

Hydrogen peroxide generated by over-expression of cytosolic superoxide dismutase in transgenic plums enhances bacterial canker resistance and modulates plant defence responses.

H2O2 generated during the oxidative burst, plays important roles in plant defenses responses against pathogens. In this study we examined the role of H2O2 on bacterial canker resistance in transgenic plums over-expressing cytosolic superoxide dismutase. Three transgenic lines (C64, C66 and F12) with elevated levels of H2O2 accumulation showed enhanced resistance against bacterial canker disease caused by Pseudomonas syringae pv. syringae, when compared to the non-transformed control. Analysis of the expression of several genes involved in the plant-pathogen interaction showed that the expression of those involved in SA pathway (pr1 and npr1) and JA (lox3) were activated earlier and transiently in transgenic lines C66 and F12 when compared to the wild type. However, the expression of genes involved in anthocyanin synthesis (chi, chs, f3h, dfr, atcs, myb10) and ethylene (acs) was induced at very low levels whereas it was activated by the pathogen at exaggerated levels in the non-transformed line. These results suggest that resistance observed in transgenic lines over-producing H2O2 is correlated with an early and transient induction of defense genes associated with the SA and JA pathways and inhibition of gene expression associated with ethylene and anthocyanin biosynthesis.

Genome Wild Analysis and Molecular Understanding of the Aquaporin Diversity in Olive Trees (Olea Europaea L.).

Cellular aquaporin water channels (AQPs) constitute a large family of transmembrane proteins present throughout all kingdoms of life, playing important roles in the uptake of water and many solutes across the membranes. In olive trees, AQP diversity, protein features and their biological functions are still largely unknown. This study focuses on the structure and functional and evolution diversity of AQP subfamilies in two olive trees, the wild species Olea europaea var. sylvestris (OeuAQPs) and the domesticated species Olea europaea cv. Picual (OleurAQPs), and describes their involvement in different physiological processes of early plantlet development and in biotic and abiotic stress tolerance in the domesticated species. A scan of genomes from the wild and domesticated olive species revealed the presence of 52 and 79 genes encoding full-length AQP sequences, respectively. Cross-genera phylogenetic analysis with orthologous clustered OleaAQPs into five established subfamilies: PIP, TIP, NIP, SIP, and XIP. Subsequently, gene structures, protein motifs, substrate specificities and cellular localizations of the full length OleaAQPs were predicted. Functional prediction based on the NPA motif, ar/R selectivity filter, Froger's and specificity-determining positions suggested differences in substrate specificities of Olea AQPs. Expression analysis of the OleurAQP genes indicates that some genes are tissue-specific, whereas few others show differential expressions at different developmental stages and in response to various biotic and abiotic stresses. The current study presents the first detailed genome-wide analysis of the AQP gene family in olive trees and it provides valuable information for further functional analysis to infer the role of AQP in the adaptation of olive trees in diverse environmental conditions in order to help the genetic improvement of domesticated olive trees.

Silencing of Agrobacterium tumefaciens oncogenes ipt and iaaM induces resistance to crown gall disease in plum but not in apricot.

BACKGROUND: In this study, two vectors with short-length chimeric transgenes were used to produce Prunus rootstocks resistant to crown gall disease through RNA-interference-mediated gene silencing of the Agrobacterium tumefaciens oncogenes ipt and iaaM. RESULTS: Transgenic plum and apricot lines were produced with efficiencies of up to 7.7 and 1.1% respectively. An in vitro evaluation method allowed identification of susceptible lines and reduction in the number of lines to be evaluated in the greenhouse. Five transgenic plum lines, expressing transgene-derived small interfering RNA (siRNA) and low levels of transgene hairpin RNA (hpRNA), showed a significant reduction in the development of the disease after infection with Agrobacterium strains C58 and A281 under greenhouse conditions. However, unexpectedly, all transgenic apricot lines were gall susceptible. The infection of apricot plants with a binary vector containing only the 6b oncogene demonstrated that the expression of this gene is involved in the induction of tumours in the apricot species. CONCLUSION: RNAi-mediated gene silencing can be used for inducing crown gall resistance in plum rootstocks. These could be used to graft non-genetically modified commercial fruit cultivars reducing, or eliminating, the disease symptoms. © 2017 Society of Chemical Industry.

A new strategy to enhance the biosynthesis of trans-resveratrol by overexpressing stilbene synthase gene in elicited Vitis vinifera cell cultures.

In this work, transgenic lines of suspension cultured cells of Vitis vinifera cv. Monastrell containing the plasmid pMOG800-sts have been obtained. The cell growth of these transgenic cell lines decreased slightly as compared to non-transgenic suspension cultured cells, while cell viability was not affected. In addition, the elicitation with cyclodextrins and methyl jasmonate enhanced the production of trans-resveratrol, observing the highest levels of this compound in sts-expressing transgenic Vitis suspension cultured cells with the sts expression cassette in the forwards orientation. Moreover, the forwards 2 (F2) transgenic cell line produced the greater levels of trans-resveratrol in comparison with the non-transgenic cell line. In fact, when suspension cultured cells were treated with both elicitors, the accumulation of trans-resveratrol outside the cells in the F2 transgenic suspension cultured cells increased twice (1458 mg.L-1) as compared to non-transgenic cell lines (724 mg.L-1). In both cases, the levels of trans-resveratrol detected in the treatment with cyclodextrins and methyl jasmonate were greater than the sum of the individual treatments, and therefore we observed a synergistic effect in the presence of both elicitors. Moreover, the expression profile of sts gene in transgenic V. vinifera cell lines was similar to the expression profile detected for the endogenous sts gene in non-transgenic V. vinifera cell lines, being the expression levels greater in the treatment with methyl jasmonate and cyclodextrins, which was related to the high levels of trans-resveratrol found in the presence of both elicitors.

Antimicrobial Activity of two Semisynthetic Triterpene Derivatives from Euphorbia officinarum Latex against Fungal and Bacterial Phytopathogens.

This study evaluated the in vitro antimicrobial effect of 3ß-acetoxy-norlup-20-one (1) and 3-chloro-4a,14a-dimethyl-5a-cholest-8-ene (2), triterpene derivatives from Euphorbia officinarum latex against fungal and bacterial phytopathogens. Results showed that although mycelial growth of several strains of Vericillium dahlia, and Fusarium oxysporum fsp. melonis and Penicillium expansum was affected only moderately, the two compounds were able to reduce highly conidia formation and germination, suggesting that they act as fungistatic compounds. Their antibacterial activity was tested against Pseudomonas syringae pv. syringae (Pss), P. syringae pv. tabacci (Pst), Erwinia amylovora (Ea) and Agrobacterium tumefaciens (At) using disc diffusion method. Results showed that compound 2 was more effective in inhibiting the growth of Pss, Pst and Ea than compound 1.

Transformation of plum plants with a cytosolic ascorbate peroxidase transgene leads to enhanced water stress tolerance.

BACKGROUND AND AIMS: Water deficit is the most serious environmental factor limiting agricultural production. In this work, the tolerance to water stress (WS) of transgenic plum lines harbouring transgenes encoding cytosolic antioxidant enzymes was studied, with the aim of achieving the durable resistance of commercial plum trees. METHODS: The acclimatization process was successful for two transgenic lines: line C3-1, co-expressing superoxide dismutase (two copies) and ascorbate peroxidase (one copy) transgenes simultaneously; and line J8-1, harbouring four copies of the cytosolic ascorbate peroxidase gene (cytapx). Plant water relations, chlorophyll fluorescence and the levels of antioxidant enzymes were analysed in both lines submitted to moderate (7 d) and severe (15 d) WS conditions. Additionally, in line J8-1, showing the best response in terms of stress tolerance, a proteomic analysis and determination of the relative gene expression of two stress-responsive genes were carried out. KEY RESULTS: Line J8-1 exhibited an enhanced stress tolerance that correlated with better photosynthetic performance and a tighter control of water-use efficiency. Furthermore, this WS tolerance also correlated with a higher enzymatic antioxidant capacity than wild-type (WT) and line C3-1 plum plants. On the other hand, line C3-1 displayed an intermediate phenotype between WT plants and line J8-1 in terms of WS tolerance. Under severe WS, the tolerance displayed by J8-1 plants could be due to an enhanced capacity to cope with drought-induced oxidative stress. Moreover, proteomic analysis revealed differences between WT and J8-1 plants, mainly in terms of the abundance of proteins related to carbohydrate metabolism, photosynthesis, antioxidant defences and protein fate. CONCLUSIONS: The transformation of plum plants with cytapx has a profound effect at the physiological, biochemical, proteomic and genetic levels, enhancing WS tolerance. Although further experiments under field conditions will be required, it is proposed that J8-1 plants would be an interesting Prunus rootstock for coping with climate change.

Ectopic expression of cytosolic superoxide dismutase and ascorbate peroxidase leads to salt stress tolerance in transgenic plums.

To fortify the antioxidant capacity of plum plants, genes encoding cytosolic antioxidants ascorbate peroxidase (cytapx) and Cu/Zn-superoxide dismutase (cytsod) were genetically engineered in these plants. Transgenic plum plants expressing the cytsod and/or cytapx genes in cytosol have been generated under the control of the CaMV35S promoter. High levels of cytsod and cytapx gene transcripts suggested that the transgenes were constitutively and functionally expressed. We examined the potential functions of cytSOD and cytAPX in in vitro plum plants against salt stress (100 mm NaCl). Several transgenic plantlets expressing cytsod and/or cytapx showed an enhanced tolerance to salt stress, mainly lines C5-5 and J8-1 (expressing several copies of sod and apx, respectively). Transformation as well as NaCl treatments influenced the antioxidative metabolism of plum plantlets, including enzymatic and nonenzymatic antioxidants. Transgenic plantlets exhibited higher contents of nonenzymatic antioxidants glutathione and ascorbate than nontransformed control, which correlated with lower accumulation of hydrogen peroxide. Overall, our results suggest that transformation of plum plants with genes encoding antioxidant enzymes enhances the tolerance to salinity.

Cu/Zn superoxide dismutase and ascorbate peroxidase enhance in vitro shoot multiplication in transgenic plum.

In this study we examined the role of antioxidant metabolism in in vitro shoot multiplication. We generated transgenic plum plantlets overexpressing the cytsod and cytapx genes in cytosol under the control of the constitutive promoter CaMV35S. Three transgenic lines with up-regulated sod at transcriptional levels that showed silenced cytapx expression displayed an elevated in vitro multiplication rate. By contrast, a transgenic line harboring several copies of cytapx and with elevated APX enzymatic activity did not show any improvement in plant vigor, measured as the number of axillary shoots and shoot length. All of the lines with elevated micropropagation ability exhibited intensive H2O2 accumulation, monitored by 3,3'-diaminobenzidine (DAB) staining as well as by colorimetric analysis, providing direct in vitro evidence of the role of H2O2 and antioxidant genes in in vitro shoot multiplication.

Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation.

BACKGROUND: The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently. RESULTS: We show here that chimeras are a very frequent phenomenon observed after regenerating transgenic plants. Spatial and temporal analyses of transformed tobacco and apricot plants with a quantitative, real-time PCR amplification of the neomycin phosphotransferase (nptII) transgene as well as of an internal control (beta-actin), used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the nptII transgene differed greatly along with the sub-cultivation period of these plants and was dependent on the localisation of the analysed leaves; being higher in roots and basal leaves, while in the apical leaves it remained at lower levels. These data demonstrate that, unlike the use of the gus marker gene, real-time PCR is a powerful tool for detection of chimeras. Although some authors have proposed a consistent, positive Southern analysis as an alternative methodology for monitoring the dissociation of chimeras, our data show that it does not provide enough proof of uniform transformation. In this work, however, real-time PCR was applied successfully to monitor the dissociation of chimeras in tobacco plants and apricot callus. CONCLUSIONS: We have developed a rapid and reliable method to detect and estimate the level of chimeras in transgenic tobacco and apricot plants. This method can be extended to monitor the dissociation of chimeras and the recovery of uniformly-transformed plants.

Acibenzolar-s-methyl-induced resistance to Japanese pear scab is associated with potentiation of multiple defense responses.

ABSTRACT This study reports the mode of action of acibenzolar-S-methyl (ASM) against Japanese pear scab, caused by Venturia nashicola. Pretreatment of potted Japanese pear trees with ASM reduced scab symptoms and potentiated several lines of plant defense response. This included transcripts encoding polygalacturonase-inhibiting protein (PGIP) that were highly and transiently promoted after scab inoculation of plants pretreated with ASM, suggesting a possible role for defenses involved in direct interaction with the pathogen. The activity of the key enzyme of phenylpropanoid pathway, phenylalanine ammonia lyase (PAL), was enhanced in scab-inoculated leaves pretreated with ASM only 7 days after inoculation, suggesting that it may play a minor role in induced resistance. In this work, salicylic acid (SA) accumulation was enhanced in ASM-treated leaves for the first time, according to an equivalent time course to that of PAL activity. However, a delayed induction of SA accumulation in ASM-treated leaves compared with kinetics of induction of several pathogenesis- related (PR) proteins or their encoding genes suggested that resistance triggered by ASM may be SA-independent. Among these PR proteins, PR-1, chitinase and PR-10 were promoted early by ASM after scab inoculation. Peroxidase, as well as enzymes involved in the oxidative burst such as superoxide dismutase, catalase, and ascorbate peroxidase were weakly activated with ASM treatment alone or pathogen inoculation alone and highly enhanced in ASM pretreated plants upon challenge inoculation, suggesting the occurrence of priming phenomenon during the interaction of Japanese pear-ASM-V. nashicola. An early potentiation of the activity of these enzymes after scab inoculation of leaves pretreated with ASM suggested that active oxygen species may be involved as a signal for the activation of PR proteins or genes.