Can Ethylene Inhibitors Enhance the Success of Olive Somatic Embryogenesis?

An efficient in vitro morphogenesis, specifically through somatic embryogenesis, is considered to be a crucial step for the application of modern biotechnological tools for genetic improvement in olive (Olea europaea L.). The effects of different ethylene inhibitors, i.e., cobalt chloride (CoCl2), salicylic acid (SA), and silver nitrate (AgNO3), were reported in the cyclic somatic embryogenesis of olive. Embryogenic callus derived from the olive immature zygotic embryos of the cultivar Leccino, was transferred to the expression ECO medium, supplemented with the ethylene inhibitors at 20 and 40 µM concentrations. Among these, the maximum number of somatic embryos (18.6) was obtained in media containing silver nitrate (40 µM), followed by cobalt chloride (12.2 somatic embryos @ 40 µM) and salicylic acid (40 µM), which produced 8.5 somatic embryos. These compounds interfered on callus traits: white friable embryogenic calli were formed in a medium supplemented with 40 µM cobalt chloride and salicylic acid; in addition, a yellow-compact embryogenic callus appeared at 20 µM of all the tested ethylene inhibitors. The resulting stimulatory action of silver nitrate among all the tested ethylene inhibitors on somatic embryogenesis, clearly demonstrates that our approach can efficiently contribute to the improvement of the current SE protocols for olive.

Response of Olive Shoots to Salinity Stress Suggests the Involvement of Sulfur Metabolism.

Global warming has two dangerous global consequences for agriculture: drought, due to water scarcity, and salinization, due to the prolonged use of water containing high concentrations of salts. Since the global climate is projected to continue to change over this century and beyond, choosing salt-tolerant plants could represent a potential paramount last resort for exploiting the secondary saline soils. Olive is considered moderately resistant to soil salinity as compared to other fruit trees, and in the present study, we investigated the influence of NaCl solutions (ranging from 0 to 200 mM) in a salt-tolerant (cv Canino) and two of its transgenic lines (Canino AT17-1 and Canino AT17-2), overexpressing tobacco osmotin gene, and in a salt-sensitive (Sirole) olive cultivar. After four weeks, most of the shoots of both Canino and Sirole plants showed stunted growth and ultimate leaf drop by exposure to salt-enriched media, contrary to transgenic lines, that did not show injuries and exhibited a normal growth rate. Malondialdehyde (MDA) content was also measured as an indicator of the lipid peroxidation level. To evaluate the role of the S assimilatory pathway in alleviating the adverse effects of salt stress, thiols levels as well as extractable activities of ATP sulfurylase (ATPS) and O-acetyl serine(thiol)lyase (OASTL), the first and the last enzyme of the S assimilation pathway, respectively, have been estimated. The results have clearly depicted that both transgenic lines overexpressing osmotin gene coped with increasing levels of NaCl by the induction of S metabolism, and particularly increase in OASTL activity closely paralleled changes of NaCl concentration. Linear correlation between salt stress and OASTL activity provides evidence that the S assimilation pathway plays a key role in adaptive response of olive plants under salt stress conditions.

Osmotin: A Cationic Protein Leads to Improve Biotic and Abiotic Stress Tolerance in Plants.

Research on biologically active compounds has been increased in order to improve plant protection against various environmental stresses. Among natural sources, plants are the fundamental material for studying these bioactive compounds as their immune system consists of many peptides, proteins, and hormones. Osmotin is a multifunctional stress-responsive protein belonging to pathogenesis-related 5 (PR-5) defense-related protein family, which is involved in inducing osmo-tolerance in plants. In this scenario, the accumulation of osmotin initiates abiotic and biotic signal transductions. These proteins work as antifungal agents against a broad range of fungal species by increasing plasma membrane permeability and dissipating the membrane potential of infecting fungi. Therefore, overexpression of tobacco osmotin protein in transgenic plants protects them from different stresses by reducing reactive oxygen species (ROS) production, limiting lipid peroxidation, initiating programmed cell death (PCD), and increasing proline content and scavenging enzyme activity. Other than osmotin, its homologous proteins, osmotin-like proteins (OLPs), also have dual function in plant defense against osmotic stress and have strong antifungal activity.

Influence of Continuous Spectrum Light on Morphological Traits and Leaf Anatomy of Hazelnut Plantlets.

Light spectra influence growth, development, and quality of plants and seedlings, that is one of the main aspects engaging the interests of private and public researchers and nursery industries. Propagation of hazelnut (Corylus avellana L.), which in the past has been held in low consideration because of the widespread use of rooted suckers directly collected in the field, today is taking on increasing interest due to the strong expansion of hazelnut cultivation. In order to improve the quality of plants and seedlings in greenhouse acclimatization, the effects of light emitting diodes (LED) lights during the ex vitro growth of two hazelnut cultivars (Tonda di Giffoni and Tonda Gentile Romana) were investigated. Plantlets were maintained in a growth chamber and exposed to three different continuous spectrum LED systems as a primary source of illumination and to fluorescent lamps used as control. LEDs differed in the percentage of some wavelength ranges in the spectrum, being AP673L rich in green and red wavelengths, NS1 in blue and green light, G2 in red and far red wavelengths. After a 4-week experimental period, morphometric, biochemical, and histological analyses were carried out. Shoot and leaf growths were influenced by LEDs more than by fluorescent lamps in both cultivars. G2 positively affected biomass increment more than the other LEDs, by inducing not only cell elongation (increase in shoot length, new internodes length, leaf area) but also cell proliferation (increase in new node number). G2 exposure had negative effects on total chlorophyll content but positively affected synthesis of flavonoids in both varieties; therefore, plants grown under this LED showed the lowest nitrogen balance index. Leaf morpho-anatomical analyzed traits (thickness, palisade cell height, number of chloroplasts, number of palisade cells), were influenced especially by G2 and, to a less extent, by NS1 light. Significant differences in some parameters were observed between the two cultivars in response to a same light source. The results obtained underline the importance of light modulation for hazelnut, providing useful information for ex vitro growth of hazelnut plantlets.

Plant Cellular and Molecular Biotechnology: Following Mariotti's Steps.

This review is dedicated to the memory of Prof. Domenico Mariotti, who significantly contributed to establishing the Italian research community in Agricultural Genetics and carried out the first experiments of Agrobacterium-mediated plant genetic transformation and regeneration in Italy during the 1980s. Following his scientific interests as guiding principles, this review summarizes the recent advances obtained in plant biotechnology and fundamental research aiming to: (i) Exploit in vitro plant cell and tissue cultures to induce genetic variability and to produce useful metabolites; (ii) gain new insights into the biochemical function of Agrobacterium rhizogenes rol genes and their application to metabolite production, fruit tree transformation, and reverse genetics; (iii) improve genetic transformation in legume species, most of them recalcitrant to regeneration; (iv) untangle the potential of KNOTTED1-like homeobox (KNOX) transcription factors in plant morphogenesis as key regulators of hormonal homeostasis; and (v) elucidate the molecular mechanisms of the transition from juvenility to the adult phase in Prunus tree species.

Somatic Embryogenesis in Olive (Olea europaea L. subsp. europaea var. sativa and var. sylvestris).

Protocols for olive somatic embryogenesis from zygotic embryos and mature tissues have been described for both Olea europaea sub. europaea var. sativa and var. sylvestris. Immature zygotic embryos (no more than 75 days old), used after fruit collection or stored at 12-14 °C for 2-3 months, are the best responsive explants and very slightly genotype dependent, and one single protocol can be effective for a wide range of genotypes. On the contrary, protocols for mature zygotic embryos and for mature tissue of cultivars are often genotype specific, so that they may require many adjustments according to genotypes. The use of thidiazuron and cefotaxime seems to be an important trigger for induction phase particularly for tissues derived from cultivars. Up to now, however, the application of this technique for large-scale propagation is hampered also by the low rate of embryo germination; it proves nonetheless very useful for genetic improvement.

Mutation scanning and genotyping by high-resolution DNA melting analysis in olive germplasm.

The application of high-resolution melting (HRM) analysis of DNA is reported for scanning and genotyping Olea europaea germplasm. To test the sensitivity of the method, a functional gene marker, phytochrome A (phyA), was used, since this gene is correlated with important traits for the ecology of the species. We have designed a set of oligos able to produce amplicons of 307 bp to scan for the presence of single polymorphic mutations in a specific phyA fragment encompassing the chromophore attachment site (Cys323). The presence of mutations for substitution, either homozygous or heterozygous, was easily detected by melting curve analysis in a high-resolution melter. It has been established that the sensitivity of the HRM analysis can be significantly improved designing specific primers very close to the mutation sites. All SNPs found were confirmed by sequence analyses and ARMS-PCR. The method has also been confirmed to be very powerful for the visualization of microsatellite (SSR) length polymorphisms. HRM analysis has a very high reproducibility and sensitivity for detecting SNPs and SSRs, allowing olive cultivar genotyping and resulting in an informative, easy, and low-cost method able to greatly reduce the operating time.

Salicylic acid induces H2O2 production and endochitinase gene expression but not ethylene biosynthesis in Castanea sativa in vitro model system.

Salicylic acid (SA), ethylene (ET), and wounding are all known to influence plant defense response. Experiments attempting to determine SA's relation to ET biosynthesis and defense gene expression have shown conflicting results. To confront this, we developed an in vitro model system to investigate how SA affects ET biosynthesis, hydrogen peroxide (H(2)O(2)) production and endochitinase gene expression in the European chestnut. ET measurements of in vitro shoots indicated a critical time point for SA exogenous application, enabling us to study its effects independent of ET. In addition, ET measurements demonstrated that its own increased biosynthesis was a response to wounding but not to SA treatment. Application of the ET biosynthesis inhibitor, aminoethoxyvinylglycine (AVG), on wounded and SA-treated shoots blocked wounding-induced ET production. Interestingly, SA inhibited ET production, but to a lesser extent than AVG. Additionally, SA also induced the accumulation of endochitinase transcript level. Likewise, a sensitive tissue-print assay showed that SA further increased the level of H(2)O(2). Yet, SA-induced endochitinase gene expression and SA-enhanced H(2)O(2) production levels were independent of ET. The cumulative results indicate that SA acts as an inducer of endochitinase PR gene expression and of H(2)O(2) oxidative burst. This suggests that SA is a component of the signal transduction pathway leading to defense against pathogens in chestnut. Further, the model system developed for this experiment should facilitate the deciphering of defense signaling pathways and their cross-talk. Moreover, it should also benefit the study of trees of long generation time that are known to be recalcitrant to in vitro studies.

Pectic enzymes as a selective pressure tool forin vitro recovery of strawberry plants with fungal disease resistance.

A novel strategy in selecting strawberry (Fragaria xananassa L.) plants with resistance toRhizoctonia fragariae andBotrytis cinerea was developed. Purified pectic enzymes produced byR. fragariae were usedin vitro to select morphogenetic calluses. Both regenerated shoots and plants were testedin vitro andin vivo withR. fragariae andB. cinerea. Thein vitro resistance of shoots regenerated under selection pressure was confirmed byin vivo tests with runner plants either by root immersion in a suspension ofR. fragariae mycelium before potting the plants in sterile soil, or by spraying the leaves with several strains ofB. cinerea spores. The increase of resistance against pathogens was correlated to the increase of phenolic compounds, particularly orthodibydroxyphenols.