Genetsko oplemenjivanje masline

In the last decade significant progress has been made in developing successful olive cloning techniques, although some difficulties still remain such as that with establishing sterile cultures in vitro and morphogenesis from mature tissue of cultivars. More work is required, although significant advances have also been made in shoot regeneration from petioles of in vitro grown shoots of several cultivars (Mencuccini and Rugini, 1993). However, the regeneration ability is still low for use in biotechnological applications. The novel strategy of the «double regeneration», developed to achieve somatic embryos in olive cv. Canino and Moraiolo may also be applicable in other cultivars. This technique can be generalised since at present it is essential for inducing and maintaining shoot morphogenic callus in other species such as cherry, apple, and pear (Gutiérrez Pesce et al., 1998; Rugini e Muganu 1998; Abdollahi et al., 2005). Gene transfer techniques offer a more powerful strategy for genetic improvement in respect to traditional breeding methods. It allows the introduction into one genotype, one or a few pieces of genetic information without drastic modifications of the general characteristics of the plant. Transformation techniques have been developed, by using somatic embryogenesis, and transgenic plants, with some desirable agronomic traits, have already been generated in one cultivar. At present field trials, approved by the Italian Health Minister, are conducted on transgenic rolABC, and osmotin plants. From transgenic olive plants, similar to kiwi transgenic plants with rolABC genes we expect plants with large root systems, compact vegetative habitus, smaller number of flowers per plant, and high rooting ability of cuttings. In plants over-expressing osmotin gene, we expect a higher tolerance to some fungi. Many genes have already been isolated from several species, which may be introduced in olive singly or associated with others. Transformation experiments with multiple genes (chitinase + osmotin + PR1) are in progress in our laboratory in order to increase fungal resistance. Antibacterial genes (thionin, cecropin, attacin, etc.) against Pseudomonas syringae and genes for modifying the pattern of fruit ripening (ethylene, PG) are only a few examples of the potential of genetic manipulation to improve olive. A high content of di-hydroxiphenols could confer the valuable bitter taste in the olive oil. Agrobacterium-mediated gene transfer seems to be the most efficient method in olive. The molecular techniques should not aim only to make clear the phylogenesis of the genus, but also to clone useful genes and promoters in olive. To facilitate this work, biotechnology, long-term breeding programs and biochemical research should be closely linked to achieve the objectives quickly.U zadnjem desetljeću učinjen je znatan napredak u razvijanju uspješnih metoda kloniranja maslina iako će neke poteškoće ostati, kao utvrđivanje sterilnih kultura in vitro i morfogeneza iz zrelog tkiva kultivara. Potrebno je još raditi, makar je učinjen znatan napredak i u regeneraciji izbojaka iz petiola uzgojenih in vitro od nekoliko kultivara (Mencuccini i Rugini, 1993). Međutim, mogućnost regeneracije još je uvijek mala za biotehnološku primjenu. Nova strategija "dvostruke regeneracije", razvijena kako bi se dobili somatski embriji masline cv. Ganino i Moraiolo mogu se primijeniti i u drugim kultivarima. Ova se tehnika može generalizirati jer je danas bitna za induciranje i održavanje morfogenskog kalusa izbojka u drugim vrstama kao što su trešnja, jabuka i kruška (Gutierrez Pesce et al. 1998; Rugini e Muganu, 1998; Abdollahi et al., 2005). Tehnike transfera gena pružaju snažniju strategiju za genetsko oplemenjivanje u odnosu na tradicionalne uzgojne metode. One omogućuju uvođenje u jedan genotip jedne ili više genetskih informacija bez drastičnih modifikacija općih značajki biljke. Razvijene su tehnike transformacije primjenom somatske embriogeneze i transgenske biljke određenih poželjnih agronomskih svojstava i već su proizvedene u jednom kultivaru. Upravo se provode pokusi na terenu, koje je odobrio talijanski Ministar zdravstva, na transgenskim rolABC i osmotinskim biljkama. Od transgenskih biljaka masline, slično transgenskim biljkama kivija s genima rolABC, očekujemo biljke velikog sustava korijena, zbijenog vegetativnog habitusa, manjeg broja cvjetova po biljci i velike sposobnosti sadnica za ukorijenjenjem. U biljaka preizraženog osmotinskog gena očekujemo veću tolerantnost na neke gljive/gljivice. Izolirani su već mnogi geni iz nekoliko vrsta, što se mogu introducirati pojedinačno ili zajedno s drugima. Pokusi transformacije s mnogostrukim genima (chitinase + osmotin + PRL) su u tijeku u našem laboratoriju, kako bi se povećala otpornost na gljivice. Antibakterijski geni (tionin, cecropin, atacin itd.) protiv Pseudomonas syringae i geni za modificiranje uzorka zriobe voća (etilen, PG) samo su neki primjeri potencijala genetske manipulacije za oplemenjivanje masline. Visok sadržaj dihidroksifenola mogao bi prenijeti dragocjen gorki okus maslinovog ulja. Prijenos gena posredstvom agro-bakterija čini se najdjelotvornijom metodom u masline. Cilj molekularnih tehnika ne bi trebao biti samo razjašnjenje filogeneze gena nego i kloniranje korisnih gena i stimulatora u maslini. Da bi se taj posao olakšao potrebno je usko povezati biotehnologiju, dugoročne uzgojne programe i biokemijska istraživanja radi brzog postizanja ciljeva

Olea

The genus Olea contains about 30 species were grouped into three subgenera, Tetrapilus, Paniculatae, and Olea (cultivated olive and wild relatives), found in Asia, Australia and Asia, Africa and Europe, respectively. The species O. europaea L. includes six subspecies: Olea europaea L. ssp. europaea (the Mediterranean olives); O. e. laperrinei (distributed in Saharan massifs of Hoggar, Aïr, Jebel Marra in Algeria); O. e. cuspidata (which moved from South Africa to Egypt, East Australian areas and Hawaii, and from Arabia to northern India and Southwest China); O. e. guanchica (Canary Islands); O. e. maroccana (southwestern Morocco); and O. e. cerasiformis (Madeira). Using molecular markers, it has been ascertained that the Mediterranean olives include the cultivated types (O. europaea L. ssp. europaea var. sativa), the true wild oleaster (O. e. e. var. sylvestris), and the feral form olevaster from seedlings raised from seeds of the cultivated types. The oleaster has a narrow range of distribution and it is often mistaken for olevaster. Recolonization of the Mediterranean basin by Oleaster occurred after the last glacial event, from refuges located in both eastern and western Mediterranean basin areas toward southern Europe. Oleaster is a source of rootstock for propagating new improved cultivated varieties. Cultivated and wild forms have the same diploid chromosome number (2n = 46) and are fully interfertile. Triploid and tetraploid genotypes have been isolated from cultivated O.e.e., but polyploid forms have been found in endangered natural populations of O. e. guancica (tetraploid) and O. e. maroccana (hexaploid). Individual oleaster trees showing superior performance for size and/or oil content of fruit were selected empirically during olive domestication and propagated vegetatively as clones using cuttings that were planted directly or, more recently, grafted onto indigenous oleasters. Genetic markers linked for most important agronomic traits, such as size of the tree, content of secondary products of fruit, flowering induction, oil quality, and biotic and abiotic resistance, will help introgression by conventional breeding of oleaster trait-enhancing genes into cultivated olive. Successful results were difficult to achieve due to both the complex genetic basis of the traits to be improved and the long juvenile period of the progenies that delays the expression of the target traits. In vitro techniques to regenerate doubled haploids from hybrids or somaclonal variation induction may complement classical breeding procedures. Genetic transformation could speed up the development of new genotypes, and transgenic olive plants with modified growth habit and putative induced disease resistance are being tested under filed conditions. However, the development of an efficient regeneration method from mature tissue is the limiting factor for the routine application of this technology to olive genetic improvement.La pubblicazione originale è disponibile sul sito dell'editore http://www.springerlink.co

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Influence of plant growth regulators, carbon sources and iron on the cyclic secondary somatic embryogenesis and plant regeneration of transgenic cherry rootstock ‘Colt’ (Prunus avium x P. pseudocerasus)

The frequency of long-term secondary somatic embryogenesis and shoot meristem development from embryogenic masses of the cherry rootstock ‘Colt’ (Prunus avium x P. pseudocerasus), differentiated from transgenic roots containing the T-DNA of Agrobacterium rhizogenes, has opened the way for genetic improvement by biotechnological techniques. Whole plants were produced by stimulating shoot meristem development from somatic embryos. The combination of 4 mg 1¯¹ of kinetin and 2% of maltose under illumination stimulated shoot development and, subsequently, whole plants have been recovered by applying 1.5 mg 1¯¹ kinetin to the rooting medium. Although numerous treatments have been tested involving both embryogenic masses and whole embryos, normal embryo germination was observed sporadically. Cold treatment was effective in stimulating secondary somatic embryogenesis with embryo development to the cotyledonary stage, but did not promote their germination. Similarly, a higher concentration (44–55 mg 1¯¹) of chelated iron than that commonly used in tissue culture media (36.7 mg 1¯¹) produced, after 3 weeks in culture, almost a 50% increase in the number of embryos at the cotyledonary stage per embryogenic mass. Among the cytokinins tested, 1 mg 1 ¯¹ of 6-benzylaminopurine and 0.1 mg 1¯¹ of thidiazuron were effective in inducing secondary somatic embryogenesis; however, each of them expressed highest efficiency with specific medium and environmental conditions. Furthermore, application of 1 mg 1¯¹ thidiazuron reverted morphogenic callus to non-morphogenic callus, particularly in medium containing 2% sucrose. Finally, hormone free medium with 2% maltose enhanced maturation of the embryos to the normal cotyledonary stage. This paper has improved knowledge of embryo culture and plant production in this important genotype, opening the way for genetic improvement by biotechnological techniques, mainly with the aim of modifying the growth pattern of the canopy of sweet cherry grafted on it.L'articolo è disponibile sul sito del nuovo editore http://www.springerlink.co

Olive

Biochemical and molecular procedures have been developed for olive genotyping, for taxonomy between cultivated and wild olives, for phylogenetic studies of cultivars, and for identification of usable markers linked to the most important agronomic traits, such as size of the tree, flowering induction, apical dominance, productivity, self-fertility, quantity and quality of oil and secondary products of fruit, biotic and abiotic resistance. These characters could be introgressed into cultivars by conventional breeding or through transgeny. Successful results by classical breeding are difficult to obtain due to the different flower fertility and the long juvenile period of the progenies. The low efficiency of modern genetic improvement techniques suggests that genetic transformation techniques could speed up the development of new genotypes; however, the development of efficient regeneration methods from mature tissue is the limiting factor for applying this technology, in olive. Transgenic olive plants with modified growth habit and putative induced disease resistance are under filed conditions to test their phenotype. Other attempts of genetic improvement such as clonal selection, mutation through -irradiation, protoplast technology, haploid, changes in ploidy level, somaclonal variation, and somatic hybridization are discussed and reviewed.L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com

Risk of recurrence after discontinuing anticoagulation in patients with COVID-19- associated venous thromboembolism: a prospective multicentre cohort studyResearch in context

Background: The clinical relevance of recurrent venous thromboembolism (VTE) after discontinuing anticoagulation in patients with COVID-19-associated VTE remains uncertain. We estimated the incidence rates and mortality of VTE recurrences developing after discontinuing anticoagulation in patients with COVID-19-associated VTE. Methods: A prospective, multicenter, non-interventional study was conducted between March 25, 2020, and July 26, 2023, including patients who had discontinued anticoagulation after at least 3 months of therapy. All patients from the registry were analyzed during the study period to verify inclusion criteria. Patients with superficial vein thrombosis, those who did not receive at least 3 months of anticoagulant therapy, and those who were followed for less than 15 days after discontinuing anticoagulation were excluded. Outcomes were: 1) Incidence rates of symptomatic VTE recurrences, and 2) fatal PE. The rate of VTE recurrences was defined as the number of patients with recurrent VTE divided by the patient-years at risk of recurrent VTE during the period when anticoagulation was discontinued. Findings: Among 1106 patients with COVID-19-associated VTE (age 62.3 ± 14.4 years; 62.9% male) followed-up for 12.5 months (p25-75, 6.3–20.1) after discontinuing anticoagulation, there were 38 VTE recurrences (3.5%, 95% confidence interval [CI]: 2.5–4.7%), with a rate of 3.1 per 100 patient-years (95% CI: 2.2–4.2). No patient died of recurrent PE (0%, 95% CI: 0–7.6%). Subgroup analyses showed that patients with diagnosis in 2021–2022 (vs. 2020) (Hazard ratio [HR] 2.86; 95% CI 1.45–5.68) or those with isolated deep vein thrombosis (vs. pulmonary embolism) (HR 2.31; 95% CI 1.19–4.49) had significantly higher rates of VTE recurrences. Interpretation: In patients with COVID-19-associated VTE who discontinued anticoagulation after at least 3 months of treatment, the incidence rate of recurrent VTE and the case-fatality rate was low. Therefore, it conceivable that long-term anticoagulation may not be required for many patients with COVID-19-associated VTE, although further research is needed to confirm these findings. Funding: Sanofi and Rovi, Sanofi Spain