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Somatic embryogenesis, as a promising biotechnological tool for many conifer trees, has never been applied for the Abies nebrodensis species. Although all the encouraging results previously obtained by the EU LIFE (European LIFE program) funded projects in over ten years, the critically endangered Sicilian fir remains alarmingly close to extinction. In this study, we reported the first protocol of somatic embryogenesis obtained from mature zygotic embryos of the Abies nebrodensis. Seeds from Abies adult trees with specific identification numbers (IN) were collected and full seeds were identified by X-ray. Different experiments were carried out for callus initiation, from both zygotic immature and mature embryos, testing different culture media. The immature embryos did not give embryogenic tissue (ET). Embryogenic callus (EC) was successfully induced from mature embryos with variable frequencies (0–40%). Schenk and Hilderbrandt (SH) was the most suitable initiation medium where the obtained callus initiation rate reached up to 40% for IN7 (first experiment). 6-benzylaminopurine (BAP) showed to be essential to induce EC (second experiment). IN8 presented the highest callus initiation rate (40%) among all tested donor trees, whereas IN13 recorded the lowest rate with 4% (third experiment). ET maturation from each singular embryo of IN7, IN8, IN10 and IN21 was successfully achieved in SH medium containing 37,83 µM abscisic acid (ABA), 8% of polyethylene glycol (PEG-4000) and 4% maltose. The encapsulation technology was assessed on the obtained ET and its proliferation was observed after encapsulation.

Abies nebrodensis (Lojac.) Mattei is an endemic species of the north-west of Sicily located in an 84 ha area in the Madonie Regional park. The current population is limited to 30 relic adult trees and a fluctuating number of juveniles of natural regeneration. The species is defined as “Critically Endangered” in the Italian list of threatened plants and is classified as CR-D in the 2000 IUCN Red List of Threatened Species. This article reports the key action undertaken by the LIFE4FIR project aimed at preserving A. nebrodensis, and the results obtained so far in three years of activity. OpenArrays SNPs genotyping revealed a high rate of inbreeding in the natural population and that the adult trees are genetically related. Controlled cross-pollination was consequently performed to increase the genetic variability of the progeny. Outbred offspring are currently being grown in the nursery. Reforestation has been planned by using 4000 selected outbred seedlings in 10 areas within Madonie Park to create re-diffusion cores. Support and protection of the relic population have been implemented through regular phytosanitary surveys, as well as new fencing and video surveillance systems against grazing and wild herbivores. A seedbank and cryobank for the long-term germplasm conservation have been established.

First developed for biomedical and industrial applications, nanovectors have recently been extended to agriculture. Therefore, innovative plant growing procedures making use of nanoparticles should be adapted to sustainable processes and materials. This work aims at proposing newly synthetized polymeric nanocapsules (NCs) to be used as biocompatible vectors for delivery of bioactive compounds to plants. Nanoparticles were fabricated from lignin, which is the main byproduct of wood processing and is currently a waste material. Lignin can thus find a virtuous fate and be reused in the context of circular economy. Specifically, we loaded lignin NCs with Gibberellic Acid (GA), assessing that stable and reproducible nanoparticles could be obtained in a range of GA content that is relevant for delivery purposes, i.e. 0.5-1.5 mg ml-1. Plain and GA-loaded NCs were characterized by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM). Assays of cytotoxicity and cargo release were carried out in two model plants, Eruca vesicaria and Solanum lycopersicum. These experiments, conducted both in vitro and in vivo, included the investigations of the percentage of germination, the stem and primary root lengths, as well as the fresh and dry weight of treated plants vs non-treated ones. Furthermore, NCs were loaded with Fluorol Yellow 088 to track their entrance and accumulation in seeds and seedlings.

Investigations on the lithobiontic colonization of the stone cultural heritage in (semi-)arid regions are needed to address conservation strategies. In this work, lithobiontic communities were examined on the carbonate rock surfaces of the heritage site of the Tomb of Lazarus. We aimed to evaluate their distribution and interaction with the lithic substrate, together with the efficacy of biocidal treatments for their control. Diversity and abundance of lithobionts were surveyed on the Jerusalem stone blocks of three architectural elements. Observations at the lichen-rock interface were carried out by reflected light and scanning electron microscopy. The efficacy against lichens of the widely used biocide benzalkonium chloride (BZC) was compared for different concentrations and application methods, and evaluated by epifluorescence microscopy. Chlorolichens were the dominant component of lithobiontic communities, more thoroughly adapted to the semi-arid conditions of the site than mosses and black biofilms of cyanobacteria and dematiaceous fungi. A different structural organization, in terms of thallus thickness and depth of the hyphal penetration component, characterized epilithic and endolithic lichen species, responsible for different deteriogenic activities. Biocidal assays showed that even the methodologies that are usually effective in temperate conditions (as the application of BZC 1.5% by poultice) may not completely devitalize lichens adapted to the stress conditions of semi-arid climates, unless a pervasive biocide diffusion through metabolically active thalli is carefully guaranteed. Lithobionts act as biodeteriogens on the semi-arid surfaces of the investigated heritage site. Their removal is thus recommendable, but it needs to be adequately supported with a careful calibration of devitalization strategies.

The olive is a long-lived evergreen tree, capable of regenerating itself from any part of the plant (pollard tree). This characteristic has allowed the olive trees to survive across the centuries until they become real natural monuments. The aim of this work has been to study the monumental “Olivo della Strega” (“Witch’s Olive tree”) olive tree, located in the olive orchard of Santissima Annunziata church, in Magliano (Grosseto, Italy), a tree with a special place in local history and folklore and with estimated age over 700 years. The morphometric data of leaf, fruit, stone and pollen were used to characterize the phenotype, while genetic diversity was assessed on the basis of microsatellite markers. The morphological and genetic analysis pointed to two clearly distinguishable parts in the “Olivo della Strega” tree, called sample left (SL) and sample right (SR). This ancient plant consists of two different genotypes: one part (SL) was identified as cv Frantoio and the other (SR) as an unknown genotype. Moreover, traditional (in vivo conservation) and advanced techniques (cryopreservation) have made conservation achievable to preserve its genetic heritage. In particular “Olivo della Strega” pollen was stored for 1 year in liquid nitrogen. The morphology of pollen grains, viability and germinability of fresh and long term cryopreserved pollen were observed at different times during preservation, without revealing significant differences.

Cryopreservation is a useful tool for the long-term storage of plant genetic resources, and different cryogenic procedures have recently been developed. The present study focused on the use of the Droplet-vitrification (DV) and V cryo-plate protocol for the cryopreservation of Stevia rebaudiana in vitro-derived apical shoot tips and axillary shoot tips. A preliminary test showed that 90 and 120 min PVS2 (Plant Vitrification Solution 2) treatment significantly reduced the regrowth of the explants before immersion in liquid nitrogen (LN). For both procedures tested, the best osmoprotective condition for obtaining a higher regrowth of cryopreserved explants occurred when explants were PVS2 treated for 60 min. After direct immersion in LN, thawing and plating, the highest regrowth recorded was 80% with DV and 93% with V cryo-plate. Moreover, shoot tips proved to be a more suitable material for Stevia cryopreservation. A satisfactory vegetative regrowth was observed in the subcultures following cryopreservation by DV and V cryo-plate cryogenic procedures.

Nanoscience has allowed outstanding progress in many fields of research. Concerning the transport and delivery of active principles (drugs, contrast agents, sensitizers etc.), the improvements obtained by miniaturized carriers have recently been extended from medicine and pharmacy to agriculture. However, when coming to crop production, issues such as scalability, eco-compatibility, and sustainability represent a veritable challenge, so far only partially addressed. In this study, we propose lipid-based nanoformulations for the administration of root-promoting phytohormones to different cultivars of Olea europaea. In order to maximize the efficiency of these novel carriers, we devised nanovectors made by lipids extracted from olive pomace, a material derived from the plant itself and representing a waste in oil processing. This allowed combining cost-effectiveness and environmentally friendly procedures. The implementation obtained using adjuvants, such as purified natural lipids forming stable and well-defined nanoobjects, was also investigated. Controlled and reproducible formulations were achieved after extensive study of the obtained systems, in particular by physicochemical characterization through advanced methods. Trials in vivo and in vitro showed that rooting was enhanced with respect to conventional treatments, thus indicating that the innovative formulations here fabricated have significant potentiality for the large-scale administration of agrochemicals in the context of sustainable economy.

BACKGROUND: Vaccinium myrtillus L. is a valuable wild fruit species of conservation concern in the Tuscan Apennines (Italy). OBJECTIVE:The development of a micropropagation protocol for the conservation of this species in in vitro-banks for the year-round mass production of specific selected superior bilberry genotypes, maintaining the genetic variability of natural populations, avoiding genetic erosion. METHODS:Nodal segments from seedlings were cultured on mMS medium with zeatin or 2iP for multiplication and elongation stage, afterwards shoots were rooting in vitro and in vivo. The RAPD analysis was applied to lines obtained and slow growth storage (4°C - darkness) was evaluated for the conservation. RESULTS:Zeatin concentration 9.1?M was the most favorable for shoot proliferation. The activated charcoal was essential for in vitro rooting. Both in vitro and ex vitro methods were successfully used to root and acclimatize bilberry shoots. The RAPD analysis indicated a large genetic diversity among the micropropagated genotypes. Shoots were successfully in vitro preserved for up to 6 months with no apparent deterioration effect. CONCLUSIONS: In vitro propagation and conservation are fundamental tools for exploiting the whole potential of this wild crop species for breeding and genetic improvement purposes.

Aquatic ornamentals are generally used for aquarium decorations because of their colored foliage and attractive appearance. Cryptocoryne species are popular due to their easy growth and capacity to thrive for a long time. Cryptocoryne lutea is an excellent mid-ground plant with bright green or bronzy oblong leaves. Rotala rotundifolia is characterized by a rapid growing stem and beautiful growth pattern; for these features it is considered the ideal aquarium plant for beginners. These aquatic plants are usually easily propagated by cutting, but previous studies allowed to develop a suitable protocol of micropropagation. A renewed interest is due to the possibility to obtain synthetic seeds by in vitro-derived propagules to be used in aseptic or in ex vitro conditions for propagation system.

Experiments were performed to determine the influence of various dehydration and vitrification treatment times on the ‘one-step freezing’ cryopreservation of embryonic axes (EAs), composed of zygotic embryos and cotyledon residuals, from mature seeds of a Georgian provenance of chestnut (Castanea sativa Mill.). Dehydration was carried out in laminar flow hood from 1 to 5 h, and vitrification experiments were carried out by immersion of EAs in PVS2 vitrification solution up to 120 min, both followed by direct immersion in liquid nitrogen. Both systems resulted in inducing specimen tolerance to ultra-rapid freezing, although to a different extent. Full germination of cryo-stored EAs after 5 h of dehydration (reducing moisture content from initial 66% to 21%) has been increased from 0% to 66.7%. A pre-treatment of EAs in PVS2 vitrification solution for 30 min produced fully developed plantlets at a rate of 55.6% in post-cryopreservation. Plantlet regrowth from cryopreservation was faster in EAs that underwent the dehydration/‘one-step freezing’ procedure. All the plantlet from cryopreserved EAs could be easily acclimatized, producing healthy potted plants. Finally, the TTC test showed to be useful for a fast evaluation of specimen survival after thawing and, as a consequence, to speed up the development of optimized cryo-protocols. ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. *********

Olea europaea L. cv Canino shoots were micropropagated to test two different culture systems: (1) on conventional semi-solid medium in glass jars and (2) in liquid culture in a Plantform(TM) bioreactor. The temporary immersion system, Plantform(TM), is a new propagation approach that uses liquid culture, where shoots undergo periodic immersion in liquid medium alternated with dry periods, avoiding gas accumulation through forced ventilation. This study proposes a protocol to improve in vitro propagation of olive reducing production costs. Our findings revealed that olive shoots propagated in Plantform(TM), with an immersion frequency of 8 min every 16 h and additional ventilation, showed good adaptability and better growth rates than those cultured in conventional system. Overall, the Plantform(TM) improves in vitro culture of 'Canino', showing higher proliferation, shoot length and better vigour of shoots. Moreover, the study found no significant differences in shoot length when 5 or 10 µM zeatin was applied in Plantform(TM) (3.04 and 3.13 cm, respectively); it is, therefore, possible to achieve efficient olive proliferation also with half hormone concentration. The positive performance of the bioreactor approach was also confirmed by Relative Growth Rate index. This is the first documented study of the Plantform(TM) technique for olive propagation.

У раду су приказани резултати анализе садржаја мангиферина, (1,3,6,7- тетрахидроксиксантон-C2-β-D-гликозид) у различитом биљном материјалу балканске ендемичне перунике (Iris reichenbachii Heuffel). Помоћу методе течне хроматографије под великим притиском (HPLC), садржај мангиферина анализиран је у биљном материјалу сакупљеном на природним стаништима, током гајења у култури in vitro, као и у регенерисаним биљкама гајеним у условима ex vitro (стакленик и башта института). Kод биљака сакупљених са природних станишта садржај мангиферина у биљном ткиву зависио је од биљног органа, као и од места где су биљке сакупљене. Током индукције регенерације биљака у култури in vitro применом културе зрелих зиготских ембриона на хранљивим подлогама обогаћеним са 2,4-дихидрофенокси сирћетном киселином продукција мангиферина у биљном ткиву зависила је од састава хранљиве подлоге као и од степена диференцијације ткива. Највећа продукција мангиферина током гајења у условима in vitro постигнута je током индукције формирања изданака док је најмања продукција мангиферина добијена у ембриогеним калусним културама где је забележена синтеза мангиферина само у траговима. После криопрезервације врхова изданака методoм витрификацијe у капљици добијен је исти ниво синтезе мангиферина у изданцима, као и пре криопрезервације. Највећи садржај мангиферина уочен је у надземним деловима биљака регенерисаних у култири ткива и аклиматизованих на спољашње услове. Садржај мангиферина код биљака добијених применом ове методе био је знатно већи него код биљака сакупљених из природних станишта. У овом раду представљен је потенцијал техника културе ткива и криопрезервације у циљу ex situ заштите једне ендемичне биљне врсте као и потенцијал ових техника за производњу секундарних метаболита као што је мангиферин. Применом ових техника може се произвести овај секундарни метаболит за потребе фармацеутске индустрије, без уништавања биљног материјала са природног станишта.

The present study investigated the effects of gas- tight and gas-permeable culture containers and different sucrose concentrations, as well as sucrose and mannitol combinations on the development of an effective in vitro slow growth storage protocol (at 4 °C, in darkness) for 'Gisela®5' shoot cultures. 'Gisela®5' is the most widely used cherry rootstock in Europe. This dwarf triploid hybrid has many advantages over the conventional cherry root- stocks. Optimizations for the cold storage of 'Gisela®5' in vitro shoot cultures included use of storage medium supplemented with 10, 20, 30, 45, and 60 g L-1 sucrose and sucrose (15, 30 g L-1) and mannitol (15 g L-1) combinations, contained in gas-tight glass jars and gas- permeable 'Star PacTM' bags. Cold storage was prolonged to 12 months, during which in every 3 months, cultures were evaluated. Possibility of 16 month-cold storage in gas-tight glass jars was also explored, during which gas chromatographic analysis was performed for the detection of CO2 and ethylene accumulation for the first 5 months of cold storage. Our results showed that both the 12- and 16-month conservations were possible, especially when 45 or 60 g L-1 sucrose was supplemented to storage medium, contained in glass jars. Mannitol inclusion to the storage medium was also effective to reduce the metabolic activity of the shoot cultures during storage; however, it did not have a significant positive influence on shoot quality in post-conservation.

Artichoke is of great economic significance in the Mediterranean basin. Although Italy has the richest primary cultivated gene pool, only a limited number of varietal types are vegetatively propagated to fit market demand. This could lead to the erosion of genetic resources. The development of cryopreservation procedures for the ex situ conservation of the genetic resources of artichoke is advisable not only to counter genetic erosion, but also to develop a certified phytosanitary germplasm collection. In this study, several parameters were tested in order to set up a simple and efficient procedure for the cryopreservation of artichoke shoot tips, based on vitrification/one-step cooling, which was successfully applied to two spring cultivars ‘Grato 1’ and ‘Campagnano’. Cold acclimation prior to cooling was essential for tissue recovery and regrowth. The size of the shoot tips was also critical, and medium size tips (3–4 mm in length) proved to be the best responding starting material. The best survival percentage of cryopreserved shoot tips was 61% for ‘Grato1’ and 55% for ‘Campagnano’. Cryopreserved shoot tips developed directly into plantlets, which proved to be stable in terms of chromosome numbers. The cryopreservation procedure was notably effective in eliminating the endemic Artichoke Latent virus (ArLV) from both cultivars, irrespective of the shoot tip size used, which were up to 10 times the size used in meristem cultures for virus elimination. All plantlets analyzed for ArLV by RT-PCR were virus-free eight months after regeneration. An analysis of ArLV elimination and chromosome numbers of cryopreserved artichoke plants is presented here for the first time.

Diplomazia2 is framed within the program "Science for Diplomacy: Multi-disciplinary Training Program", and it is the result of closer and more mature collaboration between the Directorate General for Cooperation on Development of the Ministry for Foreign Affairs and International Cooperation (MAECI), the Italian Agency for Cooperation on Development (AICS) and the National Research Council (CNR). The implementation of joint projects such as DIPLOMAzia2 highlights the effectiveness of the diplomacy-science partnership as the driving force behind the achievement of relevant goals jointly pursued by the international community.

In this study, the genes of the Aux/IAA family were used as functional markers to characterise bud break stages in a white poplar clone. In the first experiment, under greenhouse conditions, the sprouting of repressed sylleptic buds was obtained by pruning the shoot tip during growing season. Buds were collected at 0, 6, 24 and 48 h after pruning for molecular analyses. A decrease in transcript level of IAA4 and IAA8 genes was observed in the first and second bud below the cut after 6 and 24 h, respectively. In the second experiment, bud break of post-dormant proleptic buds was induced by forcing in climatic chamber. The first 6 and the following 6 buds below the apical one were sampled every 48 h during forcing. Anatomical studies were also carried out on buds and plants were equipped with stem and bud radial growth sensors to check their swelling. In both experiments, gene expression patterns showed a decrease and a successive increase in expression of IAA4 and IAA8 genes during bud break. The transient down-regulation of these genes was observed only in buds that formed new branches. Thus, similar molecular mechanisms are involved in bud break of both sylleptic and proleptic buds.

A morphological and anatomical study of regenerants obtained from mandarin anther culture was carried out. Beside morphologically normal somatic embryos, abnormal structures were originated in the course of somatic embryogenesis. Anatomical anomalies can be found at several growth stages, causing the formation of slender stems, stubby structures, non-functional leaves. When too long, some structures are subject to shedding, with the formation of various abscission zones. Most of them are subject to degeneration, although many are capable of further, localized, morphogenesis. A thorough knowledge of morphology and anatomy of normal and abnormal regenerants could make possible to select and subculture the lines considered most suitable for conversion into plantlets.

The cryogenic technology can be regarded as an important biotechnological approach, offering unrivalled opportunities for cost-effective long-term germplasm preservation of plants. New cryopreservation techniques are now available, aiming at the direct immersion in liquid nitrogen (“one-step freezing”) of explants, with a considerable simplification of procedures. This technology is based on the induction of cell vitrification during a very fast decrease of their temperature and in this report it has been applied on shoot tips, excised from dormant buds of Diospyros kaki. Shoot tips of cvs. ‘Hirathanenashi’ and ‘Triumph’, showed a survival of 86% and 80%, respectively, after they were incubated at 25°C in the Plant Vitrification Solution (PVS2) and plunged in liquid nitrogen. Afterwards, in order to validate the procedure for the preservation of this germplasm, the RAPDs analysis was carried out to ascertain the genetic stability of the cryopreserved shoots.

The application of three different vitrification-based freezing strategies for the cryostorage of white poplar (Populus alba L.) and hybrid aspen (P. tremula L. × P. tremuloides Michx.) have been assessed. The PVS2 vitrification protocol was successfully applied to two white poplar in vitro clones stored for more than 6 months in slow-growth conditions (4 °C, in darkness) and showing clear signs of explant etiolation and decay. After 60 min of PVS2 treatment, P. alba L. (cv. Villafranca) explants isolated from axillary buds demonstrated significantly better potential for post-freeze regrowth (64%) compared to those obtained from apical buds (17%). Similarly, a high level of survival (78%) of the frozen hybrid aspen shoot tips was recorded following the application of the same technique. Using the ‘encapsulation-vitrification’ procedure, no toxic effects of the PVS2 treatment were noticed after 120 min exposure, however none of the cryopreserved (poplar and aspen) explants survived after 3 weeks. In contrast, the ‘droplet-vitrification’ technique appeared to be very efficient in the cryopreservation of white poplar shoot tips, which increases the opportunities for wider application of this method in other woody species.

Red chicory (Cichorium intybus L.) is one of the most economically important vegetables produced in the Italian region of Veneto. Here, a programme for the medium-term (by slow-growth storage) and long-term (by cryopreservation) conservation of red chicory selected lines was initiated in 2002. As regards slow growth storage, shoots from the proliferation stage were stored in one of the following conditions: (i) at 4°C, in darkness or under a 8-h photoperiod (with 15 μmol m -2 sec -1 of light intensity), (ii) on a storage medium added of osmotically active compounds (OACs), i.e., NaCl (4.48 g L -1 ) or mannitol (40 g L -1 ), and (iii) at 4°C and in darkness, comparing the storage in gas-tight plastic cylinders, in slow gas-permeable glass jars or in gas-permeable plastic bags ('StarPac'). After 9 months, best storage conditions for the 'Treviso precoce' line in terms of shoot survival (95%) were obtained with the conservation at 4°C, with the shoots maintained in the dark showing a lower relative growth rate (RGR). As for the storage in OACs, a higher shoot survival (70%) and a lower RGR was obtained with the use of mannitol. A general lower adaptability to SGS was evidenced by the 'Chioggia' line, as 100% shoot survival was recorded after 6 months of conservation at 4°C in darkness, but this value dramatically dropped to 45% after 9 months. All the tested containers allowed a very satisfactory conservation of shoots at 4°C in the dark for the whole period (9 months). However, higher RGR values were always recorded from shoots stored in the 'StarPac' gas-permeable plastic bags. As regards cryopreservation, red chicory shoot tips survived and regrew satisfactorily from the storage at -196°C when a procedure of vitrification/one-step freezing, consisting in loading the explants in the PVS2 vitrification solution, was applied.

This paper presents the advances made over the last decade in cryopreservation of economically important vegetatively propagated fruit trees. Cryopreservation protocols have been established using both dormant buds sampled on field-grown plants and shoot tips sampled on in vitro plantlets. In the case of dormant buds, scions are partially dehydrated by storage at -5 degrees C, and then cooled slowly to -30 degrees C using low cooling rates (c.a. 1 degrees C/h) before immersion in liquid nitrogen. After slow rewarming and rehydration of samples, regrowth takes place either through grafting of buds on rootstocks or excision of apices and inoculation in vitro. In the case of shoot tips of in vitro plantlets, the cryopreservation techniques employed are the following: controlled rate cooling procedures involving slow prefreezing followed by immersion in liquid nitrogen or vitrification-based procedures including encapsulation-dehydration, vitrification, encapsulation-vitrification and droplet-vitrification. The current status of cryopreservation for a series of fruit tree species including Actinidia, Diospyros, Mobs, Olea, Prunus, Pyrus and Vitis is presented. Routine application of cryopreservation for long-term germplasm storage in genebanks is currently limited to apple and pear, for which large cryopreserved collections have been established at NCGRP, Fort Collins (USA), using dormant buds and in vitro shoot tips, respectively. However, there are a growing number of examples of pilot scale testing experiments under way for different species in various countries. Progress in the further development and application of cryopreservation techniques will be made through a better understanding of the mechanisms involved in the induction of tolerance to dehydration and ctyopreservation in frozen explants.

In vitro preservation of the "Kober 5BB" rootstock (Vitis berlandieri × Vitis riparia) was assessed with the encapsulation technique and slow growth storage. Shoot tips and nodal segments excised from in vitro cultures were encapsulated in calcium-alginate beads. A 30 min ion exchange time proved optimal for forming proper beads. The encapsulated and naked explants were stored at 4 °C in the dark or light. After 9 months of cold storage, the highest regrowth, 83.3%, was recorded for the encapsulated shoot tips maintained in darkness. The development of the encapsulated nodal segments was 55.6% under the same storage conditions. The encapsulated explants had a better regrowth capacity after storage than the naked explants.

Persimmon (Diospyros kaki Thunb.) is a temperate fruit tree species diffused in all continents. The traditional propagation method adopted by the nursery industry is based on budding/grafting scion cultivars on seedlings from D. kaki, Diospyros lotus, and Diospyros virginiana, the most important species used as rootstock, reproduced by seeds since they are not easy to root. Furthermore, most of nonastringent cultivars of persimmon are not compatible with D. lotus, a rootstock largely utilized because of its hardiness and frost resistance. The main in vitro tissue culture techniques, developed for persimmon, deal with direct regeneration (from dormant buds and root tips), and indirect regeneration through callus from dormant buds, apexes, and leaves. The bottlenecks of micropropagation are (1) the recalcitrance of many cultivars to in vitro establishment, (2) the low multiplication ratio of D. kaki compared to other fruit tree species, (3) the very low rooting ability of ex novo microcuttings both from direct and indirect regeneration, (4) the high sensitivity to transplant from in vitro to in vivo conditions. The development of reliable in vitro regeneration procedures is likely to play a key role for production of both clonal rootstocks and self-rooted cultivars. The general protocol for micropropagation of persimmon reported here is based on the establishment of winter dormant buds in vitro, shoot development, multiplication and elongation, and shoot rooting, using cytokinins (BA or zeatin) in a MS media along with an auxinic pretreatment for rooting induction.

A three-day pretreatment of olive somatic embryos (SE) with 0.75 M sucrose, combined with cryoprotection (0.5 M DMSO, 1 M sucrose, 0.5 M glycerol and 0.009 M proline) and controlled rate cooling, supported regrowth (as 34.6% fresh weight gain) and resumption of embryo development after cryopreservation. Pretreatment with mannitol or sorbitol did not support regrowth. Profiles of sugars, proline, antioxidant enzymes, Reactive oxygen species (ROS), secondary oxidation products and ethylene were constructed for the most successful (0.75 M) pretreatment series. Sucrose was the optimal pretreatment for supporting recovery, it also elevated glutathione reductase (GR) activity compared to controls, whereas superoxide dismutase (SOD), catalase and guaiacol peroxidase activities remained relatively unchanged. Superoxide dismutase activity was higher in SE pretreated with sucrose, compared with those pretreated with polyols; H2O2 was enhanced in SE pretreated with sorbitol and sucrose compared to mannitol. The overall trend for ethylene and OH production revealed their levels were highest in SE pretreated with polyols albeit, for individual treatments this was not always the case. Generally, pretreatments did not significantly change embryo secondary oxidation profiles of T hio B arbituric A cid R eactive S ubstances (TBARS) and Schiff's bases. In combination these studies suggest oxidative processes may influence regrowth of cryopreserved olive SE and that optimal pretreatments could, in part, increase tolerance by an overall enhancement of endogenous antioxidants (particularly GR), proline and sugars.