Anna De Carlo

Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis

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 °C, and then cooled slowly to -30 °C using low cooling rates (c.a. 1 °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, Malus, 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 cryopreservation in frozen explants.

Application of Tissue Culture to the Germplasm Conservation of Temperate Broad-Leaf Trees

The recovery and conservation of a broad genetic pool of wild and domesticated species are essential for the maintenance of plant biodiversity. Traditionalex-situconservation systems (i.e., seed and field collections), alongsidein-situconservation, make a fundamental contribution to the preservation of plant genetic resources. Up to now these approaches have been the only reliable option for the long-term germplasm preservation of woody species, although they have several drawbacks. Numerous forest Angiosperms (e.g.,Acerspp.,Quercusspp., chestnut, horsechestnut, many tropical species) have non-orthodox (recalcitrant) seeds that quickly loose their viability or are subject to a rapid decrease in germinability during storage (Pence, 1995). Moreover, recalcitrant seeds do not tolerate the level of dessication that is commonly required to prolong conservation (Roberts, 1973). As regards temperate fruit trees, the majority of the species belonging to this group are reproduced by vegetative propagation, thus requiring the conservation in clonal orchards of huge numbers of accessions, including old and newly selected cultivars, local varieties and wild material. Table 1, for instance, gives a clear idea of the efforts required today for the preservation of the EuropeanPrunusgermplasm, kept in the field repositories of 21 countries.

Xylem morphology determines the drought response of two Arundo donax ecotypes from contrasting habitats

Arundo donax exhibits rapid growth and requires little nutrient input, making it an ideal perennial biomass crop species. However, this growth is accompanied by high rates of water use, potentially restricting the use of A. donax in rain‐fed marginal lands. Here, we investigated the physiological and morphological responses to drought in two ecotypes of A. donax from contrasting habitats: one from an arid environment in Morocco, and the second from a warm humid sub‐Mediterranean climate in central Italy. Prolonged drought resulted in identical reductions in leaf‐level photosynthesis (PN) and stomatal conductance (Gs) in the two ecotypes. However, water deficit induced an increase in xylem vessel diameter in the Moroccan plants, improving the movement of water along the stem, but also likely reducing the resistance to embolism. In contrast, the Italian ecotype reduced xylem vessel area, thus increasing resistance to water transport and xylem embolism. The increased xylem vessel size and associated vulnerability to embolism in the Moroccan plants may have contributed to an increase in the loss of leaf numbers, but also to higher relative water content (RWC) in the remaining leaves in comparison to the Italian ecotype, where a greater number of leaves persisted. Despite the Moroccan plants possessing stems with a lower basal area than their Italian counterparts, both ecotypes exhibited identical leaf to supporting stem area ratios under both control and water deficit conditions. This may account for the similarities observed in leaf area measures of PN and Gs in this and previous studies of different A. donax ecotypes. Selection of A. donax ecotypes on the basis of xylem responses to drought may facilitate the development of varieties suited to arid environments prone to severe drought and wetter habitats where prolonged droughts occur less frequently.

Cryogenic Technologies for the Long-Term Storage of Citrus Germplasm

With its beautiful trees, Citrus species have long been valued by humanity. The tasteful fruits, extensively used for nutrition, are also good for health due to the high content in vitamins, minerals, and dietary fibers. Like majority of the woody fruit plants, Citrus germplasm is conserved mainly as field collections in clonal orchards. However, such a traditional approach presents several difficulties, among which are the high cost, manual labor, and extensive land required to maintain the collections, as well as the necessity of a careful protection of plants from diseases and extreme environmental conditions. As many species in the genus have seeds recalcitrant to desiccation, conservation in seed banks is also inadequate. On the other hand, cryopreservation, i.e., the storage of specimens at ultra-low temperatures (usually in liquid nitrogen, at -196°C) where reactions within the cells are minimized, presents a unique alternative for the safe storage of such germplasm. The present contribution outlines the cryopreservation techniques applied to seeds, zygotic and somatic embryos, embryogenic callus cultures of Citrus spp. and provides sample protocols to be used for Citrus conservation.

The Impact of Heat Stress and Water Deficit on the Photosynthetic and Stomatal Physiology of Olive (Olea europaea L.)—A Case Study of the 2017 Heat Wave

Heat waves are predicted to increase in frequency and duration in many regions as global temperatures rise. These transient increases in temperature above normal average values will have pronounced impacts upon the photosynthetic and stomatal physiology of plants. During the summer of 2017, much of the Mediterranean experienced a severe heat wave. Here, we report photosynthetic leaf gas exchange and chlorophyll fluorescence parameters of olive (Olea europaea cv. Leccino) grown under water deficit and full irrigation over the course of the heat wave as midday temperatures rose over 40 °C in Central Italy. Heat stress induced a decline in the photosynthetic capacity of the olives consistent with reduced ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity. Damage to photosystem II was more apparent in plants subject to water deficit. In contrast to previous studies, higher temperatures induced reductions in stomatal conductance. Heat stress adversely affected the carbon efficiency of olive. The selection of olive varieties with enhanced tolerance to heat stress and/or strategies to mitigate the impact of higher temperatures will become increasingly important in developing sustainable agriculture in the Mediterranean as global temperatures rise.

Drought response of Mucuna pruriens (L.) DC. inoculated with ACC deaminase and IAA producing rhizobacteria

Drought is one of the major constraints limiting agricultural production worldwide and is expected to increase in the future. Limited water availability causes significant effects to plant growth and physiology. Plants have evolved different traits to mitigate the stress imposed by drought. The presence of plant growth-promoting rhizobacteria (PGPR) could play an important role in improving plant performances and productivity under drought. These beneficial microorganisms colonize the rhizosphere of plants and increase drought tolerance by lowering ethylene formation. In the present study, we demonstrate the potential to improve the growth of velvet bean under water deficit conditions of two different strains of PGPR with ACCd (1-Aminocyclopropane-1-Carboxylate deaminase) activity isolated from rainfed farming system. We compared uninoculated and inoculated plants with PGPR to assess: a) photosynthetic performance and biomass; b) ACC content and ethylene emission from leaves and roots; c) leaf isoprene emission. Our results provided evidence that under drought conditions inoculation with PGPR containing the ACCd enzyme could improve plant growth compared to untreated plants. Ethylene emission from roots and leaves of inoculated velvet bean plants was significantly lower than uninoculated plants. Moreover, isoprene emission increased with drought stress progression and was higher in inoculated plants compared to uninoculated counterparts. These findings clearly illustrate that selected PGPR strains isolated from rainfed areas could be highly effective in promoting plant growth under drought conditions by decreasing ACC and ethylene levels in plants.

КУЛТУРА ТКИВА И КРИОПРЕЗЕРВАЦИЈА КАО МЕТОДЕ ЗА ПОВЕЋАЊЕ ПРОДУКЦИЈЕ МАНГИФЕРИНА КОД БАЛКАНСКЕ ПЕРУНИКЕ (IRIS REICHENBACHII HEUFFEL)

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.