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“Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015

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“Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 COST Action FA1104 Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 An overview on Prunus spp. cryopreservation with focus on cherry Emilia Caboni*, Simona Monticelli, Adele Gentile, Andrea Frattarelli Fruit Tree Research Center (CREA-FRU) Rome, Italy Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 The genus Prunus (Rosaceae family; Amygdaloideae subfamily) comprises 5 sub-genera (the most widely accepted classification) Prunus plums - apricots Amygdalus peaches - almonds Cerasus cherries Laurocerasus Padus Because of their considerable value as both food and ornamental plants, many Prunus species have been introduced to parts of the world to which they are not native and became naturalized. Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 AGROBIODIVERSITY LOSS Since the 1900s, about 75% of plant genetic agrobiodiversity has been lost since farmers worldwide have left their multiple local varieties and landraces for genetically uniform, high- yielding varieties. Today, 75% of the world’s food is generated from only 12 plants. Of the 4% of 300,000 known edible plant species, only 150 - 200 are used by humans. From www.fao.org/biodiversity/index.asp?lang=en Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 GERMPLASM CONSERVATION - In situ: in natural conditions - Ex situ: - seed collections - in field collections - in vitro culture based collections • Standard axillary and apical buds based culture • Slow growth • Cryopreservation Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 PRUNUS SEED COLLECTIONS • Seeds are considered intermediate tolerant to desiccation, but loose germinability quickly • Problems with breaking dormancy • Prunus spp. are genetically heterozygous and genetic integrity of varieties can be maintained through vegetative propagation Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 IN FIELD COLLECTION Conservation in clonal orchards needs huge space and it is risky due to climate avversities and diseases: phytoplasmas and viruses such as Sharka (Plum Pox Virus) are very common in Prunus spp. IN VITRO CULTURE Cost of sub-culturing and risks of somaclonal variation. SLOW GROWTH Cost of sub-culturing reduced compared to previous methods but only medium term conservation. Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 CRYOPRESERVATION: long term storage of living tissues at ultra-low temperatures (-196°C) (Sakai, 1960, on silver birch twigs) • Cryopreservation procedures are available for about 150-200 different plant species • For each species and explant type, the cryopreservation protocol needs to be adapted in function of their • natural freezing resistance • explant size and type • water content Cryopreservation of shoot tips or axillary buds is suitable for conservation of clonal material in high heterozigotic species, such as Prunus spp. Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 CRYOPRESERVATION IS BASED ON VITRIFICATION Vitrification is a physical process by which a highly concentrated solution supercools to very low temperatures and finally solidifies without undergoing crystallization. Thus, samples are cryopreserved without detrimental intracellular crystalline ice formation. Reviewed by Sakay and Engelman, 2006 Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 ENCAPSULATION DEHYDRATION Encapsulation dehydration (ED) was originally described for cryopreservation of in vitro grown pear shoot tips by Dereuddre (1990), improved by Nino and Sakai (1992). Applied to cryopreservation of shoot tips of several fruit tree species such as Actinidia, Malus and Vitis vinifera (reviewed by Engelmann et al., 2008). Advantage: explants are protected in beads during handling. Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 De Boucaud et al. (2002) applied ED to Prunus hybrids with regrowth of 47, 55 and 40%, respectively with 0.50, 0.75 and 1M sucrose dried in a laminar air flow for 3-4 h. Helliot et al. (2002) treated Prunus “Ferlenain” for 3 d with increasing concentrations of sucrose (0.25–1M) and desiccated them for 2–4 h in sterile air flow. Recovery was 50% regardless of the time of desiccation and the concentration of sucrose. Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 Soliman (2013) used ED on shoot tips of apricot: the highest regrowth percentage (60.4%) was obtained with two days in medium containing 0.75M sucrose and 2 h of dehydration. Shatnawi et al. (2007) applied ED to sweet cherry shoot tips: growth recovery up to 76.3% was obtained when encapsulated shoot tips were pre-grown for 3 d in liquid medium containing 0.75M sucrose, desiccated by to 20-22% moisture content and immersed in liquid nitrogen (LN). Ružić et al. (2013) applied ED to tips of cherry plum (Prunus cerasifera Ehrh.): 0.75M sucrose followed by 8 h desiccation gave the highest regrowth (60%) of explants encapsulated in 3% and 5% alginate beads. Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 VITRIFICATION Vitrification (V) has been applied to cryopreservation of in vitro grown shoot tips of several species included fruit tree species such as Actinidia, Musa, Prunus spp., Pyrus, Vitis (reviewed by Sakai and Engelman, 2007). Advantage: easy to be performed. Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 Niino et al. (1997) applied V to in vitro grown shoot tips of Prunus jamasakura Seib. cv. Sendaiya and 7 cherries: 0.7M sucrose (1 d) and PVS2 for 105 or 120 min gave survival rate higher than 70% in all the cultivars. Shatnawi et al. (1999) found V less effective than ED in almond, while Channuntapipat et al. (2000) found V effective in this species. Shatnawi et al. (2007) used V on shoot tips of P. avium from Jordan: high regrowth (up to 77.8%) was found when shoot tips were exposed to PVS2 for 60 min. Barraco et al. (2012) successfully cryopreserved by V sour cherry (Prunus cerasus L.) (cv. Montmorency and Schattenmorelle): the highest recovery percentages were between 41 and 63%. Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 Ružić et al. (2014) applied V to shoot tips of the cherry rootstock Gisela 5® (Prunus cerasus × Prunus canescens): the application of the PVS A3 (modified PVS2: 22.5% sucrose, 37.5% glycerol, 15% ethylene glycol and 15% DMSO in MS medium) resulted in considerably higher survival rates (81–92%), as well as higher regrowth rates (39–56%) after cryopreservation, than PVS2. De Carlo et al. (2000) applied V to plum (Prunus domestica) shoot tips in comparison with ED: with the optimised procedure V gave higher regrowth in respect to ED. DROPLET VITRIFICATION ‘‘Droplet freezing method’’ was established for potato (Schäfer- Menuhr et al., 1997) using a cryoprotectant solution containing 10% dimethylsulfoxide (DMSO). Later on, droplet vitrification (DV), with the ‘classical’ PVS2 solution, was used in Musa: the explants were included in individual micro- droplets of vitrification solution on small pieces of aluminum foils which were immersed rapidly in LN (Panis et al., 2005). Advantage: DV technique reaches cooling rates considerably higher compared to the other vitrification methods, thus reducing intracellular ice crystal formation during freezing. Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 De Boucaud (2002) managed to cryopreserve with DV Japanese plum (cv. Ogden), P. domestica (cv. Torinel) and 2 almond cultivars: the size of explant was critical to obtain high re-growth. Ružić et al. (2013) applied DV to shoot tips of the cherry rootstock Gisela 5 ® (Prunus cerasus × Prunus canescens): regrowth (max 40%) was not influenced by the type of PVS: PVS3 (Nishizawa et al., 1993) or PVS A3. Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 National Collection of fruit germoplasm (CNGF) Fruit Tree Research Center (CREA- FRU) - Rome, Italy: National repository for fruit species (8000 accessions). Activities: collection, characterisation and valorisation of fruit agrobiodiversity RGV – FAO project financed by the Italian Agricultural Ministry for conservation of agrobiodiversity Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 Accessions conserved at CNGF Peach/Nectarine: 24% Malus: 21% Pyrus: 18% Cherry: 14% Plum: 5% Apricot: 5% Old varieties Almond: 3% and “wild Walnut: 2% relatives” Hazelnut: 2% Kiwi: 2% other: 4% In vitro: 180 accessions partially in standard culture and partially in slow growth Workshop “Long Term Preservation of Woody Species by Cryo-Techniques”. Florence, Italy, 26-27 March 2015 Cryopreservation by ED in Prunus spp. at CREA-FRU Cryopreservation of apices of in vitro plantlets of almond (Prunus dulcis Mill.). CryoLetters, 20:13-20. Mohamad A. Shatnawi1,2, Florent Engelmann3, Andrea Frattarelli2 and Carmine Damiano2 1: Plant Tissue Culture Laboratory,
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