Development of an Efficient ‘One-Step Freezing’ Cryopreservation Protocol for a Georgian Provenance of Chestnut (Castanea sativa Mill.) Zygotic Embryos
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.
Barstow M, Khela S (2018). Castanea sativa. The IUCN Red List of Threatened Species, e.T202 948A67740523 from http:// dx.doi.org/ 10.2305/IUCN.UK.2018-1.RLTS. T202948A67740523.en.
Benelli C, De Carlo A, Engelmann F (2013). Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis. Biotechnology Advances 31(2):175-185.
Berjak P, Dumet D (1996). Cryopreservation of seeds and isolated embryonic axes of neem (Azadirachta indica). CryoLetters 17:99-104.
Berjak P, Pammenter NW (1997). Progress in the understanding and manipulation of desiccation-sensitive (recalcitrant) seeds. Basic and Applied Aspects of Seed Biology 30:689-703.
Berjak P, Walker M, Watt MP, Mycock DJ (1999). Experimental parameters underlying failure or success in plant germplasm cryopreservation: A case study on zygotic axes of Quercus robur L. CryoLetters 20:251-262.
Capuana M, Di Lonardo S (2013). In vitro conservation of chestnut (Castanea sativa) by slow growth. In Vitro Cellular & Developmental Biology -Plant 49(5):605-610.
Conedera M, Jermini M, Sassella A, Sieber TN (2004). Récolte, traitement et conservation des châtaignes [Collection, treatment and conservation of chestnut fruits]. Notice pour le praticien, WSL, Birmensdorf 38:1-12.
Corredoira E, San-Jose MC, Ballester A, Vieitez AM (2004). Cryopreservation of zygotic embryo axes and somatic embryos of European chestnut. CryoLetters 25:33-42.
Gagnidze R (1975). Fagaceae. In: Ketskhoveli N, Kharadze A, Gagnidze R (Eds). Sakartvelos flora [Flora of Georgia], 2nd ed, vol III, Metsniereba, Tbilisi, pp 40-43.
Giacalone G, Bounous G (1993). Tradizioni ed innovazioni nella trasformazione e nell’utilizzo delle castagne [Tradition and innovation in the transformation and utilization of chestnut fruits]. Monti Boschi 44(5):33-41.
Gulisashvili VZ (1961). Dendroflora Kavkaza [Dendroflora of the Caucasus]. Izdatelstvo AN GSSR, Tbilisi.
IUCN Red List of Threatened Species (2019). IUCN Red List version 2019-1, Sweet Chestnut (Castanea sativa) from https://www.iucnredlist.org/species/202948/67740523.
Kim HH, No NY, Shin DJ, Ko HC, Kang JH, Cho EG, Engelmann F (2011). Acta Horticulturae 908:181-186.
Lakon G (1949). The topographical Tetrazolium method for determining the germinating capacity of seeds. Plant Physiology 24(3):389-394.
Lambardi M, Fabbri A, Caccavale A (2000). Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips. Plant Cell Reports 19(3):213-218.
Lambardi M, Benelli C, De Paoli G, Battistini A (2001). Biotecnologie per la Conservazione del Castagno [Biotechnology for the conservation of chestnut]. Proc. “Convegno Nazionale Castagno 2001”. Marradi (Firenze), 25-27 October, pp 86-91.
Leprince O, Buitink J, Hoekstra FA (1999). Axes and cotyledons of recalcitrant seeds of Castanea sativa Mill. exhibit contrasting responses of respiration to drying in relation to desiccation sensitivity. Journal of Experimental Botany 50(338):1515-1524.
Lloyd G, McCown BH (1981). Woody plant medium (WPM)-a mineral nutrient formulation for microculture of woody plant species. HortScience 16:453-453.
MacKay DB (1972). The measurement of viability. In: Roberts EH (Ed). Viability of seeds. Syracuse University Press, NY, pp 172-208.
Matsumoto T, Sakai A, Nako Y (1998). A novel preculturing for enhancing the survival of in vitro-grown meristems of wasabi (Wasabia japonica) cooled to -196 °C by vitrification. CryoLetters 19(1):27-36.
Nakhutsrishvili G (2013). Forest vegetation of Georgia. In: Nakhutsrishvili G (Ed). The vegetation of Georgia (South Caucasus). Springer, pp 35-87.
Normah MN, Makeen AM (2008). Cryopreservation of excised embryos and embryonic axes. In: Reed BM (Ed). Plant cryopreservation. A practical guide. Springer, New York, pp 211-240.
Ozden-Tokatli Y, Ozudogru EA, Gumusel F, Lambardi M (2007). Cryopreservation of Pistacia spp. seeds by dehydration and one-step freezing. CryoLetters 28(2):83-94.
Ozudogru EA, Ozden-Tokatli Y, Gumusel F, Benelli C, Lambardi M (2009). Development of a cryopreservation procedure for peanut (Arachis hypogaea L.) embryonic axes and its application to local Turkish germplasm. Advances in Horticultural Science 23(1):41-48.
Panis B, Lambardi M (2006). Status of cryopreservation technologies in plants (crops and forest trees). The role of biotechnology in exploring and protecting agricultural genetic resources. FAO, Rome, pp 61-78.
Pence VC (1990). Cryostorage of embryo axes of several large-seeded temperate tree species. Cryobiology 27(2):212-218.
Pence VC (1992). Desiccation and survival of Aesculus, Castanea and Quercus embryo axes through cryopreservation. Cryobiology 29(3):391-399.
Pritchard HW, Nadarajan J (2008). Cryopreservation of orthodox (desiccation tolerant) seeds. In: Reed BM (Ed). Plant cryopreservation. A practical guide. Springer, New York, pp 485-501.
Pridnya MV, Cherpakov VV, Paillet FL (1996). Ecology and pathology of European chestnut (Castanea sativa) in the deciduous forests of the Caucasus Mountains in southern Russia. Bulletin of the Torrey Botanical Club 123(3):213-222.
Red List of Georgia (2006). The decree of the president of Georgia on approval of the Red List of Georgia. Tbilisi from: https:// www.matsne.gov.ge/ka/document/view/97288?publication=0.
Reed BM (2008). Plant cryopreservation. A practical guide. Springer, New York.
Sakai A, Kobayashi S, Oiyama I (1990). Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9(1):30-33.
Sakai A (2000). Development of cryopreservation techniques. In: Engelmann, F and Takagi H (Eds.). Cryopreservation of tropical plant germplasm. Current research progress and application. IPGRI, Rome, Italy, pp 1-7.
San-José MC, Jorquera L, Vidal N, Corredoira E, Sánchez C (2005). Cryopreservation of European chestnut germplasm. In: III International Chestnut Congress 693, pp 225-232.
Stanwood PC, Bass LN (1981). Seed germplasm preservation using liquid nitrogen. Seed Science and Technology 9:423.
Tavadze B, Supatashvili A, Kapanadze G, Mamukashvili TS (2012). Pathological status of chestnut stands in Tkibuli region (Georgia). Annals of Forestry 5:21-32.
Vertucci CW, Farrant JM (1995). Acquisition of desiccation tolerance. In: Kigel J, Galili G (Eds). Seed development and germination. Marcel Dekker Press Inc., New York, pp 237-271.
Vidal N, Sanchez C, Jorquera L, Ballester A, Vieitez AM (2005). Cryopreservation of chestnut by vitrification of in vitro-grown shoot tips. In Vitro Cellular & Developmental Biology - Plant 41(1):63-68.
Vidal N, Vieitez AM, Fernandez MR, Cuenca B, Ballester A (2010). Establishment of cryopreserved gene banks of European chestnut and cork oak. European Journal of Forest Research 129(4):635-643.
Walters C, Wesley-Smith J, Crane J, Hill LM, Chmielarz P, Pammenter NW, Berjak P (2013). Cryopreservation of recalcitrant (i.e. desiccation-sensitive) seeds. In: Reed BM (Ed). Plant cryopreservation. A practical guide. Springer, New York, pp 465-484.
Westengen OT, Jeppson S, Guarino L (2013). Global ex-situ crop diversity conservation and the Svalbard global seed vault: assessing the current status. PloS One 8(5):e64146.
Copyright (c) 2019 Notulae Botanicae Horti Agrobotanici Cluj-Napoca
This work is licensed under a Creative Commons Attribution 4.0 International License.
Open Access Journal:
The journal allows the author(s) to retain publishing rights without restriction. Users are allowed to read, download, copy, distribute, print, search, or link to the full texts of the articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author.