Phytophthora cinnamomi is a threat to chestnut fruit production. For improving resistance to this pathogen, a breeding program was implemented based on controlled crosses between Castanea species (Europe x Asia), using the latter as donor of resistant genes. The offspring with altered susceptibility to Phytophthora have been micropropagated. Plantlets were acclimatized to obtain copies of each genotype for phenotyping to Phytophthora susceptibility through inoculation tests. In this work, different methods of in vitro conservation, slow-growth storage and synthetic seed, during 9 and 12 months (at 4 ºC, darkness) were compared. For slow-growth storage, different types and concentrations of carbon sources (sucrose and mannitol at 0.09, 0.16, 0.22 M) were compared. Conversely, for synseeds, nodal segments were encapsulated using different concentrations of Na-alginate (2.75, 3%) and released in a solution of CaCl22H2O (50, 75, 100 mM). For conservation, three conditions were tested: empty tubes; tubes filled (15 mL) with sterile distilled water, and tubes with a 30% glycerol solution. Both methods tested were found suitable for medium-term conservation (12 months). For slow-growth storage, the best results of survival (93.3%) and multiplication rate (2.10, at 2nd subculture) were achieved with 0.22 M sucrose. To the best of our knowledge, our studies show for the first time that the storage of the synseeds in tubes with sterile distilled water was the most effective method for conservation, allowing higher survival (97.5%) and germination (92.5%). The use of the highest concentration of Na-alginate demonstrated to promote the multiplication rate (3.18, at 2nd subculture).
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Bamberg JB (2016) In vitro technology at the US Potato Genebank .In Vitro Cell Dev Biol Plant 52: 213–225 https://doi.org/10.1007/s11627-016-9753-x
Bekheet S, Taha H, El-Bahr M (2005) Preservation of date palm cultures using encapsulated somatic embryos. Arab J Biotechnol 8: 319–328
Capuana M, Lonardo Di S (2013) In vitro conservation of chestnut (Castanea sativa) by slow growth. In Vitro Cell Dev Biol Plant 49: 605–610. Doi: https://doi.org/10.1007/s11627-013-9536-6
Charoensub R, Phansiri S (2004) In vitro conservation of Rose coloured leadwort: effect of mannitol on growth of plantlets. Kasetsart J (Nat Sci) 38: 97–102
Cordeiro SZ, Simas NK, Henriques AB, Sato A (2014) In vitro conservation of Mandevilla moricandiana (Apocynaceae): short-term storage and encapsulation–dehydration of nodal segments. In Vitro Cell Dev Biol Plant 50: 326–336. Doi: https://doi.org/10.1007/s11627-014-9600-x
Fossard De RA (1974) Tissue culture of Eucalyptus. Aust For 37: 43–54: Doi: https://doi.org/10.1080/00049158.1974.10675595
Duncan DB (1955) Multiple range and multiple F tests. Biometrics 11: 1–42. Doi: https://doi.org/10.2307/3001478
Engelmann F (2011) Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell Dev Biol Plant 47: 5–16. Doi: https://doi.org/10.1007/s11627-010-9327-2
Engelmann F (2014) Cryopreservation of clonal crops a review of key parameters. ISHS Acta Horticult 1039: 31–39. Doi: https://doi.org/10.17660/ActaHortic.2014.1039.2
Ganapathi TR, Srinivas L, Suprasanna P, Bapat VA (2001) Regeneration of plants from alginate-encapsulated somatic embryos of banana cv. Rasthali (Musa SPP. AAB Group). In Vitro Cell Dev Biol Plant 37: 178 181. Doi: https://doi.org/10.1007/s11627-001-0031-0
Gomes F, Clemente M, Figueiredo P, Plácito F, Machado H, Santos C, Costa R (2015) Castanea spp hybrid clones in vitro conservation: Synthetic seeds vs slow growth storage. ISHS Acta Horticulturae 1039: 37–44. Doi: https://doi.org/10.17660/ActaHortic.2017.1155.4
Jain SM (2011) Prospects of in vitro conservation of date palm genetic diversity for sustainable production. Emir J Food Agric. Doi: https://doi.org/10.9755/ejfa.v23i2.6344
Jourdan P (2018) Short- and medium-term in vitro conservation and management of germplasm within the USDA's national plant germplasm system. ISHS Acta Horticulturae 1224: 31–38. Doi: https://doi.org/10.17660/ActaHortic.2018.1224.5
Lambardi M, Benelli C, Carlo De A, Previati A, Re Da F, Giannini M (2004) Biotechnologies for the preservation of selected red chicory (Cichorium intybus L.) lines. ISHS Acta Horticulturae 725: 311–318. Doi: https://doi.org/10.17660/ActaHortic.2006.725.38
Lambardi M, Benelli C, Ozudogru EA, Ozden-Tokatli Y (2006) Synthetic seed technology in ornamental plants. In: Silva J eds. Floriculture ornamental and plant biotechnology, vol II, 1st edn, pp 347–354. Kagawa: Global Science Books Ltd
Lata H, Moraes RM, Bertoni B, Pereira AMS (2010) In vitro germplasm conservation of Podophyllum peltatum L. under slow growth conditions. In Vitro Cell Dev Biol Plant 46: 22–27. Doi: https://doi.org/10.1007/s11627-009-9243-5
Matsumoto KC, L.D. (2016) Effects of sucrose and incubation temperature on in vitro conservation of Cassa and Brazilian Ginseng germplasms. Institute ISfHSHR (ed), 83–84
Muñoz M, Díaz O, Reinún W, Winkler A, Quevedo R (2019) Slow growth in vitro culture for conservation of Chilotanum potato germplasm. Chilean J Agric Res 79: 26–35. Doi: https://doi.org/10.4067/S0718-58392019000100026
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15: 473–497. Doi: https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Nin S, Benelli C, Petrucci WA, Turchi A, Pecchioli S, Gori M, Giordani E (2019) In vitro propagation and conservation of wild bilberry (Vaccinium myrtillus L.) genotypes collected in the Tuscan Apennines (Italy). J Berry Res 9: 411–430. Doi: https://doi.org/10.3233/JBR-180379
Ozden-Tokatli Y, Carlo De A, Gumusel F, Pignattelli S, Lambardi M (2008) Development of encapsulation techniques for the production and conservation of synthetic seeds in ornamental plants. Propag Ornam Plant 8: 17–22
Ozudogru A, Silva da DP, Ergun K, Dradi G, Paiva R, Lambardi M (2013) In vitro conservation and cryopreservation of Nandina domestica, an outdoor ornamental shrub. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 41: 638–645. Doi: https://doi.org/10.15835/nbha4129335
Ozudogru EA, Benelli C, Dradi G, Lambardi M (2017) Effect of culture container and carbohydrate content on in vitro slow growth storage of the cherry rootstock ‘Gisela®5’. Acta Physiol Plant 39: 94. Doi: https://doi.org/10.1007/s11738-017-2372-2
Ozudogru EA, Previati A, Lambardi M (2010). In vitro conservation and cryopreservation of ornamental plants. Protocols In Vitro Propag Ornam Plant. Doi: https://doi.org/10.1007/978-1-60327-114-1_28
Prewein C, Wilhelm E (2003) Plant regeneration from encapsulated somatic embryos of pedunculate oak (Quercus robur L.). In Vitro Cell Dev Biol Plant 39: 613–617. Doi: https://doi.org/10.1079/ivp2003453
Reddy MC, Murthy KSR, Pullaiah T (2012) Synthetic seeds: a review in agriculture and forestry. Afr J Biotech 11: 14254–14275. Doi: https://doi.org/10.5897/AJB12.770
Richter DL, Kangas LC, Smith JK, Laks Pe 2010 Comparison of effectiveness of wood decay fungi maintained by annual subculture on agar and stored in sterile water for 18 years Can J Microbiol 56 268 271 https://doi.org/10.1139/W10-001
H Rihan F Kareem M El-Mahrouk M Fuller 2017 Artificial seeds (Principle, aspects and applications) Agronomy 7 71 https://doi.org/10.3390/agronomy7040071
Ruta C, Lambardi M, Tagarelli A, De Mastro G (2016) Tissue culture technologies to preserve Chickpea (Cicer arietinum L.) germplasm. International Society for Horticultural Science (ISHS) & Horticulture Research Institute (HRI) 85–86
Santos C, Machado H, Correia I, Gomes F, Gomes-Laranjo J, Costa R (2015). Phenotyping Castanea hybrids for Phytophthora cinnamomi resistance Plant Pathol 64: 901–910. Doi: https://doi.org/10.1111/ppa.12313
Sarkar D, Naik PS (1998) Factors affecting minimal growth conservation of potato microplants in vitro. Euphytica 102: 275–280. Doi: https://doi.org/10.1023/A:1018309300121
R Shibli M Shatnawi W Subaih M Ajlouni 2006 In vitro conservation and cryopreservation of plant genetic resources: a review World J Agric Sci 2 372 382
Singh AK, Sharma M, Varshney R, Agarwal SS, Bansal KC (2006) Plant regeneration from alginate-encapsulated shoot tips of Phylianthus amarus schum and thonn, a medicinally important plant species. In Vitro Cell Dev Biol Plant 42: 109–113. Doi: https://doi.org/10.1079/IVP2005735
Singh SK (2017) Ex situ conservation of fungi: a review Developments in fungal biology and applied mycology, p 543–562. Singapore: Springer. Doi: https://doi.org/10.1007/978-981-10-4768-8_27
Slama I, Ghnaya T, Hessini K, Messedi D, Savouré A, Abdelly C (2007) Comparative study of the effects of mannitol and PEG osmotic stress on growth and solute accumulation in Sesuvium portulacastrum. Environ Exp Bot 61: 10–17. Doi: https://doi.org/10.1016/j.envexpbot.2007.02.004
Thakur S, Tiwari KL, Jadhav SK (2015) In vitro approaches for conservation of Asparagus racemosus Willd. In Vitro Cell Dev Biol Plant 51: 619–625. Doi: https://doi.org/10.1007/s11627-015-9706-9
Walters C, Berjak P, Pammenter N, Kennedy K, Raven P (2013) Preservation of recalcitrant seeds. Science 339 915 916 https://doi.org/10.1126/science.1230935
West TP, Ravindra M, Preece JE (2006) Encapsulation, cold storage, and growth of Hibiscus moscheutos nodal segments. Plant Cell Tissue Organ Cult 87: 223–231. Doi: https://doi.org/10.1007/s11240-006-9155-6
Yahyaoui EC, Frasheri D, D’Onghia AM, Germanà A. (2016) Encapsulation and evaluation of some growth regulators effects on in vitro-derived micro-cuttings of three Italian Ficus carica L. genotypes. International Society for Horticultural Science (ISHS) & Horticulture Research Institute (HRI) 87
Zar J (1996) Biostatistical analysis 3. London: Prentice Hall International
This work was supported by the project ProDeR, co-financed by the FEADER / European Union, Cooperation for Innovation, Ref. N. 53.590. We thank to Rosemeyre Cordeiro for the contribution to the manuscript.
Project ProDeR, co-financed by the FEADER / European Union, Cooperation for Innovation, Ref. N. 53.590.
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Gomes, F., Clemente, M., Figueiredo, P. et al. Medium-term in vitro conservation of Castanea spp. hybrid clones. Vegetos 34, 127–137 (2021). https://doi.org/10.1007/s42535-020-00184-9
- Artificial seeds
- Slow growth storage
- Storage conditions
- Sucrose concentration