Droplet-vitrification and morphohistological studies of cryopreserved shoot tips of cultivated and wild pineapple genotypes |
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Authors: | Souza Fernanda Vidigal Duarte Kaya Ergun de Jesus Vieira Lívia de Souza Everton Hilo de Oliveira Amorim Vanusia Batista Skogerboe Dianne Matsumoto Tracie Alves Alfredo Augusto Cunha da Silva Ledo Carlos Alberto Jenderek Maria M |
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Institution: | 1.Embrapa Cassava and Fruits, Cruz das Almas, Bahia, CEP 44380-000, Brazil ;2.National Center for Genetic Resources Preservation, USDA-ARS, Fort Collins, CO, 80521, USA ;3.Molecular Biology and Genetics Department, Faculty of Science, Mugla Sitki Kocman University, 48000, Koteki, Mugla, Turkey ;4.Coordination for the Improvement of Higher Education Personnel, CAPES, Cruz das Almas, Bahia, CEP 44380-000, Brazil ;5.Daniel K. Inouye U.S. Pacific Basin Agriculture Research Center, USDA-ARS, Hilo, HI, 96720, USA ; |
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Abstract: | Germplasm conservation of pineapple Ananas comosus (L.) Mer.] is crucial to preserve the genus’ genetic diversity, to secure material for genetic improvement and to support innovative and new research. Long-term conservation is accomplished through cryopreservation, that is done by storing cells or tissues at ultra-low temperature in liquid nitrogen (?196 °C). Droplet-vitrification, a combination of droplet freezing and solution-based vitrification, was used to establish a protocol for cryopreservation of pineapple genetic resources. This protocol was tested on cultivated and wild pineapple genotypes to establish a long-term germplasm security duplicate as well as to investigate cryo-injuries in the tissues by means of histological techniques. Excised shoot tips (0.5–1 mm with one primordial leaf) of different pineapple genotypes were precultured for 48 h on solid MS medium containing 0.3 M of sucrose. Three PVS2 exposure times (30, 45 and 60 min) were tested. The results showed high post cryopreservation survival for all genotypes evaluated. The best PVS2 exposure time varied according to genotype, although 45 min gave the best survival for the majority of genotypes. The technique was highly efficient in cryopreserving meristem shoot tips of different pineapple genotypes, and was also less laborious than techniques previously reported. This is a first report on application of the droplet-vitrification technique to diverse genotypes of cultivated and wild pineapples and the first report on histological changes occurring in cryopreserved Ananas tissue.
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