Ultra-low temperature conservation of Brazilian Pine embryogenic cultures

Demarchi, Grasiela; Stefenon, Valdir Marcos; Steiner, Neusa; Vieira, Felipe N.; Vesco, Lírio Luiz Dal; Guerra, Miguel Pedro

doi:10.1590/0001-3765201420130405

Cited by 7 publications

(5 citation statements)

References 18 publications

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“…In these species, PVS2 solution ( Sakai et al., 1990 ) or its modifications were used as cryoprotectant. This solution has also been tested in Araucaria angustifolia ( Demarchi et al., 2014 ) and Larix kaempefri × L. gmelinii embryo-genic tissues ( Ma et al., 2023 ); however, in these cases, the samples were cooled slowly before LN. In other species, vitrification versus slow cooling methods have been compared for cryopreservation of embryogenic tissues ( Lambardi et al., 2018 ) and, as a general trend, the slow-cooling method has given the best results.…”

Section: Cryopreservation Of Embryogenic Materials In Woody Speciesmentioning

confidence: 99%

Current status of the cryopreservation of embryogenic material of woody species

Ballesteros,

Martínez,

Sánchez-Romero

et al. 2024

Front. Plant Sci.

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Cryopreservation, or the storage at liquid nitrogen temperatures (-196°C), of embryogenic cells or somatic embryos allows their long-term conservation without loss of their embryogenic capacity. During the last decade, protocols for cryopreservation of embryogenic material of woody species have been increasing in number and importance. However, despite the large experimental evidence proved in thousands of embryogenic lines, the application for the large-scale conservation of embryogenic material in cryobanks is still limited. Cryopreservation facilitates the management of embryogenic lines, reducing costs and time spent on their maintenance, thus limiting the risk of the appearance of somaclonal variation or contamination. Somatic embryogenesis in combination with cryopreservation is especially useful to preserve the juvenility of lines while the corresponding clones are being field-tested. Hence, when tree performance has been evaluated, selected varieties can be propagated from the cryostock. The traditional method of slow cooling or techniques based on vitrification are mostly applied procedures. For example, slow cooling methods are widely applied to conserve embryogenic lines of conifers. Desiccation based procedures, although simpler, have been applied in a smaller number of species. Genetic stability of the cryopreserved material is supported by multiloci PCR-derived markers in most of the assayed species, whereas DNA methylation status assays showed that cryopreservation might induce some changes that were also observed after prolonged subculture of the embryogenic lines. This article reviews the cryopreservation of embryogenic cultures in conifers, fruit species, deciduous forest species and palms, including a description of the different cryopreservation procedures and the analysis of their genetic stability after storage in liquid nitrogen.

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Section: Cryopreservation Of Embryogenic Materials In Woody Speciesmentioning

confidence: 99%

Current status of the cryopreservation of embryogenic material of woody species

Ballesteros,

Martínez,

Sánchez-Romero

et al. 2024

Front. Plant Sci.

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“…Contrary, large vacuolated cells exhibit a high level of plasmolysis, and the loss of water from protoplasts may be lethal leading to much damage and death of cells (Volk and Caspersen, 2007). The FDA test to evaluate cell survival after thawing is a frequently used procedure in cryopreserved conifer embryogenic cultures (Häggman et al, 1998;Ford et al, 2000;Salaj et al, 2010;Demarchi et al, 2014). However, this test may not be the only reliable indicator of successful tissue recovery and subsequent growth (Perez et al, 1997).…”

Section: Relationship Between Cryotolerance and Genotypementioning

confidence: 99%

“…The samples should be rapidly thawed in a water bath at a higher temperature (30 to 40 °C) to avoid recrystallization and ensure cell/tissue recovery. Another technique of cryopreservation -vitrification very often used for herbaceous plants (Sant et al, 2008) or hardwood trees (Lambardi et al, 2005;Guzman Garcia et al, 2013;San Jose et al, 2015), for conifer embryogenic tissues has been used exceptionally, e.g., in Picea sitchensis (Touchell et al, 2002) or Araucaria angustifolia (Demarchi et al, 2014).…”

Section: Introduction Introduction Introduction Introductionmentioning

confidence: 99%

Cryopreservation of Abies alba embryogenic tissues by slow-freezing method

Salaj

Panis

Klubicová

et al. 2022

Not Bot Horti Agrobo

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Embryogenic tissues of Abies alba Mill. were cryopreserved using the slow-freezing approach. Four cell lines were incubated for 24 h on a medium with 0.5 M sorbitol and pre-treated with 5% DMSO. Subsequently, the tissues were frozen at a cooling rate of 1 °C min-1 to -40 °C and transferred to liquid nitrogen for 72 hours. After thawing in a water bath at 40 °C, the tissues were cultivated on a proliferation medium. All tested lines recovered, but variations in regrowth frequencies across cell lines were noticed (91.66 to 100%). The recovered tissues showed similar features to the control 2 (non-pre-treated and non-cryopreserved tissues). In the accumulation of fresh and dry mass, no statistically significant differences were observed between cryopreserved cultures and control 2. The cryopreserved tissues produced cotyledonary somatic embryos capable of germination. Microscopic observations revealed considerable structural changes as a consequence of the cryopreservation procedure. The long vacuolated suspensor cells were disrupted, and mostly the meristematic cells of the embryonal region survived. The typical bipolar structure of early somatic embryos has been regained during the post-thaw period. Differences in cryotolerance across cell lines were also observed.

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“…Cryopreservation has already been established for A. angustifolia embryogenic cultures but never for embryonic axes (EA) (Demarchi et al, 2014;Fraga et al, 2016). The mature embryos of this conifer are large (about 2.5 cm in length), contain more than 1 g H 2 O g -1 dry mass and are killed by drying (Pieruzzi et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Viability of embryonic axes of Araucaria angustifolia after freezing using two cryopreservation methods

Frizzo

Quoirin

2018

Pesq. flor. bras.

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Araucaria angustifolia (Bertol.) O. Kuntze é uma das espécies nativas do Brasil com maior importância na Região Sul. No entanto, suas sementes são recalcitrantes, constituindo um obstáculo para a sua conservação em longo prazo, sendo a criopreservação uma alternativa viável para o armazenamento de seu germoplasma. Os eixos embrionários (EE) excisados das sementes de araucária foram encapsulados, desidratados e submetidos a dois métodos de criopreservação: o resfriamento rápido, congelando em nitrogênio líquido (NL) durante 2 h, e o pré-resfriamento a - 40 °C, seguido do congelamento em nitrogênio líquido durante 2 h. Posteriormente, os EE foram descongelados rapidamente e avaliados quanto à integridade do DNA pelo teste bioquímico do tetrazólio, germinação in vitro e ocorrência de oxidação. Tanto o DNA dos EE que não foram criopreservados quanto o daqueles que foram criopreservados mantiveram sua integridade. Os resultados do teste de tetrazólio indicaram que a maioria dos EE desidratados pelo congelamento rápido permaneceu viável. Após 15 dias de cultivo in vitro, os EE não germinaram e apresentaram sinais de oxidação. Os métodos de encapsulamento-desidratação seguidos de congelamento rápido são promissores para a criopreservação de EE de araucária, como demonstrado pelo teste de tetrazólio e da manutenção da integridade total do DNA.

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Ultra-low temperature conservation of Brazilian Pine embryogenic cultures

Cited by 7 publications

References 18 publications

Current status of the cryopreservation of embryogenic material of woody species

Current status of the cryopreservation of embryogenic material of woody species

Cryopreservation of Abies alba embryogenic tissues by slow-freezing method

Viability of embryonic axes of Araucaria angustifolia after freezing using two cryopreservation methods

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