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High-efficiency cryopreservation of Araucaria angustifolia (Bertol.) Kuntze embryogenic cultures: ultrastructural characterization and morpho-physiological features

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Abstract

Here we evaluated and characterized the growth dynamics of A. angustifolia embryogenic cultures (EC) submitted to different cryotreatment incubation times through morphological and time-lapse cell tracking analyzes. The EC submitted to cryopreservation protocol were evaluated by regrowth rates, and ultrastructural characterization by transmission electron microscopy (TEM). The results indicated that A. angustifolia EC support all the cryoprotection times evaluated, without cell proliferation inhibition, but with noticeable genotype-dependent response in all tested cell lines. The use of 1M DMSO showed non-inhibitory effects to EC regrowth independent of cell line or cryotreatment incubation time. However, after cryopreservation, Cr01 cell line regrowth was 100 % for all cryotreatments incubation times evaluated (30, 60, 120, 240 min), while Cr02 cell line only showed 100 % regrowth in 240 min of cryotreatment. The 100 % cell regrowth obtained in both cell lines indicates that the proposed protocol can be successful applied to A. angustifolia EC cryopreservation. Cell tracking analysis showed a survival and initial proliferation of embryogenic cells, with the first cell regrowth signs after 30 days in culture. TEM analysis revealed a conspicuous cell wall thickening in embryogenic cells after cryotreatment and after thawing, which may be related to osmotic stress response caused by the cryopreservation process. An increased heterochromatin presence was also observed in cryotreated or after thawing cells, may possibly be acting as a cell defense mechanism, decreasing the DNA vulnerability to cleavage and preserving the cell integrity.

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Acknowledgments

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Proc. 478393/2013-0, and 306126/2013-3), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina (FAPESC, Proc. 3770-2012, 2780/2012-4, and 14848/2011-2). The authors are grateful to Dr. Lirio Luiz Dal Vesco, Dr. Marisa Santos and Dr. Rubens Onofre Nodari for all contributions that improved the manuscript quality.

Author contribution statement

Conceived and designed the experiments: HPFF, LNV and MPG; Performed the experiments: HPFF, LNV, CCP, JMS, KGA; Performed the transmission electron microscopy analysis: HPFF, LNV and EMO; Contributed reagents/materials/analysis tools: MPG; Wrote the paper: HPFF, LNV and MPG.

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  1. Graduate Program in Plant Genetic Resources, Plant Developmental Physiology and Genetics Laboratory, Federal University of Santa Catarina, Rod. Admar Gonzaga, Km 3, Florianópolis, SC, 88034-001, Brazil

    Hugo P. F. Fraga, Leila N. Vieira, Catarina C. Puttkammer, Jamily M. da Silva, Karina G. dos Anjos & Miguel P. Guerra

  2. Central Laboratory of Electron Microscopy, Federal University of Santa Catarina, Florianopolis, SC, Brazil

    Eliana M. Oliveira

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  1. Hugo P. F. Fraga
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  2. Leila N. Vieira
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  3. Catarina C. Puttkammer
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  5. Karina G. dos Anjos
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  6. Eliana M. Oliveira
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  7. Miguel P. Guerra
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Correspondence to Miguel P. Guerra.

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Fraga, H.P.F., Vieira, L.N., Puttkammer, C.C. et al. High-efficiency cryopreservation of Araucaria angustifolia (Bertol.) Kuntze embryogenic cultures: ultrastructural characterization and morpho-physiological features. Plant Cell Tiss Organ Cult 124, 307–318 (2016). https://doi.org/10.1007/s11240-015-0895-z

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