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Seedling Development and Evaluation of Genetic Stability of Cryopreserved Dendrobium Hybrid Mature Seeds

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Abstract

Vitrification, a simple, fast, and recommended cryopreservation method for orchid germplasm conservation, was evaluated for Dendrobium hybrid “Dong Yai” mature seeds. The genetic stability of regenerated seedlings was also evaluated using flow cytometry. Mature seeds from this hybrid were submitted to plant vitrification solution (PVS2) for 0, 0.5, 1, 2, 3, 4, 5, or 6 h at 0 °C. Subsequently, they were plunged into liquid nitrogen (LN) at −196 °C for 1 h and recovered in half-strength Murashige and Skoog culture medium (1/2 MS), and seed germination was evaluated after 30 days. Seeds directly submitted to LN did not germinate after cryopreservation. Seeds treated with PVS2 between 1 and 3 h presented the best germination (between 51 and 58 %), although longer exposure to PVS2 returned moderated germination (39 %). Germinated seeds were further subcultured in P-723 culture medium and developed whole seedlings in vitro after 180 days, with no abnormal characteristics, diseases, or nutritional deficiencies. Seedlings were successfully acclimatized under greenhouse conditions with over 80 % survival. Flow cytometry analysis revealed no chromosomal changes on vitrified seedlings, as well as seedlings germinated from the control treatment (direct exposure to LN). These findings indicate that vitrification is a feasible and safe germplasm cryopreservation method for commercial Dendrobium orchid hybrid conservation.

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Abbreviations

LN:

Liquid nitrogen

PVS2:

Plant vitrification solution

1/2 MS:

Half-strength Murashige and Skoog culture medium

TTC:

2,3,5-Triphenyl tetrazolium chloride

FCM:

Flow cytometry

DMSO:

Dimethyl sulfoxide

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Acknowledgments

The authors thank the National Council of Research and Technology Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq), Ministry of Science and Technology, Brazil, and the University of Florida for providing support for this study. Thanks also to Mrs. Alba Myers for the technical assistance and Dr. Allan Meerow, ARS-USDA and Mr. Deal Neal, UF/ICBR for the assistance with flow cytometry analysis.

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Author notes

  1. Renato Fernandes Galdiano Jr.

    Present address: Department of Technology, Univ Estadual Paulista, Via de Acesso Prof. Dr. Paulo Donato Castellane, s/n, Jaboticabal, SP, 14884-900, Brazil

Authors and Affiliations

  1. Department of Technology, Univ Estadual Paulista, Via de Acesso Prof. Dr. Paulo Donato Castellane, s/n, Jaboticabal, SP, 14884-900, Brazil

    Eliana Gertrudes de Macedo Lemos

  2. Depto. de Ciências Agrárias, Universidade Estadual de Londrina, Caixa Postal 6001, Londrina, PR, 86051-990, Brazil

    Ricardo Tadeu de Faria

  3. Tropical Research and Education Center, University of Florida, 18905 SW 280th St, Homestead, FL, 33031-3314, USA

    Wagner Aparecido Vendrame

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  1. Renato Fernandes Galdiano Jr.
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  2. Eliana Gertrudes de Macedo Lemos
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  3. Ricardo Tadeu de Faria
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Correspondence to Renato Fernandes Galdiano Jr..

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Galdiano, R.F., de Macedo Lemos, E.G., de Faria, R.T. et al. Seedling Development and Evaluation of Genetic Stability of Cryopreserved Dendrobium Hybrid Mature Seeds. Appl Biochem Biotechnol 172, 2521–2529 (2014). https://doi.org/10.1007/s12010-013-0699-8

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  • DOI: https://doi.org/10.1007/s12010-013-0699-8

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