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Cryopreservation, early seedling development, and genetic stability of Oncidium flexuosum Sims

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Abstract

An efficient cryopreservation protocol was developed for mature seeds of Oncidium flexuosum Sims. Seed morphology, protocorm formation, and early seedling development were also assessed. The effects of phloroglucinol and Supercool X-1000® as cryoprotectant additives in the vitrification solution were investigated. Dehydration using the plant vitrification solution 2 (PVS2) for 60 and 120 min prior to immersion in liquid nitrogen promoted the highest frequency of in vitro seed germination 6 weeks following culture on half-strength Murashige and Skoog (½ MS) medium. Mature seeds submitted to vitrification for 120 min in PVS2 and 1 % phloroglucinol at 0 °C enhanced germination by 68 %, whereas in PVS2 and 1 % Supercool X-1000® germination was just moderately enhanced (26 %). In vitro-germinating seedlings developed healthy shoots and roots without the use of plant growth regulators. After 6 months of growth, there were no differences between in vitro- and ex vitro-grown seedlings for various phenotypic characteristics, including shoot length, number of leaves, number and length of roots, and fresh and dry weight. Seedlings were transferred to greenhouse conditions and successfully acclimatized, further developing into normal plants with over 90 % survival. Comparative analysis of seedlings from control and vitrified seeds using flow cytometry indicated that no change in ploidy levels occurred as a result of cryopreservation, therefore maintaining seedlings genetic stability. In this study, vitrification with PVS2 for 120 min with the addition of 1 % phloroglucinol offers a simple, safe, and feasible protocol for cryopreservation of O. flexuosum mature seeds.

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Abbreviations

DMSO:

Dimethyl sulfoxide

FCM:

Flow cytometry

LN:

Liquid nitrogen

LS:

Loading solution

½ MS:

Half-strength Murashige and Skoog (1962) culture medium

PVA:

Polyvinyl alcohol

PVS2:

Plant vitrification solution 2

TTC:

2,3,5-triphenyl tetrazolium chloride

References

  • Ai P, Lu L, Song J (2012) Cryopreservation of in vitro-grown shoot-tips of Rabdosia rubescens by encapsulation-dehydration and evaluation of their genetic stability. Plant Cell Tissue Organ Cult 108:381–387

    Article  Google Scholar 

  • Alvarez MR, Sagawa Y (1965) Histochemical study of embryo development in Vanda (Orchidaceae). Caryologia 18:251–261

    Google Scholar 

  • Arditti J (1992) Fundamentals of orchid biology. Wiley, New York

    Google Scholar 

  • Bae JS (2012) Barrier protective activities of phloroglucinol on lipopolysaccharide (LPS)-induced barrier disruption in human endothelial cells. Inflammation 35:920–926

    Article  PubMed  CAS  Google Scholar 

  • Chase MW, Williams NH, de Faria AD, Neubig KM, Amaral MC, Whitten WM (2009) Floral convergence in Oncidiinae (Cymbidieae; Orchidaceae): an expanded concept of Gomesa and a new genus Nohawilliamsia. Ann Bot 104:387–402

    Article  PubMed  Google Scholar 

  • Chen JT, Chang WC (2000) Plant regeneration via embryo and shoot bud formation from flower-stalk explants of Oncidium ‘Sweet Sugar’. Plant Cell Tissue Organ Cult 62:95–100

    Article  CAS  Google Scholar 

  • Chugh S, Guha S, Rao IU (2009) Micropropagation of orchids: a review on the potential of different explants. Sci Hortic 122:507–520

    Article  CAS  Google Scholar 

  • Cribb PJ (1999) Morphology. In: Pridgeon AM, Cribb PJ, Chase MW, Rasmussen FN (eds) Genera Orchidacearum: volume 1- general introduction, Apostasioideae, Cypripedioideae. Oxford University Press, Oxford

    Google Scholar 

  • Ding F, Jin S, Hong N, Zhong Y, Cao Q, Yi G, Wang G (2008) Vitrification–cryopreservation, an efficient method for eliminating Candidatus Liberobacter asiaticus, the citrus Huanglongbing pathogen, from in vitro adult shoot tips. Plant Cell Rep 27:241–250

    Article  PubMed  CAS  Google Scholar 

  • Dolezel J, Greilhuber J, Suda J (2007) Flow cytometry with plants: an overview. In: Dolezel J, Greilhuber J, Suda J (eds) Flow cytometry with plant cells: analysis of genes, chromosomes and genomes. Wiley-VCH Verlag Gmbh & Co, Weinheim, pp 41–66

    Chapter  Google Scholar 

  • Engelmann F (2004) Plant cryopreservation: progress and perspectives. In Vitro Cell Dev Biol Plant 40:427433

    Google Scholar 

  • Flashsland E, Terada G, Socchi A, Ray H, Mroginski L, Engelmann F (2006) Cryopreservation of seeds and in vitro-cultured protocorms of Oncidium bifolium Sims. (Orchidaceae) by encapsulation-dehydration. Cryo Lett 27:235–242

    Google Scholar 

  • Galdiano RF Jr, Lemos EGM, Faria RT, Vendrame WA (2012) Cryopreservation of Dendrobium hybrid seeds and protocorms as affected by phloroglucinol and Supercool X1000. Sci Hort 148:154–160

    Article  CAS  Google Scholar 

  • Gaspi FOG, Foglio MA, Carvalho JE, Santos GMT, Testa M, Passarini JE, Moraes, CP, Esquisatto MAM, Mendonça JS, Mendonça FAS (2011) Effects of the topical application of hydroalcoholic leaf extract of Oncidium flexuosum Sims (Orchidaceae) and microcurrent on the healing of wounds surgically induced in wistar rats. Evidence Based Complement Altern Med 1–9

  • Harding K (2004) Genetic integrity of cryopreserved plant cells: a review. Cryo Lett 25:3–22

    Google Scholar 

  • Hazubska-Przbyl T, Chmielarz P, Michalak M, Bojarczuk K (2010) Cryopreservation of embryogenic tissues of Picea omorika (Serbian spruce). Plant Cell Tissue Organ Cult 102:35–44

    Article  Google Scholar 

  • Hirano T, Godo T, Mii M, Ishikawa K (2005) Cryopreservation of immature seeds of Bletilla striata by vitrification. Plant Cell Rep 23:534–539

    Article  PubMed  CAS  Google Scholar 

  • Hirano T, Godo T, Miyoshi K, Ishikawa K, Ishikawa M, Mii M (2009) Cryopreservation and low-temperature storage of seeds of Phaius tankervilleae. Plant Biotechnol Rep 3:103–109

    Article  Google Scholar 

  • Hirano T, Yukawa T, Miyoshi K, Mii M (2011) Wide applicability of cryopreservation with vitrification method of seeds of some Cymbidium species. Plant Biotechnol 28:99–102

    Article  Google Scholar 

  • Hoekstra FA, Golovina EA, Buitink J (2001) Mechanisms of plant desiccation tolerance. Trends Plant Sci 6:431–438

    Article  PubMed  CAS  Google Scholar 

  • Hosomi ST, Custódio CC, Seaton PT, Marks TR, Machado-Neto NB (2012) Improved assessment of viability and germination of Cattleya (Orchidaceae) seeds following storage. In vitro Cell Dev Biol Plant 48:127–136

    Article  Google Scholar 

  • Jeeja G, Ansari R (1994) Taxonomic significance of seed surface morphology in Orchidaceae. Rheedea 4:48–59

    Google Scholar 

  • Kang KA, Lee KH, Chae S, Zhang R, Jung MS, Ham YM, Baik JS, Hyun JW (2006) Cytoprotective effect of phloroglucinol on oxidative stress induced cell damage via catalase activation. J Cell Biochem 97:609–620

    Article  PubMed  CAS  Google Scholar 

  • Kaviani B (2011) Conservation of plant genetic resources by cryopreservation. Aust J Crop Sci 5:778–800

    Google Scholar 

  • Kim MM, Kim SK (2010) Effect of phloroglucinol on oxidative stress and inflammation. Food Chem Toxicol 48:2925–2933

    Article  PubMed  CAS  Google Scholar 

  • Kong JM, Goh NK, Chia LS, Chia TF (2003) Recent advances in traditional plant drugs and orchids. Acta Pharmacol Sin 24:7–21

    PubMed  CAS  Google Scholar 

  • Le TN, McQueen-Mason SJ (2006) Desiccation-tolerant plants in dry environments. Rev Environ Sci Biotechnol 5:269–279

    Article  CAS  Google Scholar 

  • Li DZ, Pritchard HW (2009) The science and economics of ex situ plant conservation. Trends in Plant Sci 14:614–621

    Article  CAS  Google Scholar 

  • Marie D, Brown S (1993) A cytometric exercise in plant DNA histograms, with 2C values for 70 species. Biol Cell 78:41–51

    Article  PubMed  CAS  Google Scholar 

  • Miao NH, Kaneko Y, Sugawara Y (2005) Ultrastructural implications of pretreatment for success cryopreservation of Oncidium protocorm-like body. Cryo Lett 26:333–340

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Na HY, Kondo K (1996) Cryopreservation of tissue-cultured shoot primordial from shoot apices of cultured protocorms in Vanda pumila following ABA-preculture and desiccation. Plant Sci 118:195–201

    Article  CAS  Google Scholar 

  • Nikishina TV, Popova EV, Vakhameeva MG, Varlygina TL, Kolomeitseva GL, Burov AV, Popovich EA, Shirokov AI, Shumilov VY, Popov AS (2007) Cryopreservation of seeds and protocorms of rare temperate orchids. Russ J Plant Physiol 54:121–127

    Article  CAS  Google Scholar 

  • Nishizawa S, Sakai A, Amano Y, Matsuzawa T (1993) Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Sci 91:67–73

    Article  CAS  Google Scholar 

  • Pfosser M, Amon A, Lelley T, Heberle-Bors E (1995) Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat-rye addition lines. Cytometry 21:387–393

    Article  PubMed  CAS  Google Scholar 

  • Pinto G, Louveiro T, Santos C (2004) Analysis of the genetic stability of Eucaliptus globulus Labill. somatic embryos by flow cytometry. Theor Appl Genet 109:580–587

    Article  PubMed  CAS  Google Scholar 

  • Popov AS, Popova EV, Nikishina TV, Kolomeytseva GL (2004) The development of juvenile plants of the hybrid orchid Bratonia after seed cryopreservation. Cryo Lett 5:295–300

    Google Scholar 

  • Popova EV, Nikishina TV, Kolomeitseva GL, Popov AS (2003) The effect of seed cryopreservation on the development of protocorms by the hybrid orchid Bratonia. Russ J Plant Physiol 50:672–677

    Article  CAS  Google Scholar 

  • Popova EV, Lee E, Wu C, Hahn E, Paek K (2009) A simple method for cryopreservation of Ginkgo biloba callus. Plant Cell Tissue Organ Cult 97:337–343

    Article  CAS  Google Scholar 

  • Popova EV, Kima H, Paek H (2010) Cryopreservation of coriander (Coriandrum sativum L.) somatic embryos using sucrose preculture and air desiccation. Sci Hort 124:522–528

    Article  CAS  Google Scholar 

  • Prado MJ, Rodriguez E, Rey L, Gozalez MV, Santos C, Rey M (2010) Detection of somaclonal variants in somatic embryogenesis-regenerated plants of Vitis vinifera by flow cytometry and microsatellite markers. Plant Cell Tissue Organ Cult 102:49–59

    Article  Google Scholar 

  • Pridgeon A, Morrison A (2006) The illustrated encyclopedia of orchids: over 1100 species illustrated and identified. Timber Press Inc., Oregon, p 197

    Google Scholar 

  • Reed BM (2008) Cryopreservation—practical considerations. In: Reed BM (ed) Plant cryopreservatyion—a practical guide. Springer, New York, pp 3–14

    Chapter  Google Scholar 

  • Roberts DL, Dixon KW (2008) Orchids. CurBiol 8:325–329

    Google Scholar 

  • Rustaei M, Nazeri MS, Ghadimzadeh M, Hemmaty S (2009) Effect of phloroglucinol, medium type and some component on in vitro proliferation of dwarf rootstock of apple (Mallus domestica). Int J Agric Biol 11:193–196

    CAS  Google Scholar 

  • Sakai A, Engelmann F (2007) Vitrification, encapsulation-vitrification and droplet-vitrification: a review. Cryo Lett 28:151–172

    CAS  Google Scholar 

  • Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nuclear cells of navel orange (Citrus sinensis var. brasiliensis Tanaka) by vitrification. Plant Cell Rep 9:30–33

    Article  Google Scholar 

  • Sarkar D, Naik PS (2000) Phloroglucinol enhances growth and rate of axillary shoot proliferation in potato shoot tip cultures in vitro. Plant Cell Tissue Organ Cult 60:139–149

    Article  CAS  Google Scholar 

  • Sharifian S, Vahdati K, Mirmasoumi M, Maghami SA (2009) Assessment of phloroglucinol effect on rooting of tissue cultured Persian walnut. Acta Hortic 812:189–195

    Google Scholar 

  • Sopalun K, Thammasiri K, Ishikawa K (2010) Micropropagation of the thai orchid Grammatophyllum speciosum blume. Plant Cell Tissue Organ Cult 101:143–150

    Article  Google Scholar 

  • Surenciski MR, Dematteis M, Flachsland EA (2007) Chromosome stability in cryopreserved germoplasm of Cyrtopodium hatschbachii (Orchidaceae). Ann Bot Fenn 44:287–292

    Google Scholar 

  • Teixeira da Silva JA, Singh N, Tanaka M (2006) Priming biotic factors for optimal protocorm-like bodies and callus induction in hybrid Cymbidium (Orchidaceae), and assessment of cytogenetic stability in regenerated plantlets. Plant Cell Tissue Org Cult 84:135–144

    Google Scholar 

  • Thammasiri K (2007) Cryopreservation of some Thai orchid species. Acta Hortic 788:53–62

    Google Scholar 

  • Thammasiri K, Soamkul L (2007) Cryopreservation of Vanda coerulea Griff. Ex. Lindl. Seeds by vitrification. Sci Asia 33:223–227

    Article  Google Scholar 

  • Vendrame WA, Faria RT (2011) Phloroglucinol enhances recovery and survival of cryopreserved Dendrobium nobile protocorms. Sci Hortic 128:131–135

    Article  CAS  Google Scholar 

  • Vendrame WA, Carvalho VS, Dias JMM (2007) In vitro germination and seedling development of cryopreserved Dendrobium hybrid mature seeds. Sci Hortic 114:188–193

    Article  CAS  Google Scholar 

  • Verleysen H, Samyn G, Van Bockstaele E, Debergh P (2004) Evaluation of analytical techniques to predict viability after cryopreservation. Plant Cell Tiss Org Cult 77:11–21

    Article  CAS  Google Scholar 

  • Wang Q, Valkonen JPT (2009) Cryotherapy of shoot tips: novel pathogen eradication method. Trends Plant Sci 14:119–122

    Article  PubMed  CAS  Google Scholar 

  • Wirth M, Withner CL (1959) Embryology and development in the Orchidaceae. In: Withner CL (ed) The orchids: a scientific survey. Ronald Press, New York, pp 155–188

    Google Scholar 

  • Yin M, Hong S (2009) Cryopreservation of Dendrobium candidum Wall. Ex Lindl. Protocorm-like bodies by encapsulation-vitrification. Plant Cell Tissue Org Cult 98:179–185

    Article  CAS  Google Scholar 

  • Yin LL, Poobathy R, James J, Julkifle AL, Subramaniam S (2011) Preliminary investigation of cryopreservation by encapsulation-dehydration technique on Brassidium shooting Star orchid hybrid. Afr J Biotechnol 10:4665–4672

    Google Scholar 

  • Zhao MA, Xhu YZ, Dhital SP, Khu DM, Song YS, Wang MY, Lim HT (2005) An efficient cryopreservation procedure for potato (Solanum tuberosum L.) utilizing the new ice blocking agent, Supercool X1000. Plant Cell Rep 24:477–480

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors thank the National Council of Research (Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq), of the Ministry of Science and Technology, Brazil, and the University of Florida for providing funding and 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.

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  1. Renato Fernandes Galdiano Jr.

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

Authors and Affiliations

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

    Renato Fernandes Galdiano Jr. & Eliana Gertrudes de Macedo Lemos

  2. 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. Wagner Aparecido Vendrame
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Correspondence to Renato Fernandes Galdiano Jr..

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Galdiano, R.F., de Macedo Lemos, E.G. & Vendrame, W.A. Cryopreservation, early seedling development, and genetic stability of Oncidium flexuosum Sims. Plant Cell Tiss Organ Cult 114, 139–148 (2013). https://doi.org/10.1007/s11240-013-0304-4

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