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Cryopreservation for retaining morphology, genetic integrity, and foreign genes in transgenic plants of Torenia fournieri

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Abstract

Torenia fournieri, a popular potted flower crop, is frequently used as a model plant in genetic transformation studies, and transgenic plants expressing various foreign genes have been obtained in this plant. A safe and long-term conservation of the transgenic plants is necessary. In this study, shoot tips of transgenic plants of T. fournieri were successfully cryopreserved by droplet-vitrification. Growth patterns, and morphologies of leaves and flowers were identical between the plantlets recovered from cryopreservation and the in vitro stock cultures (control). No polymorphic bands and no alternations at ploidy level assessed by ISSR and FCM, respectively, were found in the transgenic plantlets recovered from cryopreservation in comparison with the in vitro stock cultures. The transgene Cry 1Ab analyzed by PCR was maintained, and its expression levels measured by qRT-PCR did not change in the cryo-derived transgenic plantlets. Therefore, cryopreservation can be considered a promising strategy for safe and long-term conservation of the transgenes.

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References

  • Aida R (2008) Torenia fournieri (torenia) as a model plant for transgenic studies. Plant Biotechnol 25:541–545

    Article  Google Scholar 

  • Aida R, Kishimotoa S, Tanaka Y, Shibata M (2000) Modification of flower color in torenia (Torenia fournieri Lind.) by genetic transformation. Plant Sci 153:33–42

    Article  CAS  Google Scholar 

  • Benson EE (2008) Cryopreservation of phytodiversity: a critical appraisal of theory & practice. Crit Rev Plant Sci 27:141–219

    Article  CAS  Google Scholar 

  • Cao J, Earle ED (2003) Transgene expression in broccoli (Brassica oleracea var. italica) clones propagated in vitro via leaf explants. Plant Cell Rep 21:789–796

    PubMed  CAS  Google Scholar 

  • Chandler SF, Sanchez C (2012) Genetic modification; the development of transgenic ornamental plant varieties. Plant Biotechnol J 10:891–903

    Article  PubMed  Google Scholar 

  • Cheong EJ (2012) Biotechnological approaches for improvement and conservation of Prunus species. Plant Biotechnnol Rep 6:17–28

    Article  Google Scholar 

  • Cho JS, Hong SM, Joo SY, Yoo JS, Kim DI (2007) Cryopreservation of transgenic rice suspension cells producing recombinant hCTLA4Ig. Appl Microbiol Biotechnol 73:1470–1476

    Article  PubMed  CAS  Google Scholar 

  • Cornejo MJ, Wong VL, Blechl AE (1995) Cryopreserved callus: a source of protoplasts for rice transformation. Plant Cell Rep 14:210–214

    Article  PubMed  CAS  Google Scholar 

  • Corredoira E, San-José MC, Vieitez AM, Ballester A (2007) Improving genetic transformation of European chestnut and cryopreservation of transgenic lines. Plant Cell Tissue Org Cult 91:281–288

    Article  CAS  Google Scholar 

  • Elleuch H, Gazeau C, David H, David A (1998) Cryopreservation does not affect the expression of a foreign sam gene in transgenic Papaver somniferum cells. Plant Cell Rep 18:94–98

    Article  CAS  Google Scholar 

  • Fretz A, Lorz H (1995) Cryopreservation of in vitro cultures of barley (Hordeum vulgare L. and H. murinum L.) and transgenic cells of wheat (Triticum aestivum L.). J Plant Physiol 146:489–496

    Article  CAS  Google Scholar 

  • Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051

    Article  PubMed  CAS  Google Scholar 

  • Gazeau C, Elleuch H, David A, Morisset C (1998) Cryopreservation of transformed Papaver somniferum cells. CryoLett 19:147–158

    Google Scholar 

  • Hao YJ, Deng XX (2003) GUS gene remains stable in transgenic Citrus callus recovered from cryopreservation. CryoLett 24:375–380

    CAS  Google Scholar 

  • Hao YJ, Cheng YJ, Deng XX (2005) Stable maintenance and expression of a foreign gene in transgenic pear shoots retrieved from in vitro conservation. J Plant Physiol 162:237–243

    Article  PubMed  CAS  Google Scholar 

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

    Google Scholar 

  • Hitmi A, Barthomeuf Ch, Sallanon H (2000) Cryopreservation of Chrysanthemum cinerariaefolium shoot tips. J Plant Physiol 156:408–412

    Article  CAS  Google Scholar 

  • Hornung R, Holland A, Taylor HF, Lynch PT (2001) Cryopreservation of auricula shoot tips using the encapsulation/dehydration technique. CryoLett 22:27–34

    CAS  Google Scholar 

  • Ishikawa K, Harata K, Mii M, Sakai A, Yoshimatsu K, Shimomura K (1997) Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification. Plant Cell Rep 16:754–757

    Article  CAS  Google Scholar 

  • James C (2010) A global overview of biotech (GM) crops: adoption, impact and future prospects GM Crops. GM Crop 1:8–12

    Article  Google Scholar 

  • Kulus D, Zalewska M (2014) Cryopreservation as a tool used in long-term storage of ornamental species—a review. Sci Hortic 168:88–107

    Article  Google Scholar 

  • Li HH, Zhao B, Seitz C, Forkmann G (2009) In vitro plant regeneration and Agrobacterium-mediated genetic transformation of Torenia fournieri. In: Zhang QX (ed) Advances in ornamental horticulture. Forest Press of China, Beijing, pp 183–187

    Google Scholar 

  • Li ZA, Du YQ, Wang ZC (2013) Effect of cryopreservation on the efficiency of exogenous gene, genetic transformation and expression level of Arabidopsis thaliana. Electron J Biotechnol 16:6. doi:10.2225/vol16-issue6-fulltext-12

    CAS  Google Scholar 

  • Meijer EGM, Iren E, Schrijnemakers E, Hensgens LAM, van Zijderveld M, Schilperoort RA (1991) Retention of the capacity to produce plants from protoplasts in cryopreserved cell lines of rice (Oryza sativa L). Plant Cell Rep 10:171–174

    Article  PubMed  CAS  Google Scholar 

  • Menges M, Murray AH (2004) Cryopreservation of transformed and wild-type Arabidopsis and tobacco cell suspension cultures. Plant J 37:635–644

    Article  PubMed  CAS  Google Scholar 

  • Mezzalama M, Crouch JH, Ortiz R (2010) Monitoring the threat of unintentional transgene flow into maize gene banks and breeding materials. Electron J Biotechnol 13:2. doi:10.2225/vol13-issue2-fulltext-5. http://www.ejbiotechnology.info/content/vol13/issue2/full/5/

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Ogawa Y, Suzuki H, Sakurai N, Aoki K, Saito K, Shibata D (2008) Cryopreservation and metabolic profiling analysis of Arabidopsis T87 suspension-cultured cells. CryoLett 29:427–436

    CAS  Google Scholar 

  • Perlak FJ, Fuchs RL, Dean DA, Mcpherson SL, Fischhoff DA (1991) Modification of the coding sequence enhances plant expression of insect control protein genes (Bacillus thuringiensis/insect-resistant plants/synthetic genes). Proc Nat Acad Sci 88:3324–3328

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Ryynänen L, Sillanpää M, Kontunen-Soppela S, Tiimonen H, Kangasjärvi J, Vapaavuori E (2002) Preservation of transgenic silver birch (Betula pendula Roth) lines by means of cryopreservation. Mol Breed 10:143–152

    Article  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Sansavini S, Belfanti E, Costa F, Donati F (2005) European apple breeding programs turn to biotechnology. Chron Hortic 45:16–19

    Google Scholar 

  • Schmale K, Rademacher TH, Fischer R, Hellwig S (2006) Towards industrial usefulness—cryocell-banking of transgenic BY-2 cell cultures. J Biotechnol 124:302–311

    Article  PubMed  CAS  Google Scholar 

  • Sopalun K, Kanchit K, Ishikawa K (2010) Vitrification-based cryopreservation of Grammatophyllum speciosum protocorm. CryoLett 31:347–357

    CAS  Google Scholar 

  • Suzuki M, Akihama T, Ishikawa M (2005) Cryopreservation of encapsulated gentian axillary buds following 2 step-preculture with sucrose and desiccation. Plant Cell Tissue Org Cult 83:115–121

    Article  CAS  Google Scholar 

  • Teixeira da Silva JA (2004) Ornamental chrysanthemums: improvement by biotechnology. Plant Cell Tissue Org Cult 79:1–18

    Article  Google Scholar 

  • Tsai SF, Yeh SD, Chan CF, Liaw SL (2009) High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines. Plant Cell Tissue Org Cult 98:157–164

    Article  CAS  Google Scholar 

  • Valenzuela S, Balocchi C, Rodríguez J (2006) Transgenic trees and forestry biosafety. Electron J Biotechnol. http://www.ejbiotechnology.info/content/vol9/issue3/full/22/

  • Van Eck J, Keen P (2009) Continued expression of plant-made vaccines following long-term cryopreservation of antigen-expressing tobacco cell cultures. In Vitro Cell Dev Biol Plant 45:750–757

    Article  CAS  Google Scholar 

  • Wang F, Yuan QH, Shi L, Qian Q, Liu WG, Kuang BG, Zeng DL, Liao YL, Cao B, Jia SR (2006) A large-scale field study of transgene flow from cultivated rice (Oryza sativa) to common wild rice (O. rufipogon) and barnyard grass (Echinochloa crusgalli). Plant Biotechnol J 4:667–676

    Article  PubMed  CAS  Google Scholar 

  • Wang B, Zhang ZB, Yin ZF, Feng CH, Wang QC (2012a) Novel and potential application of cryopreservation to plant genetic transformation. Biotechnol Adv 30:604–612

    Article  PubMed  CAS  Google Scholar 

  • Wang QC, Wang RR, Li BQ, Cui ZH (2012b) Cryopreservation: a strategy for safe preservation of genetically transformed plant materials. Adv Genet Eng Biotechnol 1:1–2

    Article  Google Scholar 

  • Wang B, Wang RR, Cui ZH, Li JW, Bi WL, Li BQ, Ozudogru EA, Volk GM, Wang QC (2014a) Potential applications of cryobiotechnology to plant genetic transformation and pathogen eradication. Biotechnol Adv 32:583–595

    Article  PubMed  CAS  Google Scholar 

  • Wang RR, Gao XX, Chen L, Huo LQ, Li MF, Wang QC (2014b) Shoot recovery and genetic integrity of Chrysanthemum morifolium shoot tips following cryopreservation by droplet-vitrification. Sci Hortic 176:330–339

    Article  CAS  Google Scholar 

  • Ye X, Busov V, Zhao N, Meilan R, Lisa M, McDonnell LM, Heather D, Coleman HD, Shawn D, Mansfield SD, Feng CF, Li Y, Cheng ZM (2011) Transgenic populus trees for forest products, bioenergy, and functional genomics. Cri Rev Plant Sci 30:415–434

    Article  Google Scholar 

  • Zambryski P, Joos H, Genetello C, Leemans J, Montagu MV, Schell J (1983) Ti-plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J 2:2143–2150

    PubMed Central  PubMed  CAS  Google Scholar 

  • Zhang Z, Skjeseth G, Elameen A, Haugslien S, Sivertsen A, Wang QC, Blystad DR (2015) Field performance evaluation and genetic integrity assessment in Argyranthemum maderense plants recovered from cryopreserved shoot tips. In Vitro Cell Dev Biol Plant 51:505–513

    Article  Google Scholar 

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    Authors and Affiliations

    1. State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Genetic Improvement of Horticultural Crops of Northwest China of Ministry of Agriculture of China, College of Horticulture, Northwest A&F University, Yangling, 712100, Shaanxi, People’s Republic of China

      Jing-Wei Li, Ren-Rui Wang, Xiao-Xia Gao & Qiao-Chun Wang

    2. College of Forestry, Northwest A&F University, Yangling, 712100, Shaanxi, People’s Republic of China

      Hou-Hua Li

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    1. Jing-Wei Li
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    2. Hou-Hua Li
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    5. Qiao-Chun Wang
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    Correspondence to Qiao-Chun Wang.

    Additional information

    Communicated by M. Lambardi.

    J.-W. Li and H.-H. Li have contributed equally to this work.

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    Li, JW., Li, HH., Wang, RR. et al. Cryopreservation for retaining morphology, genetic integrity, and foreign genes in transgenic plants of Torenia fournieri . Acta Physiol Plant 38, 8 (2016). https://doi.org/10.1007/s11738-015-2028-z

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    • DOI: https://doi.org/10.1007/s11738-015-2028-z

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