Skip to main content

Advertisement

SpringerLink
Log in

Low-temperature preincubation enhances survival and regeneration of cryopreserved Saussurea involucrata callus

  • Micropropagation
  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Abstract

Saussurea involucrata Kar. et Kir. is one of the most well-known Chinese medicinal plants, and it is utilized for a variety of medical conditions. Due to the overexploitation of this endangered species, it is crucial to develop methods for both conservation and propagation. To address this issue, we have developed and optimized a simple and effective vitrification process for the cryopreservation of S. involucrata callus tissue. The optimized method consisted of a 3-d incubation period on medium containing 0.3 M sucrose, transfer to a plant vitrification solution (PVS2) containing 30% (v/v) glycerol, 15% (v/v) ethylene glycol, 15% (v/v) dimethylsulfoxide, and 0.4 M sucrose first at 60% PVS2 for 40 min, then at 100% PVS2 for 60 min, followed by immediate immersion and storage in liquid nitrogen. To thaw the tissue, tissues were rewarmed at 40°C for 2 min. This method resulted in a survival rate of approximately 56% and a regrowth rate of approximately 40%. Survival and regrowth were significantly improved by the addition of a low-temperature preincubation step. Incubating the calli at 4°C for 12 d prior to initiating the optimized cryopreservation protocol increased the survival rate of the tissue to 75%, increased the regrowth rate to 60%, and more than doubled the number of regenerated shoots per explant. Following cryopreservation, greater than 90% of the regenerated shoots formed complete plantlets, and 81% of the regenerated plantlets survived and grew vigorously under greenhouse conditions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.

Similar content being viewed by others

References

  • Andersone U, Levinah G (2005) In vitro regeneration of mature Pinus sylvestris buds, stored at freezing temperatures. Biologia Plant 49:281–284

    Article  Google Scholar 

  • Chang YJ, Reed BM (2000) Extended alternating-temperature cold acclimation and culture duration improve pear shoot cryopreservation. Cryobiology 40:311–322

    Article  PubMed  CAS  Google Scholar 

  • Crowe JH, Crowe LM, Carpenter JF, Wistrom CA (1987) Stabilization of dry phospholipid-bilayers and proteins by sugars. Biochem J 242:1–10

    PubMed  CAS  Google Scholar 

  • Dumet D, Engelmann F, Chabrillange N, Duval Y, Dereuddre J (1993) Importance of sucrose for the acquisition of tolerance to desiccation and cryopreservation of oil palm somatic embryos. CryoLetters 14:243–250

    Google Scholar 

  • Engelmann F (2011) Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell Dev Biol-Plant 47:5–16

    Article  Google Scholar 

  • Fahy GM, Levy DI, Ali SE (1987) Some emerging principles underlying the physical-properties, biological actions, and utility of vitrification solutions. Cryobiology 24:196–213

    Article  PubMed  CAS  Google Scholar 

  • Fu LG (1992) China plant red data book—rare and endangered plants (vol. 1). Chinese Science Press, Beijing, pp 234–235

  • Guo B, Gao M, Liu CZ (2007) In vitro propagation of an endangered medicinal plant Saussurea involucrata Kar. et Kir. Plant Cell Rep 26:261–265

    Article  PubMed  CAS  Google Scholar 

  • Harding K, Benson EE (2000) Analysis of nuclear and chloroplast DNA in plants regenerated from cryopreserved shoot-tips of potato. CryoLetters 21:279–288

    CAS  Google Scholar 

  • Hong SR, Yin MH, Shao XH, Wang AP, Xu WH (2009) Cryopreservation of embryogenic callus of Dioscorea bulbifera by vitrification. CryoLetters 30:64–75

    PubMed  CAS  Google Scholar 

  • Hou BK, Yu HM, Teng SY (1997) Effects of low temperature on induction and differentiation of wheat calluses. Plant Cell Tiss Organ Cult 49:35–38

    Article  CAS  Google Scholar 

  • Jia JM, Wu CF, Liu W, Yu H, Hao Y, Zheng JH, Ji YR (2005) Antiinflammatory and analgesic activities of the tissue culture of Saussurea involucrata. Biol Pharm Bull 28:1612–1614

    Article  PubMed  CAS  Google Scholar 

  • Kim HH, Lee YG, Shin DJ, Ko HC, Gwag JG, Cho EG, Engelmann F (2009) Development of alternative plant vitrification solutions in droplet-vitrification procedures. CryoLetters 30:320–334

    PubMed  CAS  Google Scholar 

  • Li GH, Zhao RC (1989) Studies on pharmacological actions of Saussurea involucrata Kar. et Kir. Acta Pharma Sinica 15:368–369

    Google Scholar 

  • Liu L, Xiao X, Zhang L (1985) Effect of the flavonoids from Saussurea involucrata on DNA synthesis of cancer cells. Lanzhou Univ Nat Sci 21:80–82

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Rall WF, Reid DS, Polge C (1984) Analysis of slow-warming injury of mouse embryos by cryomicroscopical and physiochemical methods. Cryobiology 21:106–121

    Article  PubMed  CAS  Google Scholar 

  • Reed B (2008) Plant cryopreservation: a practical guide. Springer, New York

    Book  Google Scholar 

  • Sakai A, Kobayashi S, Oiyama I (1991) Survival by vitrification of nucellar cells of navel orange (Citrussinensis var. brasiliensis Tanaka) cooled to −196°C. J Plant Physiol 137:465–470

    Article  Google Scholar 

  • Sarasan V, Cripps R, Ramsay MM, Atherton C, McMichen M, Prendergast G, Rowntree JK (2006) Conservation in vitro of threatened plants—progress in the past decade. In Vitro Cell Dev Biol-Plant 42:206–214

    Article  Google Scholar 

  • Scottez C, Chevreau E, Godard N, Arnaud Y, Duron M, Dereuddre J (1992) Cryopreservation of cold-acclimated shoot tips of pear in vitro cultures after encapsulation dehydration. Cryobiology 29:691–700

    Article  Google Scholar 

  • Singh R, Srivastava K, Jaiswal HK, Amla DV, Singh BD (2002) High frequency multiple shoot regeneration from decapitated embryo axes of chickpea and establishment of plantlets in the open environment. Biologia Plant 45:503–508

    Article  CAS  Google Scholar 

  • Steponkus PL, Dowgert MF, Gordon-Kamm WJ (1983) Destabilization of the plasma membrane of isolated plant protoplasts during a freeze–thaw cycle: the influence of cold acclimation. Cryobiology 20:448–465

    Article  PubMed  CAS  Google Scholar 

  • Steponkus PL, Lanphear FO (1967) Refinement of triphenyl tetrazolium chloride method of determining cold injury. Plant Physiol 42:1423–1426

    Article  PubMed  CAS  Google Scholar 

  • Uchendu EE, Leonard SW, Traber MG, Reed BM (2010) Vitamins C and E improve regrowth and reduce lipid peroxidation of blackberry shoot tips following cryopreservation. Plant Cell Rep 29:25–35

    Article  PubMed  CAS  Google Scholar 

  • Yin MH, Hong SR (2010) A simple cryopreservation protocol of Dioscorea bulbifera L. embryogenic calli by encapsulation-vitrification. Plant Cell Tiss Organ Cult 101:349–358

    Article  Google Scholar 

  • Zeliang PK, Pattanayak A, Iangrai B, Khongwir EA, Sarma BK (2010) Fertile plant regeneration from cryopreserved calli of Oryza rufipogon Griff. and assessment of variation in the progeny of regenerated plants. Plant Cell Rep 29:1423–1433

    Article  PubMed  CAS  Google Scholar 

  • Zhao Y, Qi LW, Wang WM, Saxena PK, Liu CZ (2011) Melatonin improves the survival of cryopreserved callus of Rhodiola crenulata. J Pineal Res 50:83–88

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was funded by the National Natural Science Foundation of China (no. 21150110459), National Key Technology Research and Development Program of China (no. 2012BAK25B01), the Knowledge Innovation Program of the Chinese Academy of Sciences (no. Y227051304), and the Chinese Academy of Sciences Fellowship for Young International Scientists (no. 2011Y1GA01).

Author information

Authors and Affiliations

  1. National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Science, Beijing, 100190, People’s Republic of China

    Bin Guo, Amanda R. Stiles & Chun-Zhao Liu

Authors
  1. Bin Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Amanda R. Stiles
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chun-Zhao Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chun-Zhao Liu.

Additional information

Editor: Juan Segura

Rights and permissions

Reprints and permissions

About this article

Cite this article

Guo, B., Stiles, A.R. & Liu, CZ. Low-temperature preincubation enhances survival and regeneration of cryopreserved Saussurea involucrata callus. In Vitro Cell.Dev.Biol.-Plant 49, 320–325 (2013). https://doi.org/10.1007/s11627-013-9497-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-013-9497-9

Keywords

Search

Navigation