Skip to main content

Advertisement

SpringerLink
Log in

Cryopreservation of embryogenic calli of cassava using sucrose cryoprotection and air desiccation

  • Cell Biology and Morphogenesis
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

A simplified technique which simultaneously induces and cryoprotects embryogenic calli using sucrose followed by dehydration was developed for the cryopreservation of cassava genetic resources. An initial experiment to optimise the sucrose concentration needed for both embryo production and cryoprotection showed that higher concentrations of sucrose—between 0.4 M and 0.5 M—significantly reduced the viability as well as the number of embryos produced by the embryogenic clumps in the absence of freezing. Post-thaw viability as well as embryogenic competence of clumps depended on the percentage moisture lost, duration of exposure to higher sucrose concentrations and the duration of induction of embryogenic clumps. Extending the period of cryoprotection to 21 days coupled with increased moisture loss (greater than 75%) significantly increased both post-thaw viability and the embryogenic competence of cryopreserved clumps to 95%, while reducing the duration decreased post-thaw viability. Cryopreserved callus clumps developed secondary and cyclic embryos similar to those of the non-cryopreserved controls. The optimised protocol was successfully applied to SM1-2075-1 Line 1 somatic embryos. The rate of plant recovery from cryopreserved embryos of both TME 9 and SM1-2075-1 Line 1 was comparable to that of the non-cryopreserved embryos. Successful cryopreservation of embryogenic clumps of cassava can be used to establish in vitro genebanks for long-term conservation of cassava genetic resources to complement field genebanks and other in vitro methods already being used.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5A–E

Similar content being viewed by others

References

  • Benson EE (1999) Cryopreservation. In: Benson EE (ed) Plant conservation biotechnology. Taylor and Frances, London, pp 83–107

  • Benson EE, Lynch PT, Stacey GN (1998) Advances in plant cryopreservation technology: current applications in crop plant biotechnology. AgBiotech News Inf 10:133–142

    Google Scholar 

  • Blakesley D, Percival T, Bhatti MH, Henshaw, GG (1997) A simplified protocol for cryopreservation of embryogenic tissue of sweet potato (Ipomea batatas (L) Lam) utilising sucrose preculture only. Cryo-Letters 18:77–80

    Google Scholar 

  • Côte FX, Goue O, Domergue R, Panis B, Jenny C (2000) In-field behaviour of banana plants (Musa AA sp,) obtained after regeneration of cryopreserved embryogenic cell suspensions. Cryo-Letters 21:19–24

    Google Scholar 

  • Danso KE, Ford-Lloyd BV (2002) Induction of high-frequency somatic embryos in cassava for cryopreservation. Plant Cell Rep 21:226–232

    Article  CAS  Google Scholar 

  • Dumet D, Engelmann F, Chabrillange N, Dussert S, Duval Y (2000) Cryopreservation of oil palm polyembryonic cultures. In: Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm. Current research, progress and applications. IPGRI, Rome, pp 172–177

  • Fahy GM, MacFarlene DR, Angell CA, Maryman HT (1984) Vitrification as an approach to cryopreservation. Cryobiology 21:407–426

    CAS  PubMed  Google Scholar 

  • Finer JJ (1987) Direct somatic embryogenesis and plant regeneration from immature embryos of hybrid sunflower (Helianthus annuus L) on high sucrose-containing medium. Plant Cell Rep 6:372–374

    CAS  Google Scholar 

  • Fowler J, Cohen L, Jarvis P (1998) Practical statistics for field biology, 2nd edn. John Wiley and Sons, Chichester, pp 81–89

  • Hagmann HM, Ryynanen LA, Aronen TS, Krajinakova J (1998) Cryopreservation of embryogenic cultures of Scots pine. Plant Cell Tissue Organ Cult 54:45–53

    Article  Google Scholar 

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

    Google Scholar 

  • Jitsuyama Y, Suzuki T, Harada T, Fujikawa S (2002) Sucrose incubation increases freezing tolerance of asparagus (Asparagus officinalis L) embryogenic cell suspensions. Cryo-Letters 23:103–112

    Google Scholar 

  • Kurata K, Iwabuchi K, Futaya Y (1992) Induction of carrot somatic embryogenesis by high sucrose concentration without changing the culture medium. Acta Hortic 319:195–196

    Google Scholar 

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

    CAS  Google Scholar 

  • Mycock DJ, Wesley-Smith J, Berjak P (1995) Cryopreservation of somatic embryos of four species with and without cryoprotectants pre-treatment. Ann Bot 75:331–336

    Article  Google Scholar 

  • Panis B (1995) Cryopreservation of banana (Musa spp) germplasm. PhD thesis, Katholieke Universiteit, Leuven, Belgium

  • Panis B, Thinh NT (2001) Cryopreservation of Musa germplasm. INIBAP technical guidelines, vol 5. International Network for the Improvement of Banana and Plantain, Montpellier, pp 13–16

  • Panis B, Totte N, Van Nimmen K, Withers, LA, Swennen R (1996) Cryopreservation of banana (Musa spp) meristem cultures after preculture on sucrose. Plant Sci 121:95–106

    Article  CAS  Google Scholar 

  • Raemakers CJJM (1993) Primary and cyclic embryogenesis in cassava. PhD thesis, Agricultural University of Wageningen, The Netherlands

  • Raemakers CJJM, Jacobsen E, Visser RGF (1997) Micropropagation of Manihot esculenta (Crantz) cassava. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 39. High tech and micropropagation. Springer, Berlin, Heidelberg New York

  • Schopke C, Taylor N, Carcamo R, Konan NK, Marmey P, Henshaw GG, Beachy RN, Fauquet C (1996) Regeneration of transgenic cassava plants (Manihot esculenta Crantz) from microbombarded embryogenic suspension cultures. Nat Biotechnol 14:731–735

    Google Scholar 

  • Sofiari E, Raemakers CJJM, Sofiari E, Kanju E, Danso K, van Lammeren AM, Jacobsen E, Visser RGF (1997) Comparison of NAA and 2,4-D induced somatic embryogenesis in cassava. Plant Cell Tissue Organ Cult 50:45–56

    Article  CAS  Google Scholar 

  • Stewart P, Taylor M, Mycock D (2001) The sequence of the preparative procedures affects the success of cryostorage of cassava somatic embryos. Cryo-Letters 22:35–42

    Google Scholar 

  • Suzuki T, Kaneko M, Harada T (1997) Increase in freeze tolerance of excised shoot tips of Asparagus officinalis L. by preculture on sucrose-rich media. Cryobiology 34:264–275

    Article  Google Scholar 

  • Takagi H (2000) Recent developments in cryopreservation of shoot apices of tropical species. In Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm. Current research, progress and applications. IPGRI, Rome, pp178–193

  • Taylor NJ, Masona MV, Carcamo R, Ho T, Schopke C, Fauquet CM (2001) Production of embryogenic tissues and regeneration of transgenic plants in cassava (Manihot esculenta Crantz). Euphytica 120:25–34

    CAS  Google Scholar 

  • Thierry C, Tessereau H, Florin B, Meschine M, Petiard V (1997) Role of sucrose for the acquisition of tolerance to cryopreservation of carrot somatic embryos. Cryo-Letters 18:283–292

    Google Scholar 

  • Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol 50:571–599

    Google Scholar 

  • Touchell DH, Chiang VL, Tsai CJ (2002) Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification. Plant Cell Rep 21:118–224

    Article  CAS  Google Scholar 

  • Turner S, Senarata T, Touchel D, Bunn E, Dixon K, Tan B (2001) Stereochemical arrangement of hydroxyl groups in sugars and polyalcohol molecules as an important factor in effective cryopreservation. Plant Sci 160:489–497

    Article  CAS  PubMed  Google Scholar 

  • Wang Q, Gafny R, Sahar N, Sela I, Mawassi M, Tanne E, Perl A (2002) Crypreservation of grape vine (Vitis vinifera L.) embryogenic cell suspensions by encapsulation dehydration and subsequent plant regeneration. Plant Sci 162:551–558

    Article  CAS  Google Scholar 

  • Wolfe J, Bryant G (2001) Cellular cryobiology: thermodynamic and mechanical effects. Int J Refrig 24:438–450

    Article  Google Scholar 

  • Zhang Y, Wang J, Bian H, Zhu, M (2001) Pregrowth-desiccation: a simple and efficient procedure for the cryopreservation of rice (Oryza sativa L.) embryogenic suspension cells. Cryo-Letters 22:221–228

    Google Scholar 

Download references

Acknowledgements

The authors wish to thank the Commonwealth Scholarship Commission, UK for their financial support to Mr. Kenneth Ellis Danso. We also wish to thank Dr. Bart Panis of Katholieke Universiteit, Belgium for his technical advice and Mrs. Ng and CIAT for sending us in vitro plantlets of cassava for this study.

Author information

Authors and Affiliations

  1. School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

    K. E. Danso & B. V. Ford-Lloyd

Authors
  1. K. E. Danso
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. B. V. Ford-Lloyd
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to B. V. Ford-Lloyd.

Additional information

Communicated by M.R. Davey

Rights and permissions

Reprints and permissions

About this article

Cite this article

Danso, K.E., Ford-Lloyd, B.V. Cryopreservation of embryogenic calli of cassava using sucrose cryoprotection and air desiccation. Plant Cell Rep 22, 623–631 (2004). https://doi.org/10.1007/s00299-003-0727-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-003-0727-1

Keywords

Search

Navigation