Contributors to this page: CIAT, Colombia (Daniel Debouck, Roosevelt Escobar, Graciela Mafla); IITA, Nigeria (Dominique Dumet, Badara Gueye); Bioversity International, France (Ines Van den Houwe, Bart Panis, Nicolas Roux); Bioversity International/ILRI, Ethiopia (Alexandra Jorge); INIA, Peru (Llerme Rios); independent consultants (Erica Benson, Keith Harding, Clair Hershey).
In the last 25 years, several new cryoconservation procedures like vitrification, encapsulation-dehydration, preculture-dehydration, and encapsulation/vitrification have been established, which are all based on vitrification. Vitrification can be defined as the transition of water directly from the liquid phase into an amorphous or “glassy” phase, whilst avoiding the formation of crystalline ice. Cryoconservation protocols based on vitrification techniques have been developed for different vegetatively propagated crops, including cassava (Sakai and Engelmann 2007).
Cryoconservation is the storage of germplasm at ultra low temperatures (-196oC) using cryogenic media, typically liquid nitrogen. This temperature effectively stops the biological activity of the plant cells. This technique stops the metabolism of the in vitro plant, eliminating the need to regularly rejuvenate the plant.
Cryoconservation techniques have been increasingly used for the Long Term Storage (LTS) of cassava, through the freezing of embryos or shoot tips. It is currently an additional tool to improve conservation of germplasm in genebanks. CIAT pioneered research on cassava cryoconservation in 1985, and has given varying degrees of research emphasis to improving the techniques since that time.
Cyoconservation is also used for other purposes, such as the cryoconservation of embryogenic tissues for genetic transformation and for botanic seeds.
Research on cryoconservation was initiated in the past few years in more than 600 accessions of cassava germplasm conserved at CIAT genebank. The main objective was to:
- Overcome some of the limitations of the in vitro maintenance (labour-intensive subculturing, potential for fungal and bacterial contaminants and somaclonal variation).
- To ensure the safe long-term conservation of cassava genetic resources.
This is a relatively new technique for cassava, and still with limited funding. The routine application of cryoconservation protocols for cassava shoot tip conservation has been pioneered by the CIAT genebank (Escobar et al. 1997, Gonzalez-Arnao et al. 2008, Roca 1984) modifying and adapting techniques initially developed for other crops in other laboratories. Specific cassava protocols were developed testing and optimizing a range of different cryogenic and non-cryogenic parameters. The method relies on rapid freezing of highly proliferating meristem cultures precultured for 2-3 months on 4E medium (Roca 1984). IITA recently started to test this methodology and adapt it to their conditions.
Many genebanks such as those at CIP, CGN, IPK, and USDA have also been using cryoconservation techniques for other important clonal, horticultural and herbaceous crops.
CIAT is the principal institution working on cryoconservation in cassava. New protocols for encapsulation dehydration and quick-freezing have now been developed and validated with more than 43% of the entire cassava core collection. More than 82% of the accessions tested have recovery rates of more than 30%, the minimum required for cryoconservation. However, this is still not a sufficient overall recovery rate for the entire CIAT collection: 18% of the sampled accessions fell below the minimum acceptable recovery rate. Protocols are now being adjusted for wild relatives of cassava, species of which sometimes behave very poorly in vitro or even in the field, making their conservation troublesome. Plants have been recovered for M. esculenta ssp. flabellifolia, M. esculenta ssp. peruviana and M. carthaginensis.
Cryoconservation is also being used to support transformation of cassava. Developing friable embryogenic callus cell lines is time consuming, with the inherent risks of genetic instability and low plant recovery over time.
When it should be used
- To conserve germplasm that can have more than 30% recovery. Some genotypes have very low percentages of recovery. Accessions that have been successfully recovered from cryo-conservation include some of the most common cultivated cultivars (M. esculenta) and some wild species (M. esculenta ssp. flabellifolia, M. esculenta ssp. peruviana and M. carthaginensis).
- To conserve and/or complement the conservation of materials that are not often used, or to duplicate materials maintained in tissue culture conditions.
- This is the most secure and trouble free conservation method for clonal cassava germplasm, but requires highly specialized personnel and equipment.
How it should be done
The following procedures, based on the colligative cryoprotection controlled rate cooling protocol, summarize the optimized methodologies as reported by Escobar et al. (1997):
- Explant: shoot tips 2 mm in height.
- Pre-culture: in medium (C4) comprising 1M sorbitol, 0.117M (4%) sucrose, 0.1 M DMSO for 3 days in the dark at 26 – 28ºC.
- Cryoprotection with 1M sorbitol, 0.117M (4%) sucrose, 10% DMSO for 2h on ice
- Tissue dehydration on filter paper for 1h.
- Controlled rate programmable freezing (CryoMed 1010) starting from a 5ºC chamber temperature, a rate of -0.5ºC min-1 to -15ºC, and thereafter at a rate of -1ºC min-1 to -40 ºC.
- Immersion in LN.
- Thawing at 37ºC.
- Sequential transfer recovery (2-days each) on medium containing (1) 0.75M sucrose with 0.2% activated charcoal and (2) half-strength MS medium with 0.35M sucrose and 5.5.6 x 10-3 M inositol in the dark; and standard culture medium under a light intensity of 15 μEm-2 s-1.
- Evaluation of tissue viability and shoot growth after 1 month.
There are often different genotype-dependent recovery responses.
- Apply different protocols to the same set of accessions to identify the most suitable for each type.
- Optimize cryo-conservation protocols on a case-by-case basis on very recalcitrant types.
- It is important to identify which types have high, intermediate or low response recovery rates. Protocols may need to be modified and adapted for different types.
- CIAT classified their genotype response into 3 categories:
- High response group – 70% of shooting.
- Intermediate response group – 30-70% of shooting.
- Low response group – Less than 30% of shooting.
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References and further reading
CIAT. 2001. Cryopreservation of Cassava Germplasm, Its Wild Relatives, and Cell Lines for Tissue Culture. Available from: http://webapp.ciat.cgiar.org/biotechnology/crops_cassava.htm#cryo Date accessed: 26 August 2010.
Danso KE, Ford-Lloyd BV. 2004. Cryopreservation of embryogenic calli of cassava using sucrose cryoprotection and air desiccation. Plant Cell Reports 22: 623-631.
Escobar RH, Mafla G, Roca WM. 1993. Cryopreservation of cassava shoot tips. In: Roca WM, Thro AM, editors. Proceedings First Scientific Meeting of the Cassava Biotechnology Network, 25-28 Aug. 1992, Cartagena. CIAT, Cali, Colombia, pp. 116-121.
Escobar RH, Mafla G, Roca WM. 1997. A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen. Plant Cell Reports 16: 474-478. Available here.
Escobar RH, Manrique N, Munoz L, Rios A, Debouck D, Tohme J. Cassava cryopreservation by rapid freezing methodology. CIAT, Cali, Colombia. Available from: http://isa.ciat.cgiar.org/urg/urgweb_folder/files/unitfiles/Cassava%20cryopreservation%20by%20rapid%20freezing%20methodology.pdf. Date accessed: 6 October 2010.
Gonzalez-Arnao MT, Panta A, Roca WM, Escobar RH, Engelmann F. 2007. Development and large scale application of cryopreservation techniques for shoot and somatic embryo cultures of tropical crops. Available from: http://www.springerlink.com/content/y42117245j364361/fulltext.pdf Date accessed: 26 August 2010.
Marin ML, Mafla G, Roca WM, Withers LA. 1990. Conservation of cassava (Manihot esculenta Crantz): The role of cryopreservation. In: Proceedings of the VIIth International Congress on plant tissue and cell culture, Amsterdam, The Netherlands, 24-29 June 1990, pp. 371.
Mafla G, Roca WM, Kartha K. 1987. Conservation of Cassava (Manihot esculenta Crantz) germplasm in vitro, IV: Evaluation of phenotypic estability of plants grown from cryo preserved shoot tip cultures. In: Angarita A, editor. Abstracts of International Congress of plant tissue culture tropical species. Bogotá, Colombia, pp 75.
Mycock DJ, Wesley-Smith J, Berjak P. 1995. Cryopreservation of somatic embryos of four species with and without cryoprotectant pre-treatment. Ann. Bot., 75: 331-336.
Roca WM. 1984. Cassava. In: Sharp WR, Evans DA, Ammirato RV, Yamada Y. editors. Handbook of plant cell culture: crop species, vol 2. MacMillan Publishers, New York, pp 269-301.
Szabados L, Hoyos R, Roca W. 1987. In vitro somatic embryogenesis and plant regeneration of cassava. Available for purchase online from: http://www.springerlink.com/content/r53x32l17281215h/ Date accessed: 26 August 2010.
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