Cryo bank

Page compiled by: Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge); ILRI, Addis Ababa, Ethiopia (Jean Hanson); Katholieke Universiteit Leuven, Belgium (Bart Panis).

Storage of germplasm using cryopreservation

Cryopreservation is becoming more widely used for long-term storage of seeds and in vitro cultures and is the method of choice for ensuring cost-effective and safe, long-term storage of genetic resources of species which have recalcitrant seeds or are vegetatively propagated. Storage is usually in liquid nitrogen (-196°C), whereby all metabolic processes and cell divisions are arrested.

Orthodox seeds

Seed conservation of orthodox seeds in liquid nitrogen at -196°C has been successfully achieved for a wide range of crop species. Once stored in liquid nitrogen, seeds can be kept for unlimited periods. Seed storage in base collections at -20°C is addressed under the seed conservation page.

Dessication intolerant seeds

Conservation of seeds with non-orthodox behaviour and those that are desiccation intolerant allows seeds which were previously considered as short-lived to be stored for long periods at ultra-low temperatures. This has been achieved for several species, including Citrus, coconut, coffee and areca nut through controlled desiccation of whole seeds, excised embryos or embryonic axes followed by cryopreservation.

Tissues

Preservation of dormant buds and in vitro cultures, including apical meristems and somatic embryos, at cryogenic temperatures is considered as the only suitable alternative that can ensure the long-term security of stored germplasm. Once stored in liquid nitrogen, tissues can be kept for unlimited periods. It is most appropriate for base collections of long-term storage

Advantages

The ultra low temperatures used during cryopreservation virtually stop metabolic deterioration during storage of tissues and seeds and therefore extends their longevity during long-term storage. The material is protected from contamination and very little maintenance is needed. The method relies on liquid nitrogen in self contained tanks and is not dependent on refrigeration or a constant electricity supply. It is also cost effective because of the reduced energy costs, limited space requirements and because there is no need for regular regeneration of plant material.

Disadvantages

There is no generic protocol which could be applicable to all types of explants. Therefore, each successive step of a cryopreservation protocol needs to be optimized for any new plant material to be cryopreserved.
Not all orthodox seeds are suitable for cryopreservation and hard seeded legume seeds may crack or shatter and some seeds with high oil contents loose viability. It is also a very costly method to store large seeds such as maize and beans.

Practical considerations

Until fifteen years ago, cryopreservation protocols for plant tissues were mainly based on slow freezing in the presence of cryoprotective mixtures containing dimethyl sulphoxide, sugars, glycerol and/or proline. Slow freezing results in a freeze-dehydration, leaving less water in the cells to form lethal ice crystals upon exposure to extreme low temperatures. While this method is very convenient for many plant tissues, especially undifferentiated callus or suspension cells, new methods had to be developed for plant species and tissues that were unresponsive.

During the last two decades, several new cryopreservation procedures have been established. Among them, the protocols termed encapsulation/dehydration, encapsulation/vitrification and vitrification. All these new techniques involve the extraction of freezable water from the tissue cells before cooling. As a result vitrification of internal solutes takes place during cooling. Vitrification can be defined as the transition of water directly from the liquid phase into an amorphous phase or glass, whilst avoiding the formation of lethal crystalline ice The vitrification technique involves a treatment with cryoprotective loading solutions followed by dehydration with highly concentrated vitrification solutions. A modification of this technique, that further reduces the chance for lethal ice-crystal formation through the application of ultra-fast cooling and rewarming rates called “droplet vitrification” has now been developed for different vegetatively propagated tropical crops.

Each species requires specific protocols that must be carefully followed for preparation of samples to ensure maximum survival.

General considerations

• Tissues and recalcitrant seeds generally cannot be thawed and refrozen without damage.
• Orthodox seeds and pollen behave differently and can usually be thawed and refrozen
• Transfer cryopreserved material quickly between vessels to avoid rewarming.
• Ensure good air circulation in the room where LN storage tanks are placed because nitrogen gas is constantly boiling from the tanks. Oxygen monitors should be placed around the room for detection of oxygen content of the air.
• Use emergency fans that are triggered by the oxygen monitors or emergency buttons to increase air exchange in case of build up of nitrogen gas

Storage containers

• Storage container will vary with the size of the seeds or tissues. Store seeds and tissues in 1.8 and 2.0 ml cryogenic vials for cryopreservation:
  • For meristems and shoot tips, 10-25 tissues can be placed in one vial;
  • For small seeds from 1500 to 3000 can be placed in one vial;
  • Larger vials will be needed for storage of larger seeded orthodox seeds;
• Place multiple vials into aluminum cans or metal boxes for storage.
• Liquid nitrogen storage containers vary with tissue and size of collection:
  • Small 20-30 liter tanks with no vapor phase for direct tissue storage.
  • Five-foot-diameter steel tanks that allow a vapour phase for seed storage.
• Use well insulated containers to reduce loss of liquid nitrogen. Containers with narrow necks reduce loss of liquid nitrogen but are inconvenient for access.
• Use an alarm system to indicate low liquid nitrogen levels.
• Top up storage dewars once in a week.

Storage temperature

Cryogenic vials may be stored directly in the liquid nitrogen at -196°C or suspended in trays within the vapour phase of the liquid nitrogen at -160°C

Replication

The number of replications depends on the survival, crop type, speed of propagation, stability in culture and material available for storage. Probalistic tolls have been developed to assist in the establishment and management of cryopreserved collections (see Dussert et al. 2003)

Specific crop protocols

Storage protocols have been developed for several important vegetatively propagated crops, including banana, cassava, potato, sweet potato and yam.

Banana - Cryopreservation of Musa germplasm
Cassava - http://webapp.ciat.cgiar.org/asia_cassava/pdf/proceedings_workshop_02/136.pdf
Potato - http://www.cipotato.org/csd/Materials/Tissue/Capitulo4.pdf
Sweet potato - http://www.cipotato.org/csd/materials/Sweetpotato%202-4.asp 
Yam - http://www.ejbiotechnology.info/content/vol1/issue3/full/2/bip/

References and further reading

Bajaj YPS, editor. 1995. Cryopreservation of Plant Germplasm I. Biotechnology in Agriculture and Forestry, Vol. 32. Springer, Berlin, Germany.

Batugal P, Ramanatha Rao V, Oliver J, editors. 2005. Coconut genetic resources, International Board for Plant Genetic Resources, Rome, Italy.779 p. Available here.

Benson EE. 1999. Plant Conservation Biotechnology, pp. 139–154. Taylor & Francis, London.

Benson EE, Harding K, Debouck D, Dumet D, Escobar R, Mafla G, Panis B, Panta A, Tay D, Van den houwe I, Roux N. 2011. Refinement and standardization of storage procedures for clonal crops - Global Public Goods Phase 2: Part I. Project landscape and general status of clonal crop in vitro conservation technologies. System-wide Genetic Resources Programme, Rome, Italy. Available here (2.5 MB).

Benson EE, Harding K, Debouck D, Dumet D, Escobar R, Mafla G, Panis B, Panta A, Tay D, Van den houwe I, Roux N. 2011. Refinement and standardization of storage procedures for clonal crops - Global Public Goods Phase 2: Part II. Status of in vitro conservation technologies for: Andean root and tuber crops, cassava, Musa, potato, sweet potato and yam. System-wide Genetic Resources Programme, Rome, Italy. Available as full size PDF (9 MB) and light version (3.5 MB).

Benson EE, Harding K, Debouck D, Dumet D, Escobar R, Mafla G, Panis B, Panta A, Tay D, Van den houwe I, Roux N. 2011. Refinement and standardization of storage procedures for clonal crops - Global Public Goods Phase 2: Part III. Multi-crop guidelines for developing in vitro conservation best practices for clonal crops. System-wide Genetic Resources Programme, Rome, Italy. Available here (4.5 MB).

Chandel KPS, Pandey R, Paroda RS, Arora RK. 1991. Plant genetic resources conservation: recent approaches. Plant genetic resources: conservation and management. Concepts and approaches. International Board for Plant Genetic Resources New Delhi (India). p. 247-272.

Chaudhury R, Pandey R, Malik SK, Bhag Mal, editors. 2003. In vitro conservation and cryopreservation of tropical fruit species. Proceedings of the Regional Training Course on In Vitro Conservation of Tropical Fruit Genetic Resources, organized at the National Bureau of Plant Genetic Resources (NBPGR), New Delhi, India, 4-16 February 2002. 293 p. 

Dussert S, Engelmann F, Noirot M. 2003. Development of probabilistic tools to assist in the establishment and management of cryopreserved plant germplasm collections. Cryoletters 24:149-160.

Engelmann F. 2004. Plant cryopreservation: Progress and prospects. In vitro Cellular and Developmental Biology plant 40:427-433.

Engelmann F, Dulloo ME, Astorga C, Dussert S, Anthony F, editors. 2007. Conserving coffee genetic resources: complementary strategies for ex situ conservation of coffee (Coffea arabica L.) genetic resources. A case study in CATIE, Costa Rica. Topical Reviews in Agricultural Biodiversity Rome (Italy). 61 p.

Engelmann F, Takagi H, editors. 2000. Cryopreservation of Tropical Plant Germplasm – Current Research Progress and Applications. International Plant Genetic Resources Institute, Rome, Italy.

FAO. 2013. Genebank standards for plant genetic resources for food and agriculture. Food and Agriculture Organization of the United Nations, Rome. Available in English, Spanish, French, Arabic, Russian and Chinese here.

Gonzalez-Arnao MT, Engelmann F. 2006. Cryopreservation of plant germplasm using the encapsulation-dehyrdation technique:review and case study on sugarcane. Cryoletters 27:155-168.

Harding K. 2004. Genetic integrity of cryopreserved plant cells: A review. CryoLetters 25:3-22.

Iwanaga M. 1994. Cassava genetic resources management at CIAT International network for cassava genetic resources. Report of the first meeting of the International Network for Cassava Genetic Resources held at CIAT, Cali, Colombia, 18-22 August 1992. International Crop Network Series no. 10. p. 77-86.

Kartha KK, editor. 1985. Plant Cryopreservation. CRC Press, Boca Raton, Florida, USA.

Panis B, Lambardi M, 2006. Status of cryopreservation technologies in plants (crops and forest trees). In: Ruane J, Sonnino A, editors. The role of biotechnology in exploring and protecting agricultural genetic resources. Food and Agriculture Organization of the United Nations, Rome, Italy: 61-78. Available from: http://www.fao.org/Biotech/docs/panis.pdf Date accessed: 18 April 2011.

Panis B, Piette B, Swennen R. 2005. Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Science 168:45-55.

Panis B, Thinh NT. 2001. Cryopreservation of Musa germplasm. INIBAP Technical Guideline 5 (J.V. Escalant and S. Sharrock, eds). International Network for the Improvement of Banana and Plantain, Montpellier, France. Available here.

Raja K, Palanisamy V, Selvaraju P. 2003. Desiccation and cryopreservation of recalcitrant arecanut (Areca catechu L.) embryos. Plant Genetic Resources Newsletter 133:16-18. Available from: http://www2.bioversityinternational.org/publications/pgrnewsletter/article.asp?lang=en&id_article=96&id_issue=133 Date accessed: 18 April 2011.

Pritchard HW. 2007. Cryopreservation of desiccation tolerant seeds. Methods in molecular biology, 368:185-201.

Razdan MK, editor. 1997. Conservation of Plant Genetic Resources In Vitro. Oxford and IBH Publishing Co. Pvt. Ltd., India.

Reed BM. 2008. Plant Cryopreservation: A practical guide. Springer Science and Business Media, USA. 513 pp.

Reed BM, Engelmann F, Dulloo ME, Engels JMM. 2004. Technical guidelines for the management of field and in vitro germplasm collections. IPGRI Handbook for Genebanks No.7. IPGRI, Rome, Italy. Available here.

Santos IRI, Stushnoff C. 2002. Cryopreservation of embryonic axes of Citrus species by encapsulation-dehydration. Plant Genetic Resources Newsletter 131:36-41. Available from: http://www2.bioversityinternational.org/publications/pgrnewsletter/article.asp?lang=en&id_article=71&id_issue=131 Date accessed: 18 April 2011.

Stanwood PC, Bass LN. 1981. Seed germplasm preservation using liquid nitrogen. Seed Science and Technology 9, 423-437.

Tay DCS, Liu CR. 1992. Using hard agar medium and grooved tubes for the distribution of sweet potato tissue culture. Plant Genetic Resources Newsletter 88/89:23-25.

Towill LE, Bajaj YPS, editors. 2002. Cryopreservation of Plant Germplasm II. Biotechnology in Agriculture and Forestry, Vol. 50. Springer, Berlin.

Withers LA, Paroda RS, Arora RK. 1991. Biotechnology and plant genetic resources conservation. Plant genetic resources: conservation and management. Concepts and approaches. International Board for Plant Genetic Resources, New Delhi (India). p. 273-297. 
 

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DNA bank

View publication on DNA banks by clicking on the icon above. (0.1 MB)

Page compiled by:  ILRI, Addis Ababa, Ethiopia (Jean Hanson); Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge).

Field genebank

View the guidelines on field and in vitro collection management by clicking on the icon above. (0.5 MB)

Page compiled by: Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge); ILRI, Addis Ababa, Ethiopia (Jean Hanson) including information extracted from: Reed BM, Engelmann F, Dulloo ME, Engels JMM. 2004. Technical guidelines for the management of field and in vitro germplasm collections. IPGRI Handbook for Genebanks No.7. IPGRI, Rome, Italy.

Characterization

View chapter on characterization approaches by clicking on the icon above. (0.1 MB)

Page compiled by: Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge); ILRI, Addis Ababa, Ethiopia (Jean Hanson) including information extracted from: Engels JMM, Visser L, editors. 2003. A guide to effective management of germplasm collections. IPGRI Handbooks for Genebanks No. 6. IPGRI, Rome, Italy.

What is characterization

Characterization is the description of plant germplasm. It determines the expression of highly heritable characters ranging from morphological or agronomical features to seed proteins or molecular markers.

Why is it done

Characterization of germplasm is essential to provide information on the traits of accessions assuring the maximum utilization of the germplasm collection to the final users.

The recording and compilation of data on the important characteristics which distinguish accessions within a species, enables an easy and quick discrimination among phenotypes. It allows simple grouping of accessions, development of core collections, identification of gaps and retrieval of valuable germplasm for breeding programmes, resulting in better insight about the composition of the collection and its genetic diversity.

It also facilitates a check on the trueness-to-type of homogeneous samples, allowing detection of misidentifications or duplicates and indicating possible errors made during other genebank operations. This is also important for the case of in vitro collections, to monitor the genetic stability of these collections that are susceptible to somaclonal variation.

When is it done

It can be carried out at any stage of the conservation process, as long as there are sufficient numbers of seeds or plant materials to sample. It should be done as soon as possible to add value to any collection. It is, however, very time consuming and expensive and therefore often delayed or done during regeneration in many genebanks to reduce costs.

How is it done

Normally it requires growing a representative number of plants (taking a sample from previously conserved seeds or plant tissue) in the field using a statistically sound replicated design during a full growing cycle, recording characters that are highly heritable (not much influenced by the environment), easily visible to the eye and expressed in all environments. A minimum of three replicates and preferably four with data taken from at least 10 plants across replications has been shown to be statistically acceptable for some crops. Care should be taken to select a site where the species is adapted and traits will be expressed. Measurements can be made at the plant level to capture the information about the variability between plants of the same accession. Most genetic resources collections are made up of population or landraces which are genetically variable. It may therefore be necessary to collect data at the plant level, rather than at the plot level, because knowledge of the average value of a descriptor for an accession as a whole is not always sufficient.

In order to facilitate standardization of information obtained during characterization, Bioversity International has been coordinating the development, publication and updates of various plant descriptor lists in close cooperation with crop experts and genebank curators (see also the crop descriptors on the Bioversity website by clicking here). There are descriptor lists developed for more than 90 crops.

Characterization is also increasingly done using complementary characterization methods to capture the full information. Characterization may include one or more of the following:

Morphological descriptors

A set of morphological descriptors can be used to describe the phenotype. Plant, stem, leaf, flower, pod and seed traits can all be scored or measured and expressed in numeric values. Descriptive traits such as flower colour can be expressed as a numeric value by using a standard colour chart. The descriptive traits used will vary with the species. Lists for common crops are available on the Bioversity website. Guidelines are also available for developing descriptors.

Herbarium samples

Herbarium samples are a good record of variation in a species and with care can be kept for many years and used for rechecking traits after the material is removed from the field. Sufficient detail should be captured to taxonomically identify the plant and demonstrate the traits that show variation.

• Collect plants with leaves, flowers and pods, also roots if plants are small.
• Spread plants open before pressing and arrange leaves and flowers to show both sides or any specific traits.

Digital pictures

Sufficient detail should be captured in images to taxonomically identify the plant and demonstrate the traits that show variation. The images can be stored in a database linked with the morphological data.

• Take images for character(s) which may be difficult to describe verbally.
• Images of whole plant, racemes (if applicable), flowers, pods and seeds.

Nutritional traits

Nutritional traits which include food or feed value are important for many vegetables, fruits, and forages. Cooking or utilization traits such as toxicity or flavour are important for many crops and may be part of the set of traits characterized to provide important information on potential use.

Molecular descriptors

New methods have made molecular analysis and genotyping useful techniques for studying diversity. Samples of leaf are usually collected from replicated trials. A variety of molecular techniques are used, including cytological markers, biochemical markers and molecular genetic markers such as SSR, EST-SSR, AFLP, RAPD. Their choice depends on the stage of research into molecular methodologies for the crop, facilities and expertise available in each genebank. Descriptors are available to describe a genetic marker technology and collect information about genetic markers in crops that are standardized and replicable.

Handling and analyzing data

The recording and compilation of data on the important characteristics which distinguish accessions within a species is just the first step in characterization. The analysis, interpretation and presentation of the data are key to sharing information about the diversity in the collection. There are many statistical packages available for data analysis that can be used. An analysis of variance can be done on single trait data. However, in many cases data is not normally distributed and must be transformed or analysed with statistical packages that handle this type of data for single traits. Multivariate analysis is used for data on multiple traits using using a range of different cluster, discriminant or principal components analysis methods to look for natural groupings of accessions.

Using the data

Once the data is collected it can be used to describe the diversity within and between accessions. The simplest use of characterization data is to identify accessions with specific traits to make selections for further research or plant breeding. The data can also be used to identify groups of morphologically or genetically similar accessions . The descriptive data can also be used together with spatial analysis software to match traits with collection sites and environmental adaptation.

Development of core collections

Many users find large collections difficult to work with or to know which accessions to select. The concept of core collections was introduced in 1984 to represent the diversity within a collection, a crop, a wild species or group of species in a limited number of accessions. Two ways of identifying the clusters for selection of representative accessions for a core collection have been described:

• Separate the accessions into meaningful groups using a hierarchical procedure; separating the wild from the cultivated species, and using taxonomy and knowledge about domestication, distribution, breeding history, cropping pattern and utilization.
• Create groups of similar accessions from characterization data using multivariate analysis, genetic markers, agromorphological characteristics or other characters, using a range of different cluster, discriminant or principal components analysis methods.

The methods can be used individually or together and the choice of method will depend on the information available, amount and type of genetic diversity in the collection and the crop.

The final step in establishing a core collection is choosing the actual entries from each group. This can be done randomly or systematically, based on some formal analytical procedure or on pragmatic considerations such as amount of material or information.

References and further reading

Bioversity International. 2007. Guidelines for the development of crop descriptor lists. Bioversity Technical Bulletin Series. Bioversity International, Rome, Italy. Available here.

Engels JMM, Visser L, editors. 2003. A guide to effective management of germplasm collections. IPGRI Handbooks for Genebanks No. 6. IPGRI, Rome, Italy. Available in English (1.4 MB) and Spanish (1.5 MB).

FAO/IPGRI. 2001. Multi-Crop Passport Descriptors. FAO and IPGRI, Rome, Italy. Available in English, French and Spanish.

IPGRI. 2001. The design and analysis of evaluation trials of genetic resources collections. A guide for genebank managers. IPGRI Technical Bulletin No. 4. International Plant Genetic Resources Institute, Rome, Italy. Available here.

Hodgkin T, Brown AHD, van Hintum ThJL, Morales EAV. 1995. Core collections of plant genetic resources. Wiley and Sons, UK.

van Hintum ThJL, Brown AHD, Spillane C, Hodgkin T. 2000. Core collections of plant genetic resources. IPGRI Technical Bulletin No. 3, International Plant Genetic Resources Institute, Rome, Italy. Available here.

Johnson RC, Hodgkin T. 1999. Core collections for today and tomorrow. International Plant Genetic Resources Institute, Rome, Italy.

de Vicente MC, Metz T, Alercia A. 2004. Descriptors for genetic markers technologies. International Plant Genetic Resources Institute, Rome, Italy. Available here.

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