Sample preparation in cassava field banks

Contributors to this page: IITA, Nigeria (Dominique Dumet), Bioversity International/ILRI, Ethiopia (Alexandra Jorge); INIA, Peru (Llerme Rios); independent consultant (Clair Hershey).

Source of planting material

Cassava has a typical growing cycle between 9 and 24 months, depending on the genotype and the environmental conditions.

• It is best to regenerate within 18–24 months, when most plants complete their growth cycle, to avoid lodging from excessive growth and build-up of pests and diseases.

Visual inspection of plant material

• Inspect all plants in the plot to make certain they all appear to be the same clone. If there is a mixture of clones, reconfirm through the standard descriptors, by comparison with the previously recorded genebank descriptors, which of the plants are the true accession and which are the 'contaminants'.
• Select plants as sources of planting material based on their apparent health status. Plants should be free of virus symptoms and of other pests or diseases.
• Select plants that will provide well-lignified cuttings with well-distributed, healthy buds.
• Prepare stakes from healthy plants, identified earlier in the season before leaves drop off when pest and disease (especially virus) symptoms and other foliar diseases are apparent. Also inspect roots for pest and disease symptoms.
• Select the mature portion of the stem, avoiding the top green stems and the bottom section of the plants.
• Take care to avoid mixing of genotypes.

Preparation of planting material

• Cut stakes (stem pieces) at least 20 cm long with at least 4–5 nodes with viable buds to ensure crop establishment. Use well-lignified stems, generally from the middle section of the plant. Cut them at a right angle with a machete or saw to create a smooth cut.
• Handle stems with care to prevent bruising and peeling. Do not place the stems on a hard surface to cut them, as this can damage the nodes, reduce their quality and provide entry points for pathogens and insect pests.
• Tie cuttings of each accession firmly in separate bundles. At least one cutting in each bundle is labelled, and for extra security it is best to label two stakes (with accession name and number and date of harvest). The label must be sturdy and secure and able to withstand the handling (e.g. packaging and shipping) and treatment of the stake bundles.

Pre-treatments

• Treat the bundled stakes with a mixture of broad spectrum insecticide and fungicide.
• Add zinc sulphate in regions where zinc is limited in the soil.

Disposal of contaminated material

• Incinerate or autoclave contaminated material (to avoid spreading diseases and pests).
• Rogue and burn diseased plants regularly during the growth season (if it does not compromise the survival of a specific accession).
• After harvest, destroy discarded stems and roots that have disease symptoms or pest contamination

Recording information during sample preparation in field banks

The following information should be recorded for each step:

• Site name and map/GPS reference.
• Name of collaborator.
• Field bank site name (a code to identify the site location).
• Plot reference (the plot number at the field site).
• Accession number; population identification.
• Name of staff (name of staff recording the data).
• Source of cuttings.
• Number of generations since acquisition of germplasm or date of previous multiplication (if generation is not known).
• Preparation of planting material (details of pre-treatments applied).
• Details of plants removed or destroyed (due to type mixtures or pest or disease contamination).

References and further reading

Fukuda WMG. 1996. Banco de germoplasma de mandioca: manejo, conservação e caracterização. Cruz das Almas, BA: EMBRAPA-CNPMF. 103 p. (EMBRAPA-CNPMF, Documento, 68).

Hershey C. 2008. A Global conservation strategy for cassava (Manihot esculenta) and wild Manihot species. A consultancy report to CIAT, on behalf of the Global Crop Diversity Trust, Rome. (Final report under review).

IITA Genebank Manual Series, Cassava field bank operations at the International Institute of Tropical Agriculture (IITA). International Institute of Tropical Agriculture, Ibadan, Nigeria.

Mohd SS, Rao VR, editors. 2001. Establishment and Management of Field Genebank, a Training Manual. IPGRI-APO, Serdang. 121 p. Available here.

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Slow growth storage (SGS) of cassava genetic resources

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).

Slow growth storage (SGS) was developed from the conventional in vitro techniques for cassava, to increase the length of time between culturing and rejuventation. It incorporates growth retardants to reduce the need for rejuvenation of the tissue culture plants.

Most countries with important and relevant cassava genebanks have tissue culture facilities, mostly for the elimination of pests and diseases (using also meristem and thermotherapy techniques) and for the exchange and dissemination of germplasm as well as alternative/complementary methods of conservation of clonal crops.

It is estimated that about 8100 cassava accessions are conserved in 13 tissue culture banks worldwide. However about 80% of these accessions are in the CIAT and IITA collections, and relatively few in national programmes. The other main in vitro cassava collection is held by EMBRAPA, Brazil (Hershey, 2008).

Cassava cultures under SGS can be stored for an average of a year (varying between 4 and 19 months, depending on the genotype). Advantages of SGS:

• Useful to reduce the risks of losses (due to accumulation of pests and diseases, environmental stresses) that are more prone to occur in field banks.
• Good to reduce the bulkiness and transport difficulties of field banks.
• Safer and faster way to propagate large quantities of materials for breeding or dissemination purposes and to maintain a small working collection for experimental/research purposes.
• Useful way to duplicate material already in other genebanks (in vitro or field banks).
• Essential for international exchange of germplasm (to prevent spread of pests and diseases). 

Two documents provide comprehensive guidelines for SGS of cassava and Manihot species (IITA 2007; Mafla et al. 2009). The two centers use similar techniques, with small variations based on local experiences. Anyone wishing to establish or improve a laboratory should consult these publications. The following is an overview only, and a synthesis of the procedures at CIAT and IITA, to provide general guidelines of the process and procedures, but is not intended to provide all the operational details of SGS. See here a flow chart of operations for Manihot germplasm from Mafla et. al 2009.

Sample processing for in vitro banks

Source of material

• Healthy plant materials from field, greenhouse, or screenhouse, or already growing in SGS.

Starting material

• CIAT uses nodal cuttings and apical buds.
• IITA uses explants.

Visual inspection of plant material

• Observe plantlets for vigor and phytosanitary status.

Disposal of contaminated materials

• Autoclave contaminated materials.

Recording information during sample processing

The following information should be recorded for each step:

• Passport data (accession number).
• Culture data (source of explant/place of collection).
• Date of collection (date of culture/inoculation).
• Culture media.
• Symptoms for diseases (presence/absence of diseases).

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Viability montioring for in vitro banks

Routine monitoring methods

Plant quality/viability

• Check regularly (weekly or monthly) survival and growth:
  • Viability.
  • Contamination.
  • Leaf senescence index (ratio green:dead leaves).
  • Number of green shoots suitable for further micro-propagation.
  • Number of viable green nodes (relative to green stem elongation).
  • Presence or absence of roots.
  • Occurrence of callus.
  • Necrosis.
  • Dead cultures.
• Eliminate any bacterial contamination by placing the explants in the 8S media (without agar) at low pH or antibiotics (Mafla et al., 2007).
• Check vigor and rooting after 3-4 months after initiation (especially for some wild species that might be tissue culture recalcitrant and have rooting problems).
  • Check bacterial indexation for microbial contamination.
  • Discard if results are positive.
  • Retain for culture and distribution if results are negative.
  • Identify recalcitrant accessions that might need further custom growth media developed.

Genetic integrity

• Apply phenotypic, biochemical (isozymes) and molecular (DNA fingerprinting, RAPDs, SSR, RFLPs) techniques to:
  • Assess genetic stability in the cassava world-wide collection after 10-30 years of in vitro storage.
  • Verify the genetic integrity and management practices comparing in vitro and field accessions.
  • Identify genetic duplicates and redundant accessions.

Need to rejuvenate/multiply

• Minimum quantity/viability of stocks – Regenerate every 4 to 19 months, depending on the accession (the number of plants per accession depends on the needs).

Recording information during viability monitoring

The following information should be recorded for each step:

• Accession number (unique identifier).
• Culture data (source of explants/place of collection).
• Date of collection (date of culture/inoculation).
• Culture media.
• Discarded materials and justification (due to death, loss of vigor or contamination).

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Storage for in vitro banks

Sample specifications

• Type of plant samples - apical buds and nodal cuttings.
• Size of sample (replication/accession):
  • CIAT uses 2-3 explants per tube, replicated 5 times.
  • IITA uses 1 explant per tube, replicated 10 times.
• Type and size of container:
  • CIAT uses glass tubes (25 x 150mm) capped with aluminium foil and firmly sealed with plastic wrap, containing 10ml of media.
  • IITA uses glass tubes (16 x 125 mm) or polyethylene bags.

Storage specifications

Growth media – for conservation (CIAT)

• Full strength Murashige and Skoog (MS) mineral salts, 0.01mg/L NAA, 0.1mg/L GA3, 0.02mg/L BAP, 1.0mg/L thiamine HCL, 100mg/L myo-inositol.
• Other components - 20g/L sucrose, 7g/L agar.
• Special conditions – pH = 5.7.

Growth media – for slow grow (CIAT)

• Full strength Murashige and Skoog (MS) mineral salts, 0.01mg/L NAA, 0.1mg/L GA3, 0.02mg/L BAP, 1.0mg/L thiamine HCL, 100mg/L myo-inositol, 10mg/L silver nitrate.
• Other components - 20g/L sucrose, 7g/L agar.
• Special conditions – pH = 5.0.

Growth media – for wild species (CIAT)

• Full strength Murashige and Skoog (MS) mineral salts, 1.0mg/L thiamine HCL, 100mg/L myo-inositol, 0.2mg/L kinetin, 0.48mg/L CuSO4, 1g/L charcoal activated..
• Other components - 30g/L sucrose, 7g/L agar.
• Special conditions – pH = 5.7.

Growth media – for conservation (IITA)

• Full strength Murashige and Skoog (MS) mineral salts, 0.01mg/L NAA, 0.08mg/L GA3, 0.15mg/L BAP, 100mg/L inositol.
• Other components - 30g/L saccharose, 5g/L agar.
• Special conditions – pH = 5.7?

Culture facility regimes

• Light level/intensity – 18.5µmoles m-2 s-1.
• Photoperiod – 12h light /12h dark.
• Day/night temperatures – between 18-24oC.

Storage duration (time without sub culturing)

• Average - 11 months.
• Minimum and maximum range – 4-19 months.

System for tracking material/inventory system during tissue culture storage

• Inventory the cultures every sub-culture time.

Recording information during tissue culture storage

The following information should be recorded for each step:

• Accession data.
• Source of explants.
• Date of inoculation.
• Date/Number of subculture.
• Media.
• Culture conditions.
• Plant losses.

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References and further reading

Calles T, Dulloo ME, Engels JMM, Van den Houwe I. 2003. Best Practices for Germplasm Management - A new approach for achieving genebank standards. Technial Report. International Plant Genetic Resources Institute, Global Crop Diversity Trust, Rome, Italy.

Escobar RM, Roca WM, Mafla G, Roa J. 1994. In vitro conservation of genetic resources: The case of cassava. CIAT (Internal Circulation). 23 p.

Fregene M, Ospina JA, Roca W. 1999. Recovery of cassava (Manihot esculenta Crantz) plants from culture of immature zygotic embryos. Plant Cell Reports 55:39-43.

IITA. 2007. Cassava in vitro processing and gene banking. IITA Genebank series 2007. Available here .

Mafla G. 1994. Conservación de germoplasma In vitro. In: King C, Osorio J, Salazar L, editors. Memorias I Seminario Nacional sobre Biotecnología.  Universidad del Tolima. Colombia, pp 65-77.

Mafla G. 1995. Manejo de datos e información de la colección in vitro de yuca (Manihot esculenta, Crantz). In: Memorias. Curso en Documentación de Recursos Fitogenéticos. Auspiciado por Universidad Nacional de Colombia, Bioversity y CIAT. Palmira, pp. 97-118.

Mafla G, Roa JC, Aranzales E, Debouck D. 2009. Handbook of procedures for in vitro germplasm conservation of the genus Manihot. CIAT, Cali, Colombia. 56 pp. Available here (8 MB).

Mafla G, Roa JC, Guevara CL. 2000. Advances on the in vitro growth control of cassava using silver nitrate. In: Carvalho LJCB, Thro AM, Vilarinhos AD, editors. Proceedings IV International Scientific Meeting of the Cassava Biotechnology Network, Salvador, Bahia, Brazil. November 03-07, 1998. EMBRAPA , CENARGEN and CBN. Brasilia, Brazil. Pp. 439-446.

Mafla G, Roa JC, Flor NC, Debouck DG. 2002. Conservación in vitro y utilización del germoplasma del género Manihot. Trabajo presentado en el VIII Congreso Latinoamericano de Botánica y II Congreso Colombiano de Botánica, Cartagena, Colombia, 13-18 Octubre 2002. Available from: URL: http://isa.ciat.cgiar.org/urg/urgweb_folder/files/posters/cartagenafinal.pdf Date accessed: 26 August 2010.

Mafla G, Roa JC, Ocampo C, Gallego G, Jaramillo G, Debouck DG. 2004. Efficacy of silver nitrate for slow growth conservation of cassava (Manihot esculenta Crantz). Determination of viability and genetic stability. In: Abstracts of the Sixth International Scientific Meeting of the Cassava Biotechnology Network. March 8-14 CIAT, Cali, Colombia. p. 134.

Mafla G, Roa JC, Ocampo CH, Gallego G, Jaramillo G, Debouck DG. 2004. Efficacy of silver nitrate for slow-growth conservation of cassava (Manihot esculenta Crantz). Determination of viability and genetic stability. Poster presented at CBN-IV. Available from: URL: http://isa.ciat.cgiar.org/urg/urgweb_folder/files/posters/CBN-VI.pdf Date accessed: 26 August 2010.

Mafla G, Roca WM, Reyes R, Roa JC, Muñoz L, Baca AE, Iwanaga M. 1992. In vitro management of cassava germplasm at CIAT. In: Roca WM, Thro AM,  editors. Proceedings of first international scientific meeting of the cassava Biotechnology network. Cartagena, Colombia, pp. 168-174.

Roca WM, Angel F, Sarria R, Mafla G. 1992. Future initiatives in biotechnology research for tropical agriculture: the case of cassava. In: McCorwick DK, editor. Advanceds in Gene Technology: Feeding the World in the 21st Century, 1992 Miami Bio/technology Winter Symposium, Miami, FL, USA, pp. 87.

Roca WM, Chaves R, Marin ML, Arias DI, Mafla G, Reyes R. 1989. In vitro methods of germplasm conservation. Genome 31 (2):813-817.

Roca WM, Escobar R, Angel F, Mafla G. 1991. Tissue culture methods for germplasm conservation: The case of cassava. In: Bardowell ME, editor. Tissue culture technology for improved farm production, Kingston, Jamaica. Pp 47-55.

Roca WM, Mafla G, Segovia RJ. 1991. Costo mínimo de un laboratorio de cultivo de tejidos vegetales. In: Roca WM, Mroginski LA, editors. Cultivo de tejidos en la agricultura: Fundamentos y Aplicaciones, pp. 912-920.

Roca WM, Nolt B, Mafla G, Roa JC, Reyes R. 1991. Eliminación de virus y propagación de clones en la yuca (Manihot esculenta Crantz) In: Roca WM, Mroginski LA, editors. Cultivo de tejidos en la agricultura: Fundamentos y Aplicaciones, pp. 403-421.

Szabados L, Nuñez LM, Tello LM, Mafla G, Roa JC, Roca WM. 1991. Agentes gelanitizadores en el cultivo de tejidos. In: Roca WM, Mroginski LA, editors. Cultivo de tejidos en la agricultura: Fundamentos y Aplicaciones, pp. 79-93.

Velásquez E, Mafla G. 1999. Conservación in vitro: Una alternativa segura para preservar especies silvestres de Manihot spp. (Euphorbiaceae). In: II Congreso Nacional de Conservación de la Biodiversidad. Pontificia Universidad Javeriana, Bogotá 19-22 Octubre, 1999, pp 14.

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Health diagnosis of cassava genetic resources

Contributors to this page: CIAT, Colombia (Daniel Debouck, Roosevelt Escobar, Graciela Mafla); IITA, Nigeria (Dominique Dumet); Bioversity International/ILRI, Ethiopia (Alexandra Jorge); independent consultant (Clair Hershey).

List of pests and diseases of quarantine importance for cassava

Click here for additional information on the safe transfer of germplasm of clonal crops.

The list below mentions some of the pests/diseases that are considered important worldwide, but many of them may or may not have relevance in specific countries. It also does not consider pests/diseases of limited relevance (e.g. only important in very few countries). A further disease list can be found here.

The Americas have the greatest diversity of cassava pests, followed by Africa and then Asia. Damage in Africa is often high due to the lack of natural predators of pests.

• The green mite (Mononychellus tanajoa) (Americas and Africa) and the mealybug (Phenococcus manihoti and P. Herreni) cause major damage in Africa, and recently in Asia.
• Whiteflies (Aleurotrachelus socialis and A. aepim), hornworm (Erinnyis ello), stemborers (Chilomina clarkei), burrower bugs (Sternocoelus manihoti and Tropidozineus fulveolus), thrips (Frankliniella williamsi) and lacebugs (Vatiga manihoti, V. illudens and Amblydtira machalana) are a problem in the Americas.
• Scales (Aonidomytilus albus), termites and grasshoppers are also widely reported.
• Amongst the main cassava diseases there are the complex of:
  • Cassava mosaic diseases (CMD) caused by the African cassava mosaic virus (ACMV), the East African cassava mosaic virus (EACMV) and by the South African cassava mosaic virus (SACMV).
  • The cassava brown streak virus (CBSV) in Africa.
  • In South America, the main viral diseases are caused by the cassava common mosaic virus (CsCMV and CsXV) and by the cassava frogskin virus (CFSV).
  • Other diseases like cassava bacterial blight (CBB) or those caused by fungi, like cassava anthracnose and root rot, are important worldwide.

Recommended methods to detect the presence of each pest or disease

Viruses

• ELISA, TBIA, PCR, seedling symptom test, indicator test.
• Use extra grafting techniques to test cassava materials for Frogskin disease.
  • Virus tested plants should be transplanted into sterilized soil and retested for Frogskin disease by grafting to a healthy hypersensitive clone such as cv. Secundina.
  • Plants tested negative in all tests should be available for distribution.
  • If plants are tested virus positive they can either be discarded (if more plants of the same accessions are tested virus negative) or enter again into the thermotherapy process and meristem culture.

Fungi

Blotter test, agar test, washing test, direct visual inspection.

Bacteria

Seedling symptom test, dilution plating test.

Weeds, insects and nematodes

Direct visual inspection.

Testing intervals/seasons

Testing before material goes into the genebank or to the field is important to reduce transfer of diseases or pests.

Viruses

Test seedlings before transfer to the field for regeneration or during regeneration and rogue infected material.

Fungi

Test plant propagules on entry to genebank and regularly thereafter. Rogue infected material.

Bacteria

Test plant propagules on entry to genebank and regularly thereafter. Rogue infected material.

Weeds, insects and nematodes

Test plant propagules on entry to genebank and regularly thereafter. Rogue infected material.

Recording information during health diagnosis

The following information should be recorded for each health diagnosis step:

• Site name and map/GPS reference.
• Name of collaborator.
• Field bank site name (a code to identify the site location).
• Plot reference (the plot number at the field site).
• Accession number; population identification.
• Name of staff (name of staff recording the data).
• Date of monitoring (date when data is collected).
• Date of test (the date that the test was commenced).
• Number of replications (the number of replicates in the test).
• Size of the samples per replication.
• Pre-treatments (pre treatments used for the test).
• Media (the media for the test) (e.g. for fungi).
• Material (plant part used).
• Pathogen tested (name of pathogen tested).
• Test method (method used).
• Percentage infection (% of plants or samples infected).

References and further reading

Frison EA, Feliu E, editors. 1991. FAO/IBPGR Technical Guidelines for the Safe Movement of Cassava Germplasm. Food and Agriculture Organization of the United Nations, Rome/International Board for Plant Genetic Resources, Rome.

Nolt B, Velasco AC, Pineda B. 1991. Improved purification procedure and some serological and physical properties of Cassava Common Mosaic Virus from South America. Ann. Appl. Biol. 118:105-113.

Nolt B, Pineda B, Velasco AC. 1992. Surveys of cassava plantations in Colombia for virus and virus-like diseases. Plant Pathology 41: 348-354.

Velasco AC, Nolt B, Pineda B. 1990. Comparación de tres métodos de la técnica inmunoenzimática "Elisa" para el diagnóstico de virus del mosaico común de la yuca. Fitopatología Colombiana 14(1):3-9.

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