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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Registration of cassava genetic resources

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

This section covers registration of cassava and Manihot wild species that are being newly introduced into a genebank. These genetic resources may be introduced as stakes (stem pieces) (within-country only), as in vitro material or as seeds. Wild species are typically introduced as seed samples from a population, while cassava is generally introduced as clonal material, where each accession is genetically uniform.

Verifying accompanying documentation

The following documentation should be sent with the new material

For all accessions:

• Origin and contact person.
• List of shipped accessions.
• Date of preparation of material and date of shipment.
• Details of treatments and growth conditions (e.g. in vitro media specifications).
• Post-arrival handling instructions.

 For accessions imported from another country (in addition to above):

• Import permit issued by the recipient country.
• Standard Material Transfer Agreement (SMTA), modelled upon the specifications of the International Treaty for Plant Genetic Resources for Food and Agriculture (ITPGRFA).
• Phytosanitary Certificate issued by the official phytosanitary authority of the donor country.

Verifying the consignment

Inspection of plants to determine any plant health problems should include the following:

• Visual inspection for fungi and bacteria.
• Virus indexing of material (see plant health section for more details).
• If any pathogens are detected, material should be destroyed.
• If the germplasm is rare, material may be retained, but the pathogens should be eliminated using high-confidence techniques before introducing the material into the genebank.

Assigning numbers

The introduction of cassava or Manihot material at the beginning of the conservation process can usually be accomplished in one of three different forms:

• From planting material (cuttings). These usually originate from within-country collections.
• From in vitro material. These can be from within-country or foreign collections.
• From botanic seeds, especially, in the case of wild species. Plants may need to be regenerated from embryo rescue if conventional germination fails.

Regardless of the form of introduction, a ‘temporary identifier’ must be assigned to each individual sample of the new accession until it is decided which sample, or set of derived sub-samples, will be included in the genebank and registered officially.

• This temporary identifier usually consists of the accession’s name and code, as provided by the donor, and a sample number (e.g. a serial number such as 1, 2, 3) assigned at the receiving genebank.

The official registration involves the attribution of a permanent and unique identifier code. In the case of tissue culture introductions, an accession can be officially registered once the following conditions are met:

• Tissue cultures derived from one single sample (shoot tip) are tested aseptic (i.e. free of contaminating fungi and bacteria).
• A minimal number (generally about seven) of vigorously growing tissue cultures derived from the selected sample are established.

If the conditions described above cannot be met, then a temporary accession number must be assigned and plantlets must go through the necessary disease cleaning or multiplication process until they can be assigned a permanent number.
A step by step general guideline for registration can be seen by clicking here.

Recording information during registration

New material - introduction phase

Newly introduced meristems or nodal cuttings are often processed in batches. For each batch, a series of information should be recorded in a table with the following fields (example from IITA):

• Batch number.
• Accession number (may be a number or a combination of letters and numbers).
• Date of in vitro introduction.
• Number of explants introduced.
• Contamination.
• Necrosis.
• Operator.
• Numbers of plantlets sent to multiplication 1.
• Contamination while in multiplication 1.
• Necrosis while in multiplication 1.
• Number of plantlets sent to the genebank.

On-going material - germplasm already in the genebank

Once an accession is already introduced in the bank, its permanent accession number should be added to the database. For each accession the following data should be recorded (example below is from IITA; the details of this sequence will vary according to the needs and procedures of each specific genebank):

• Accession number.
• Date of introduction in vitro (in case of replacement, all previous entries should be discarded). Type of explant (meristem/nodal cutting/other).
• Virus-free lines certified (yes/no).
• In the bank at the time of last inventory (insert date of last inventory).
• In subculture at the time of last inventory (insert date of last inventory).
• Current number of replicates in subculture.
• Total replicates (= in the bank + in subculture).
• Contamination in bank (number of tubes eliminated because of contamination).
• Necrosis in bank (number of tubes eliminated because of necrosis).
• Out 1 (number of tubes sent to subculture 1).
• Date out 1 (date of subculture 1).
• Obtained 1 (number of micro-cuttings obtained after subculture 1).
• Out 2 (number of tubes sent to subculture 2).
• Date out 2 (date of subculture 2).
• Obtained 2 (number of micro-cuttings obtained after subculture 2).
• Current number of replicates in subculture.
• Subcontamination (number of tubes eliminated from subculture due to contamination).
• Subnecrosis (number of tubes eliminated from subculture due to necrosis).
• Back 1 to bank (number of tubes sent back to the bank from subculture 1).
• Date back 1 (date when subculture 1 is sent back to the bank).
• Back 2 to bank (number of tubes sent back to the bank from subculture 2).
• Date back 2 (date when subculture 2 is sent back to the bank).
• Old cuttings discarded (number of cuttings in the bank discarded during replacement).
• Extra subcultures discarded (tubes from subculture discarded).
• Nodal cutting from the bank sent to acclimatization, other experiments, safe duplication or for multiplication and distribution.
• Computerization of all the data is advisable to facilitate germplasm management. The use of portable data recorders speeds up data collection and reduces recording mistakes. Ultimately, barcoding in vitro collections can further improve genebank management in terms of cost and data reliability.
• Establishing and maintaining a documentation system to record and manage all relevant information related to the introduction of material, for further reference. A database system is usually recommended when possible.
• Germplasm inventory. An inventory of all germplasm should be performed once a year.

References and further reading

Ceballos H. 2006. Cassava research at CIAT [poster]. Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia. Available from: http://webapp.ciat.cgiar.org/news/pdf/poster02_scmeeting_06.pdf. Date accessed: 26 August 2010.

Guevara CL, Mafla G. 1996. Manihot collections held at CIAT. In: Engelmann F, editor. Management of field and in vitro germplasm collections. CIAT, Cali, Colombia. pp. 109-112.

Mafla G, Roa JC, Debouck DG. 2004. Observations about the distribution of cassava germplasm from an international collection. Poster presented at the CBN-V. Available from: http://ciat-library.ciat.cgiar.org/Articulos_Ciat/CBN-V.%20G%20Mafla%20ppt.pdf. Date accessed: 26 August 2010.

Mafla G, Roa JC, Flor NC, Aranzales E, Debouck DG. 2006. Distribution of cassava germplasm from an international genebank: a service to the global agriculture. Poster presented at the First meeting of the Governing Body, ITPGRFA, Madrid, Spain, 12-16 June 2006. Available from: http://ciat-library.ciat.cgiar.org/Articulos_Ciat/CIAT40years.pdf. Date accessed: 26 August 2010.

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Regeneration guidelines for cassava

The information on this page was extracted from:
Jorge M.A. 2008. Regeneration guidelines: cassava. In: Dulloo M.E., Thormann I., Jorge M.A. and Hanson J., editors. Crop specific regeneration guidelines [CD-ROM]. CGIAR System-wide Genetic Resource Programme, Rome, Italy. 9 pp.

Before reading the regeneration details for this crop, read the general introduction that gives general guidelines to follow by clicking here.

This guideline provides information for the regeneration of field collections of cultivated species.

Introduction

Cassava (Manihot esculenta Crantz) is the only domesticated species of a genus which contains 98 species and belongs to the Euphorbiaceae family. It was first domesticated 7000 years ago (Allem 2002) in the lowlands of South America, probably in the Amazon Basin. Introduced into Africa by the Portuguese in the 16th century and into Asia in the 18th century (Hillocks 2002; Onwueme, 2002), today it is found all over the tropics and subtropics.
It is a very hardy perennial crop with the ability to grow on land where drought is frequent and in soils low in nutrients, where cereals and other crops do not grow well. Cassava is vegetatively propagated from nodal cuttings (stakes) in field production systems, while all of the wild species are seed propagated in nature. It is also a monoecious and predominantly cross-pollinating species with viable seeds in the genotypes that flower. Controlled pollination and seed production are mostly used for the development of new varieties in breeding programmes. Seed populations can also be collected from cassava field collections to be stored and preserved as future sources of genetic information. Wild types are more difficult to regenerate in field genebanks than cultivated landraces (M. esculenta); most of them are perennial and difficult to regenerate through cuttings. Cassava is mainly conserved as live plants in field genebanks, but it can also be maintained as seeds, in vitro and by cryopreservation. About 20 000 accessions are being conserved worldwide, the largest collections being held at CIAT (Colombia), EMBRAPA (Brazil), IITA (Nigeria), and in Ghana and India (Ng and Ng 2002).

 

Choice of environment and planting season

Climatic conditions

• Cassava grows well between 30°N and 30°S in areas where annual rainfall is more than 750 mm a year, temperatures above 18°C and low to medium altitude (1,500–2,000 m).
• It is sensitive to frost but tolerant of long dry periods, soils with low pH, high aluminium and low fertility.

Planting season

• If water is available either through irrigation or well-distributed rainfall, the crop can be planted at any time of the year, preferably at the beginning of the warm season (growth slows during cold weather).
• In places where rainfall is seasonal and irrigation is not available, delay planting until rains are reliable.

Preparation for regeneration

When to regenerate

• Cassava has a growing cycle of 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.

Preparation of planting material

• 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.
• To prepare the stakes, select the mature portion of the stem, avoiding the top green stems and the bottom section of the plants (see photo below).
• Make stakes at least 20 cm long with at least 4–5 nodes with viable buds to ensure crop establishment and cut them at a right angle. 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.
• Label stake bundles immediately after cutting with accession number and date of harvest (see photo below).
• Treat the stakes with a mixture of broad spectrum insecticide and fungicide (see photo below).
• Add zinc sulphate in regions where zinc is limited in the soil.
• Take care to avoid mixing of genotypes.
• Handle stems with care to prevent bruising and peeling.

Cassava stakes of good quality for planting. (photo: IITA genebank)	Laying out the cassava stakes for labelling (photo: INIA, Peru – Ll. Rios)	Labelling and disinfesting the cassava stakes (photo: INIA, Peru – Ll. Rios)

Field selection and preparation

• Field plots should be uniform in fertility, with light textured, deep, well-drained soil and as free as possible from noxious weeds.
• Avoid stony, clay or waterlogged soils.
• In sandy soils, apply minimum tillage to conserve soil, organic matter and moisture and reduce soil erosion.
• In shallow or hard soils, increase top-soil volume per plant for better establishment.
• In poorly drained soils, make ridges or mounds to reduce waterlogging.
• Allow an overlapping period in the field of at least six months between the ‘old’ and the newly planted field to ensure that materials that did not germinate can be replanted and provide a constant supply of planting material for research programmes.
• Secure and protect the field against theft, vandalism and damage by animals.

Method of regeneration

Planting layout, density and distance

Plot size and spacing will depend on the size and purpose of the collection, land availability and demand for planting materials.

• Group germplasm according to vigour, plant height, branching habit and tendency to lodge, establishing at least three groups: high, intermediate and low vigour.
• Establish a distance of 2.2, 1.5 and 1.0 m between plots for high, intermediate and low vigour groups respectively, to avoid mixing up planting materials at harvest.
• Space plants 1.0–1.5 m apart if evaluations are to be made simultaneously, or closer if the collection is solely for germplasm maintenance (0.75–1.0 m within the row and 1.0 m between rows) to minimize weed growth and land requirements.
• Maintain a minimum of five and an optimum of ten plants per accession to ensure adequate survival and supply of planting material for weak genotypes.

Planting method

• Plant stakes directly in the ground (so that half or two-thirds of the stake is covered) or in soil ridges or mounds, vertically or at an angle, or even bury them horizontally about 5cm below the soil surface (see photo). The local planting practice of experienced cassava growers in the area can also provide a good guide.
• Identify plots very carefully, putting a plastic tag in the first plant of the left hand row of the plot. Place an extra label on a plastic, metal or strong wood in front of the plot.
• Draw a field map of the collection immediately after planting, with each accession located on the map, including both plot numbers and accession numbers.
• Replant missing plants one month after planting.

Crop management

Fertilization

• Fertilization is usual y not required for the sole purpose of germplasm maintenance. However, add manure at land preparation (e.g. cow dung or poultry manure) if necessary.
• Apply NPK 15:15:15, depending on the soil analysis, about 8 weeks after planting, around the plant, not touching the stems or leaves.

Weed management

• Ensure adequate control of weeds pre-emergence by ploughing and harrowing the soil or applying pre-emergence herbicides before planting, and post-emergence with herbicide applications, inter-row weeders or regular manual weeding.
• Weed as often as necessary to avoid competition between plants. Often weeding will be required at least four times per season, depending on the environment.
• Critical times are during the initial four months or until leaves form a canopy and weed growth is suppressed.

Irrigation

• The soil must be moist at planting, otherwise irrigation is required.
• If irrigation is not available, it is important to plant the collection at the beginning of the wet season when rain is reliable.

Pruning

• Prune plants about nine months after planting when using high densities or if growth is excessive.
• Note that pruning can create wound entries for pests and pathogens, and can spread them from one plot to another by way of the cutting instrument and workers’ clothing.
• Dip cutting instruments in a detergent solution between plots to destroy bacterial and viral pathogens on the surface.

Rotation

• Plant germplasm on new land every generation cycle.
• Rotate with grass or leguminous crops to break the cycle of certain root pathogens and prevent land degradation.

Common pests and diseases

America has 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. Damage is greatest in the dry season or in dry areas with low or irregular rainfall. The green mite (Mononychellus tanajoa) (America and Africa) and the mealybug (Phenococcus manihoti and P. Herreni) cause major damage in Africa; 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 America, while scales (Aonidomytilus albus), termites and grasshoppers are 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); and 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 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.
Contact plant health experts to identify symptoms and recommend appropriate control measures.

Pest and disease control

• Select healthy planting material. Do not take cuttings from plants that had leaf chlorosis, shoot tip die-back, cankers, fungus patches or streaks on the stems.
• Treat cuttings with pesticides and fungicides before planting, and the plants during the growth stage when necessary.
• 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.
• Use natural enemies against cassava pests as much as possible. Complement by applying appropriate pesticides as and when necessary.
• Weed the field regularly.

Harvesting

• Harvest stakes at the end of the growing season (this guide does not refer to any root or seed harvesting, dealing only with the vegetative propagules), usually 12–18 months after planting, depending on the cultivars and environment. In some environments most of the leaves wil have dropped, but in others, a leaf canopy remains at maturity.
• Be careful to identify the stem cuttings from each plot.

Post-harvest management

• Store stakes in a well-ventilated and shaded cool place until planting or in case they need to be replanted (keep some extra planting material for a while until the collection is established).
• Take care during the harvesting and subsequent handling of the stakes not to bruise them.
• Extend storage time (not recommended for collections) with longer uncut stakes tied in bundles pre-treated in pesticide, at 70–80% RH and 20–23°C.
• Stakes can also be stored (also not recommended for collections) buried in the ground for several months, with the basal side down or horizontally; regular watering is required to avoid excessive dehydration.
• Stakes or cuttings also store well for weeks in polythene bags in drier areas and/or during the dry season.

Monitoring accession identity

• Check that varieties are as originally described. When plants reach maturity, check against the latest descriptor list (Fukuda et al., 1998) (or compiled when the accessions were first described) and, if available, photographs of the accessions.
• Check regularly for mixtures due to handling mistakes: look for uniform characteristics, such as colours of young apical leaves (expanded and non-expanded), petiole, cortex and external stem as well as pulp, cortex and external root colours; flowering and branching types, pubescence of young leaves, shape of the central lobe, internodes distance (leaf scars), storage root peduncle and surface texture.

Documentation of information during regeneration

• Regeneration site name and map/GPS reference.
• Name of collaborator.
• Field/plot reference.
• Accession number; population identification.
• Source of cuttings.
• Number of generation or previous multiplication or regeneration (if generation is not known) since acquisition of germplasm.
• Preparation of planting material (pre-treatments).
• Method of planting, date and spacing.
• Field layout used.
• Field management details (watering, fertilizer, weeding, pest and disease control, stresses recorded, others).
• Environmental conditions (altitude, precipitation, temperature, soil type and others).
• Number of plants established.
• Days from planting to flowering (note: this will only be important if seed collection is anticipated).
• Harvest date and method.
• Number of plants harvested.
• Quantity of cuttings harvested.
• Agronomic evaluation; agro-morphological traits recorded.
• Comparisons with reference materials (record any identification numbers or references of any samples taken from this regeneration plot).
• Any evaluation undertaken at harvest.
• Post harvest (describe any relevant procedures).
• Others.

References and further reading

Adekunle AA, Dixon A, Ojurongbe J, Liona P, Muthada L, Adisa S. 2004. Growing Cassava Commercially in Nigeria. USAID, ICS-Nigeria and IITA. 22 pp. Available from: http://www.cassavabiz.org/agroenterprise/ent%20images/casava%20illust%20guid%20book.pdf. Date accessed: 1 August 2008.

Allem AC. 2002. The origins and taxonomy of cassava. In: Hillocks RJ, Thresh JM, Bellotti AC, editors. Cassava: Biology, Production and Utilization. CABI Publishing, Wallingford, UK. pp. 1–16.

Alves AC. 2002. Cassava botany and physiology. In: Hil ocks RJ, Thresh JM, Bel otti AC, editors. Cassava: Biology, Production and Utilization. CABI Publishing, Wal ingford, UK. pp. 67–89.

Bellotti AC. 2002. Arthropod pests. In: Hillocks RJ, Thresh JM, Bellotti AC, editors. Cassava: Biology, Production and Utilization. CABI Publishing, Wallingford, UK. pp. 209–235.

Bioversity International. 2009. Key access and utilization descriptors for cassava genetic resources. Available here

Calvert LA, Thresh JM. 2002. The viruses and virus diseases of cassava. In: Hillocks RJ, Thresh JM, Bellotti AC, editors. Cassava: Biology, Production and Utilization. CABI Publishing, Wallingford, UK. pp. 236–260.

Cock JH. 1985. Cassava: physiological basis. In: Cock JH, Reyes JA, editors. Cassava: Research, Production and Utilization. UNDP/CIAT. pp. 33–62.

De Goes M, Sias-Costa CIR, Guedes AC, Morales EAV, Second G. 1999. Cassava – a Brazilian model for the field maintenance of germplasm. In: Engelman F, editor. Management of field and in vitro germplasm collections. IPGRI, Rome, Italy. pp. 10–12.

FAO. 2004. Production Year Book. FAO, Rome, Italy.

Fukuda WMG, Guevara CL. 1998. Descritores morfologicos e agronomicos para a caracterizacao de mandioca (Manihot esculenta Crantz). EMBRAPA-CNPMF Documentos 78. EMBRAPA-CNPMF, Cruz das Almas, Brazil. 38 pp.

Fukuda WMG, Costa IRS, Vilarinhos AD, Oliveira RP. 1996. Banco de germoplasma de mandioca: manejo, conservacao e caracterizacao. EMBRAPA-CNPMF Documentos 68 EMBRAPA-CNPMF, Cruz das Almas, Brazil. 103 pp.

Fukuda WMG, Costa IRS, Silva SO. 2005. Manejo e conservacao de recursos geneticos de mandioca (Manihot esculenta Crantz) na Embrapa Mandioca e Fruticultura Tropical. Circular Tecnica 74. 4 pp.

Hillocks RJ. 2002. Cassava in Africa. In: Hillocks RJ, Thresh JM, Bellotti AC, editors. Cassava: Biology, Production and Utilization. CABI Publishing, Wallingford, UK. pp. 41–54.

INIA. 2005. Descriptor de yuca (Manihot esculenta). Programa Nacional de Investigación en Recursos Genéticos y Biotecnología, PRONIRGEB, INIA, Lima, Peru. 52 pp.

Iglesias CA. 1999. Field maintenance of cassava germplasm. In: Engelman F, editor. Management of field and in vitro germplasm collections. IPGRI, Rome, Italy. pp. 5–9.

Lelhner D. 2002. Agronomy and cropping systems. In: Hillocks RJ, Thresh JM, Bellotti AC, editors. Cassava: Biology, Production and Utilization. CABI Publishing, Wallingford, UK. pp. 91–113.

Ng NQ, Ng SYC. 2002. Genetic resources and conservation. In: Hillocks RJ, Thresh JM, Bellotti AC, editors. Cassava: Biology, Production and Utilization. CABI Publishing, Wallingford, UK. pp. 167–177.

Onwueme IC. 2002. Cassava in Asia and the Pacific. In: Hillocks RJ, Thresh JM, Bellotti AC, editors. Cassava: Biology, Production and Utilization. CABI Publishing, Wallingford, UK. pp. 55–66.

Acknowledgement

We acknowledge the useful contributions and suggestions from Dominique Dumet, Soyode Folarin and Olaniyi Oyatomi, International Institute for Tropical Agriculture (IITA), Nigeria; Llerme Rios Lobo, Instituto Nacional de Innovación Agraria (INIA), Peru; Wania Fukuda, Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Brazil; and Francisco Morales, International Center for Tropical Agriculture (CIAT), Colombia.
These guidelines have been peer reviewed by Nyat Quat Ng, consultant, Malaysia, and Clair Hershey, private consultant, USA.

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