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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Field management 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).

Establishment

Choice of environment

• Cassava grows well between 30°N and 30°S in areas where annual rainfall is more than 750 mm a year, and mean daily temperatures are above 18°C (low to medium altitude (1500–2000 m) tropics, or low altitude subtropics).
• Well-aerated, loose and light sandy loam soil is recommended for cassava. It is sensitive to frost but tolerant of long dry periods, soils with low pH, high aluminium and low fertility.
• The area where material is maintained should be as free as possible of diseases and insect pests that could cause losses of material or create difficulties in the transfer of clean planting material to other sites.
• 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 cool weather).
• In places where rainfall is seasonal and irrigation is not available, delay planting until rains are reliable.

Field preparation

• Cassava can be maintained in field plantings as a perennial plant, but periodic renewal every one or two years is desirable to avoid problems of excessive vegetative growth, cumulative disease and insect problems and to facilitate maintenance generally.
• Allow an overlapping period in the field of at least six months between the ‘old’ and the newly planted field to ensure that material that did not germinate can be replanted and provide a constant supply of planting material for research programmes.
• As a reference, approximately 0.3 ha is needed for the maintenance of 1000 accessions (0.4 ha when cuttings are planted for germplasm characterization purposes).

Traditional field methods

• 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, shallow, hard or waterlogged soils, or manage them to correct the problems.
• In sandy soils, apply minimum tillage to conserve soil, organic matter and moisture and reduce soil erosion.
• In poorly drained soils, make ridges or mounds to reduce water logging.

Innovative method developed at CIAT

• In order to combine the benefits of lower space requirements with continual availability of planting material for experimental use, CIAT devised a slow-growth system based on restricting the root development in small planting pots (bonsai effect).
• Plants occupied only a small fraction of the space they would occupy if allowed unlimited growth in the field.
• Maintaining a cassava germplasm collection in containers has the potential advantages of space savings, better protection against pests, diseases and weather-related damage, and labour savings.
• Disadvantages can include difficulty in using plants as a source of planting material for field trials (generally small and weak stems), cost of infrastructure and cost of material.

Field planting

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

Layout

• It may be beneficial for management purposes to group germplasm according to vigour, plant height, and branching habit, 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 minimize competition while making efficient use of land area.
• 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.

Planting

• Plant the 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 5 cm below the soil surface. 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 stake in front of the plot.
• Note that three to five extra cuttings are planted behind each peg as backup in case some of the main cuttings die.
• 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.

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 Maintenance and management

 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. Weeding may be required up to 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.

Fertilization

• Fertilization is usually 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 eight weeks after planting, around the plant, not touching the stems or leaves.

Pruning

One of the problems of longer-term maintenance in the field can be excessive growth of some accessions.

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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Viability and monitoring 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).

Health diagnosis in field banks

• Preferably, test the source plants to identify healthy plants as a source of cuttings.
• If possible, do random health tests during field monitoring and checks.
• Follow recommendations from the cassava health diagnosis menu for detailed procedures.

Routine monitoring methods

Assures the viability, adequate growth and good health of the plants during field growth.

• Secure and protect the field against theft, vandalism and damage by animals.
• Weekly or bi-weekly (maximum) surveying of the collection is essential, to be aware of any problems that arise and need to be corrected.

• Check regularly for plant vigour and survival rates.
• 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, internode length (leaf scars), storage root peduncle and surface texture.
• Check regularly for pests and diseases. Contact plant health experts to identify symptoms and recommended appropriate control measures.
• Weed the field regularly.

Recording information during routine monitoring 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.
• Date of monitoring (date when data is collected).
• Date of test (the date that the test was carried out).
• Results of tests and action taken (removal of plant or application of insecticide or quarantine measures).
• Name of staff (name of staff recording the data).
• Survival rates (number of plants alive).
• Details of plants removed (due to type mixtures or pests or diseases contamination).
• Damage [a score of 1-5 (where 5 is most damaged) or qualitative assessment of damage (insect, disease etc)].
• Vigour [assessment of vigour of the plants on a scale of 1-5 (where 5 is high)].
• Field management details (watering, fertilizer, weeding, pest and disease control, stresses recorded, others).

References and further reading

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