Crop Genebank Knowledge Base

Crops

Cassava

Conservation

In vitro conservation

Slow growth storage

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.

Contents:
Sample processing
Viability monitoring
Storage

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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