Slow growth storage of banana germplasm

Contributors to this page: Bioversity International, Belgium (Ines Van den Houwe); IITA, Nigeria (Dominique Dumet, Badara Gueye).

Sample processing for tissue culture banks

Multiplication of propagules for conservation

When an accession has successfully passed the initiation phase (click here for details), then it is ready to be multiplied for storage, either for normal growth or slow growth conservation. This multiplication phase is also required for rapid propagation of selected materials for research or distribution.

Starting material

• The desired number of proliferating cultures must be obtained through repeated subculturing of propagules on proliferation medium.
• For rapid multiplication purposes, shoot tip cultures should be subcultured at 4-6 week intervals.
• Clusters of multiple shoots should be divided into individual or smaller groups of 2-3 micro-shoots and/or buds.
• Superfluous corm tissue and blackened tissue should be trimmed.
• The shoots must be shortened to a size of 5-7 mm in height.
• Each excised shoot tip or group of shoots /buds should be transferred onto a fresh pre-sterilized multiplication medium.
• The cultures should be incubated at an ambient temperature of 28±2°C and a light intensity of 64 µmol m^-2 s^-1.
• After 2-4 weeks, new lateral shoots and/or buds start to develop.
• After a further two weeks, subculturing can be repeated.

This propagation phase of an accession may vary from a few weeks to a few months as the multiplication rate strongly depends on the genomic group to which the accession belongs and is influenced by the composition of the medium (particularly the cytokinin concentration), the explant size, age of culture and the size of the culture vial.

Visual inspection of plant materials

• As the storage capacity strongly depends on the initial quality of the cultures, the general performance of each culture should be visually assessed: vigour, absence of fungal and bacterial contamination, chlorosis, blackening, necrosis of the tissue before transfer to slow growth storage.

Disposal of contaminated materials

• Contaminated and low quality cultures should be immediately discarded. If the entire set is below standard because at least one of the criteria is not met, the cultures should be re-propagated onto a new medium.
• If sufficient suitable cultures are available for storage, the culture tubes should be sealed with a few layers of parafilm and transferred to slow growth conditions.

Watch the video illustrating the aseptic subculturing process of banana shoots

Or view the video on Youtube

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Storage for tissue culture banks

• When the desired number of cultures for slow growth storage is obtained, the set of replicate cultures must be carefully observed after 2-3 weeks of growth.

Sample specifications

• An optimal sample size should be determined based on the purpose of the collection, taking in account most risks of possible losses.
• Based on statistical data, the proper sample size was determined between 12 and 24.
• An optimal number of 16-20 replicate shoot cultures per accession should be kept in storage.
• An accession tray containing an estimated reasonable number of replicate cultures should be placed in its assigned slow growth storage location within the storage growth room.

Container specifications

• Individual cultures should be stored in glass test tubes (150 mm height and 2.5 mm diameter), closed with a plastic kaput and sealed with several layers of parafilm in order to limit evaporation.

Growth media

• The test tubes should contain 20 ml of proliferation inducing culture medium composed of the MS Murashige and Skoog -mineral salts and vitamin mixture (Murashige and Skoog 1962) and be supplemented with 30 g/l sucrose; two growth regulators should be incorporated into the medium: cytokine in relatively high concentration (2.25 mg/l BAP) in order to induce multiple shoot/bud formation and an auxin (0.175 mg/l IAA) and solidified with 2 g/l Gelrite. (Van den houwe et al. 1995).
• The medium should be adjusted to pH 6.2 prior to autoclaving the medium.

Culture facility regimes

• The accession tissue cultures should be stored at an ambient temperature of 16°±1°C and at reduced light intensity of 25 µmol m² s^-1.
• A 24-hour light regime should be applied and the relative humidity of the storage room should be kept at 75%.

Storage duration

If the above physical and chemical storage conditions are followed, an average period of 12 months can be expected before re-culturing is required. These storage conditions are minimal growth conditions that proved to be acceptable for most genotypes. Not all accessions and genotypes, however, respond equally well to the applied conditions. (Further reading: Van den houwe et al. 1995).

System for tracking materials/inventory system during tissue culture storage

• The cultures should be inventoried every time subculturing is carried out.

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Monitoring the performance of tissue cultures in slow growth storage

Need for subculturing multiplication of stored cultures

Quantitative and qualitative criteria should be considered to define the moment that an accession should be recycled after the material has been maintained for a given time in storage.

• When the number of cultures maintained is reduced below the threshold value of 12, the accession should be moved to the transfer room for subculturing.
• If the number of cultures is higher than 12 but all cultures are characterized by an advanced stage of deterioration, necrosis, chlorosis, blackening or hyperhydrity, the accession should also be moved to the transfer room for subculturing (see routine monitoring methods).
• Ideally, one accession should be re-propagated into a set of 20 fresh shoot culture replicates, performing one subculture cycle. If, however, the number of newly established cultures is lower than 16, subculturing should be repeated to increase their numbers.
• In order to minimize the risk of selecting variant plant material from the remaining set of cultures, at least one shoot tip (with a maximum of three) should be isolated from each individual viable and healthy culture.
• Each individual shoot tip should be transferred to another recipient containing fresh culture medium.
• For safety reasons it is always recommended to hold 2-4 viable and healthy cultures of the previous subculture cycle as spare materials until it is known that the newly subcultured set is healthy and growing.
• Propagules should initially grow for 2-3 weeks under normal growth conditions at an ambient temperature of 28°C and under a PPF of 63 µmol m-² s^-1 (with a 24-hour photoperiod).

The spare cultures from the previous storage cycle can be discarded when the new set of cultures are transferred to the cold storage conditions described above.

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Inventory and monitoring of stored cultures

Accessions growing in tissue culture need to be inventoried and monitored on a timely basis in order to assess the number of healthy cultures remaining in storage and to determine the need for subculturing. The interval for routine visual monitoring of an active Musa collection should be about one month. Each individual culture in the accession tray should be checked visually and unsuitable cultures removed.

Checking the quality of the plantlets

The following factors should be taken into account to determine the quality of stored accessions:

• Viability.
• Vigour.
• Necrosis (of the leaves and apex).
• Chlorosis.
• Blackening of tissue / medium discoloration.
• Hyperhydricity.
• Ethiolation.
• Contamination

Plantlets of unacceptable quality should be immediately discarded.

Checking the number of replicates

• When the number of remaining healthy cultures falls below 12 or if the cultures are of unacceptable quality, the accession should be re-cultured under normal growth conditions in the following month.
• If a critical level of four or less cultures is reached, the accession must be considered at risk and immediate action should be taken to re-propagate or rescue the accession.
• In the case of contamination of all replicates, material should be transferred to the greenhouse, if plantlets are available. Otherwise, the propagules are regenerated and/or subjected to a decontamination treatment.
• If all remaining propagules are of poor quality, the material should either be immediately subcultured or rescued by being transfered into the greenhouse.
• If more than 12 cultures of the desired quality are left over in storage, the conservation cycle should continue for another month.

Monitoring genetic stability

Unless germplasm is regularly regenerated and transferred to the field for morphological observations, combined with the use of cytological techniques, genetic stability of a certain sample cannot be ascertained. Occasionally, abnormalities can be assessed in the in vitro samples.

In vitro assessment of variation

One of the criteria for efficient in vitro storage of germplasm is the maintenance of original genotypes over long periods of time. Although organized cultures (meristems, shoot and root-tip cultures) are believed to be genetically more stable than disorganized cultures (cell suspensions, protoplasts, callus, differentiated cells) variation appears to be relatively widespread in micro-propagated plants.

Factors like the culture mode, time in culture, number of subculture cycles, genotype and composition of the culture medium are known to influence the occurrence of somaclonal variation. The type and frequency of variation in micropropagated banana plants is known to be genotype and cultivar dependent.

• Monthly routine monitoring of the stored tissue culture samples should be carried out, making visual examinations for growth abnormalities.
• Some types of somaclonal variation (different degrees of dwarfism) can be observed at the tissue culture level.
• However, most types (leaf variegation, stem discoloration and particularly mutations expressed at the inflorescence and fruiting level - prolonged juvenility, small bunch, shortened fingers) cannot be assessed in vitro and require the regeneration of plants under field conditions. (Van den Houwe and Panis 2000).

Observations under greenhouse and field conditions (regeneration)

• When the accession has been continuously stored in vitro for over ten years, or when the accession is kept for more then ten subculture cycles in slow growth storage, the accession should be regenerated and observed morphologically for trueness-to-type under greenhouse and field conditions during at least two growing cycles.
• The morphological and taxonomic characteristics of the plants must be compared with those of the original accession. 

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References and further reading

Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco cell cultures. Physiologia Plantarum 15:473–497. Available for purchase here.

Strosse H, Van den houwe I, Panis B. 2004. Banana cell and tissue culture - review. Mohan Jain S, Swennen R (ed). Banana Improvement:Cellular, Molecular Biology, and Induced Mutations. Science Publishers Inc., Enfield, NH, USA:1-12. www.fao.org/docrep/007/ae216e/ae216e03.htm#bm03.1

Van den Houwe I, De Smet K, Tezenas du Montcel H, Swennen R. 1995. Variability in storage potential of banana shoot cultures under medium term storage conditions. Plant Cell, Tissue and Organ Culture 42:267-274. An abstract and full preview of the publication is available here.

Van den Houwe I, Panis B. 2000. In vitro conservation of banana: medium term storage and prospects for cryopreservation. Razdan MK, Cocking E, editors. Conservation of Plant Genetic Resources in vitro. Vol. 2. M/S Science Publishers, U.S.A. pp. 225-257.

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Field bank (regeneration) guidelines for banana

Contributors to this page: Bioversity International, France (Nicolas Roux), Bioversity International, Ethiopia (Michael Bolton, Alexandra Jorge); CIRAD,  France (Jean-Pierre Horry, Tomekpe Kodjo); IITA, Nigeria (Dominique Dumet).

Field bank for banana

When are field banks used

Many important varieties of field, horticultural and forestry species, including banana, are either difficult or impossible to conserve as seeds (i.e. no seeds are formed or if formed, the seeds are recalcitrant) or to reproduce vegetatively. Hence they can be conserved as growing plants in field genebanks or in vitro (in tissue culture or cryo banks).

Banana stored in field genebanks have a lower risk of loosing genetic integrity (due to genetic drift) if the mother plants are maintained for many years and are readily available for study and use. However, bananas maintained in field genebanks are considerably more exposed to physical risks (climate, diseases, pests) and costs are higher for storage (labour, inputs and space) than for in vitro genebanks. This balance must be considered before taking the decision to establish a field genebank for banana, if other options are also possible.

Advantages of field genebanks

• Material can be evaluated and characterized while being conserved.
• Genotypes that commonly produce variants can be more easily identified and rogued out in the field than in vitro.
• Lower risk of loosing genetic integrity.
• Easy access for research, utilization and distribution.
• Establishment and maintenance of field genebanks requires limited technology.

Disadvantages of field genebanks

• Materials are susceptible to pests, diseases, adverse weather, theft and vandalism.
• Field genebanks require large areas of land, but even then genetic diversity is likely to be restricted.
• High maintenance costs (labour, inputs and space).
• Slow multiplication of material for distribution.

Regeneration guidelines for banana

Note: These guidelines refer specifically to the regeneration of banana in field collections and include information on the establishment of a new field collection.
For regeneration or rejuvenation of banana in in vitro collections click here.

The information on this page was extracted from:
Tomekpe K. and Fondi E. 2008. Regeneration guidelines: banana. 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, please read the general introduction that gives the general guidelines to follow, by clicking here.

Introduction

Banana and plantain (Musa spp. L.) are giant monocotyledonous perennial herbs that thrive in the humid and subhumid tropics of low- and mid-altitude areas. They originate primarily from South-East Asia, with secondary centres of diversity in West and Central Africa (Plantain subgroup) and the East African highlands (Lujugira subgroup). They belong to the genus Musa, which comprises more than 1000 varieties in four sections: Australimusa, Callimusa, Rhodochlamys and Eumusa (Simmonds and Shepherd 1955). Most cultivated Musa belong to the Eumusa section and produce fruits that are a major commodity in international trade but are even more important as starchy staples in local food economies of many developing countries.
Most cultivars are derived from two species: Musa acuminata Colla (A genome) and Musa balbisiana Colla (B genome). Most edible banana are triploids (2n = 3x = 33), although there are a few diploid cultivars and even fewer tetraploid cultivars.
Bananas and plantains are also classified according to use (dessert, cooking, beer or processing/fibre) or mode of preparation of the fruit. Sweet bananas are eaten fresh or unprocessed but most bananas, and particularly plantains with orange pulp, are cooked. Cultivated bananas and plantains are parthenocarpic and are propagated vegetatively.
They are conserved in field collections as actively growing plants or in vitro as proliferating tissue. Wild relatives (normally non-parthenocarpic) are conserved in the same way. Bananas and plantains consist of an underground organ (corm) that bears roots, suckers or shoots and a pseudostem with leaves. The corm or rhizome is the true stem. Suckers develop initially as swollen buds from lateral meristems at leaf bases on the corm. A sucker has different developmental stages (Stover and Simmonds 1987): peeper, sword sucker and maiden sucker. The recommended stage for regeneration is the sword sucker (a sucker that is 50-150 cm in height with lanceolate leaves) followed by the peeper (a large green bud that has just emerged above ground). Among the followers or daughter plants, a sucker or ratoon is selected to succeed the mother plant. A group of suckers from a single parent is referred to as a stool or mat. A crop cycle is the time between planting and fruit harvest on the same mat. The second harvest from the mat is called the first ratoon crop (Gowen 1995).

Description is an important step in regeneration: plant at optimal stage for observation (photo: Emmanuel Fondi)

Choice of environment and planting

Climatic conditions

Banana grows well:

• In humid tropical lowlands between latitudes 20°N and 20°S.
• Between 100 m and 500 m altitude.
• Above 19°C mean minimum temperature (optimum average temperature is 27°C).
• In areas with >100 mm monthly rainfall (Robinson and de Villiers 2007).

However, banana can grow in a much wider range of climates within the tropics, with site selection mainly limited by soil type and rainfall.

Planting season

Planting can be done all year round if there is enough moisture. Planting should be done at the beginning of the rainy season.

Preparation for regeneration

When to regenerate

Regenerate field collections every four years to restore collections, as accumulated diseases and pests reduce plant vigour. Regeneration also allows maintenance (re-alignment) of planting pattern and optimal density since successor plants of banana emerge at variable distances away from the parent stand.

Sword sucker obtained from parent plant for regeneration (photo: Emmanuel Fondi)

Field selection and preparation

• Banana field collections are maintained indefinitely. It is necessary to have twice as much space as occupied by the collection (i.e. if a collection of 700 accessions occupies 3 ha, you need to have 6 ha available) for fallowing which is essential for proper growth of accessions.
• The best soils for banana are deep, well-drained fertile loams with high water-holding capacity and organic matter content (Purseglove 1972).
• Select fields that were not under banana during the previous two years or that were planted to a non-host crop like pineapple.
• Choose soils with adequate drainage and where water-logging is not a problem.

Method of regeneration

Both parthenocarpic edible bananas and their wild relatives are regenerated vegetatively. In this way the genetic identity remains unchanged from one cycle to the next. To safeguard the entire biodiversity, it is necessary to regenerate all the accessions.

Planting layout, density and distance

The planting layout of a banana collection should take account of the genomic constitution of the varieties and the use types of the accessions. Make a field plan (electronic and hard copy). See sample plan below.

Divide the site into main blocks separated by 6-m-wide tracks. Blocks should correspond to the genomic group or the main subgroup, e.g. plantain. Assign block names and then:

Parent plant with suckers to be used for regeneration. (photo: Emmanuel Fondi)

• Further divide the blocks into bands directed at right angles to the blocks. The bands correspond to the different subgroups.
• Plant in single rows, with five plants per row.
• Use a plant spacing of 3 m between rows and 2 m within each row.

Source of planting material

• The best planting materials are sword or maiden suckers collected from the accession to be regenerated (see photo on the right), which do not develop broad leaves until they are more than 1 m high.

Selection of planting material

• Choose plants at flowering or at the end of the growing season.
• Plants should be free of undesirable variations in the cultivar characteristics.

The following diseases must be avoided: Moko disease due to Ralstonia solanacearum Smith, phylotype II, Xanthomonas wilt (bacteria wilt) caused by Xanthomonas vasicola pv. musacearum, Panama disease (Race 1 and 2) and other vegetative compatibility groups (VGCs) of Fusarium oxysporum f. sp. cubense, Banana bunchy top virus (BBTV), Banana bract mosaic virus (BBrMV).

Other pests and diseases, especially nematodes, weevils and other viral and bacterial diseases, should be absent or have a low prevalence in the field from which planting material is extracted. Monitor by regular field inspections.

Preparing planting material and method

• Suckers removed from the mother plant must be pared in the field to remove all the roots and diseased tissue before being transported (see photo below). Remove any suspect part of a different colour. Discard sucker if darkened galleries, dead or discoloured areas or other damage make up one quarter to one third of the sucker.

Paring of sucker to eliminate soil-born pests and diseases (photo: Emmanuel Fondi)

• Disinfect the machete after each sucker in a 5% sodium hypochlorite solution or a 20% iodine solution to avoid spreading disease (bacterial wilt and Fusarium).
• Cut the pseudostem in cross section 10-15 cm above the corm to examine for any off-coloured rings, brownish spots or off-coloured liquid. Eliminate suckers or corms showing these characteristics.
• To prevent re-infection with banana weevil borer once sucker paring is complete, transport the suckers immediately to a site distant from any banana fields to limit the risk of weevils laying eggs in the planting material.
• Place identification tags on suckers and give particular care to the identity of suckers from each accession before removing them from the field.
• When the suckers are to be planted directly or into a multiplication plot, immerse them in hot water (30 seconds in boiling water or 20 minutes in water at 50°C) to kill weevil borer eggs and nematodes, if needed.
• Plant suckers directly in loose soil using hoes or spades if deep ploughing has been done.
• If deep ploughing is not done, plant suckers in square holes with sides of 40 cm and depth of 40 cm.

It is indicated to plant the suckers at once after their pulling up and treatment and if it is not possible, one or a few days afterwards suckers can however be stored for several weeks in a shaded dry area until planting is completed.

Labelling

• Place metallic identification plates at the head of rows to identify accessions (see photo below).

Metallic plate to identify accession (photo: Emmanuel Fondi)

Crop management

Fertilization

Fertilizer practices vary widely according to climate, cultivar, yield level, soil fertility and production system. Before planting, take a composite soil sample from each block or from each soil type change. Analyse the sample to determine soil pH and micronutrient levels. This should provide a reliable recommendation for pre-planting application of lime (dolomitic or calcitic), K and P. The normal pH range is 5.8 to 6.5. Below this, lime application is required. Top dressing with N and K is recommended according to the expected yield level and results of soil analysis.

Weed management

Weeds can be manually or chemically controlled.

• Apply a post-emergent, non-selective systemic herbicide (e.g. glyphosate) two weeks before planting. Clear manually once a month during early stages of crop growth.
• Apply herbicide when the plants have grown tall enough to permit direct spraying, but apply with care as the banana plants are very sensitive to herbicides.

Mulching

Mulch with organic matter to reduce evapotranspiration and to increase the organic matter content of the soil. Elephant grass (Pennisetum purpureum Schumach.) is a popular mulch in Central Africa, particularly for plantains and diploid bananas. It is rich in potassium and improves soil fertility.

Pruning and de-suckering

• Prune once a month to eliminate withered leaves and reduce leaf disease pressure.
• De-sucker plants regularly to maintain a single successor sucker at a time. De-suckering should be carried out in such a way as to maintain the initial planting layout.

Irrigation

Irrigation is required during the dry season to keep the plants vigorous.

Common pests and diseases

Major pests of banana include nematodes, banana weevil borer, tomato semi-looper (Chrysodeixis acuta) and thrips.
Major diseases include Panama wilt (Fusarium oxysporum), bacterial wilt (Xanthomonas wilt), Erwinia rhizome rot, black and yellow Sigatoka (Mycosphaerella), cucumber mosaic virus (CMV), banana bunchy top virus (BBTV) and banana streak virus (BSV). Do not distribute accessions contaminated with viruses.

Pest and disease control

• Proper cultural practices, such as fallowing, balanced nutrition and weed control, help keep disease pressure at a minimum.
• The main pesticides required are insecticides and nematicides; about two applications are required per year.
• Sigatoka diseases are controlled by pruning infected leaves and/or applying fungicides. Viral diseases are major threats to a collection and are controlled by prevention, principally by controlling the source and the quality of material introduced in to the collection. Infected plants should be uprooted and destroyed as soon as they are identified.

Harvesting

The regeneration of bananas does not involve seeds. At maturity (when first ripe fruit appears on bunch), description is carried out and the fruits taken for consumption in the case of parthenocarpic edible banana types. For wild types, the entire plant is cut down and placed in the interline space.

Regeneration of wild banana

The regeneration procedures for wild banana should be the same as for edible types.

Monitoring accession identity

Comparisons with passport or morphological data

Accessions are characterized using minimum descriptor forms adapted from‘Descriptors for bananas’ (IPGRI/INIBAP, CIRAD 1996). Typical reference materials are photos and description forms.
Reference sources for comparison include Musalogue (Daniells et al. 2001) and the Musa Germplasm Information System (MGIS) database.
Compare the following major traits in characterization data:

• General plant appearance.
• Bunch characteristics.
• Male bud characteristics.

An accession is declared true to type (TTT) if its traits match those of the known reference.
In cases where they do not match with the reference, it is declared either as miss-labelled (ML) or off type (OT). If it is mislabelled, its true identity is sought; whereas if declared off-type, it is destroyed and the right accession re-introduced in to the collection.

Documentation of information during regeneration

The following information should be collected during regeneration:

• Regeneration site name and map/GPS reference.
• Name of collaborator.
• Field/plot reference.
• Accession number, accession code in the institute, ITC code; population identification.
• Source of suckers.
• Abnormalities on the mother plant and sucker.
• Generation or previous multiplication or regeneration (if generation is not known).
• Preparation of planting materials (pre-treatments).
• Planting date and density.
• Field layout used.
• Field management details (watering, fertilizer, weeding, pest and disease control, stresses recorded, others).
• Environmental conditions (altitude, precipitation, soil type, others).
• Emergence in the field (number of plants germinated).
• Number of plants established.
• Days from planting to flowering.
• Agronomic evaluation; agromorphological traits recorded.
• Comparisons with reference materials (record any identification numbers or references of any samples taken from this regeneration plot).
• Others.

References and further reading

Daniells J, Jenny C, Karamura D, Tomekpe K. 2001. Musalogue: Diversity in the genus Musa. IPGRI/INIBAP/CTA, Rome, Italy. Available from: http://www.bioversityinternational.org/publications/publications/publication/issue/imusailogue_diversity_in_the_genus_imusai.html (16 MB). Date accessed: 23 March 2010.

IPGRI, INIBAP, CIRAD. 1996. Descriptors for Banana (Musa spp.). IPGRI, Rome, Italy; INIBAP, Montpellier, France; CIRAD, France. 55 pp. Available here.

Lassoudiere A. 2007. Le bananier et sa culture. Editions Quae, Versail es Cedex, France. 383 pp.

Purseglove JW. 1972. Tropical Crops. Monocotyledons. Vol. 2. Longman, London, UK.

Robinson JC, de Villiers EA. 2007. The cultivation of banana. ARC-Institute for Tropical and Subtropical Crops, Nelspruit, South Africa/Du Roi Laboratory, Letsitele, South Africa. 258 pp.

Simmonds, N.W. and Shepherd, K. 1955. The taxonomy and origins of the cultivated bananas. Journal of the Linnean Society (Bot.), 55(359), p. 302-312 Abstract available from: www3.interscience.wiley.com/journal/119777761/abstract?CRETRY=1&SRETRY=0. Date accessed: 23 March 2010.

Stover RH, Simmonds NW. 1987. Bananas. Longman Scientific and Technical, New York, USA. 468 pp.

Swennen, R. 1990. Plantain Cultivation under West African Conditions: A Reference Manual. IITA, Ibadan, Nigeria. 24p.

Acknowledgements

These guidelines have been peer reviewed by Sebastião de Oliveira e Silva, Centro Nacional de Pesquisa de Mandioca e Fruticultura Tropical (CNPMF), Brazil; Wayne Hancock, Bioversity International, Ethiopia; and Jeff Daniells, Department of Primary Industries & Fisheries, Queensland, Australia.

In vitro conservation of banana genetic resources

Contributors to this page: Bioversity International, Belgium (Ines Van den Houwe); IITA, Nigeria (Dominique Dumet, Badara Gueye).

What is in vitro conservation

During the last 40-50 years in vitro techniques have been increasingly used for plant propagation. They consist in growing and multiplying parts of plants in flasks or tubes in artificial media, under controlled environments and sterile conditions.

Common banana in vitro techniques used in conservation are listed below:

• Slow growth storage – using tissue culture techniques and growth retardant conditions (temperature, light, chemicals).
• Cryopreservation.

Where is it used

An increasing number of countries has invested in tissue culture facilities for the propagation of clonal crops, including banana.

Initially, traditional tissue culture techniques (shoot tip and meristem culture) were used as a tool for the elimination of pests and diseases, rapid plant propagation and for the exchange of clean germplasm. As institutes became more experienced with these techniques, and the number of accessions in collections steadily increased, the available techniques were optimized and extensively adopted for slow growth conservation of germplasm. Until now however very few genebanks have initiated banana conservation activities using cryopreservation techniques. 

When should it be used

In vitro conservation techniques should be used whenever technical expertise and facilities are available. They are generally more economic and less risky in a long-term perspective.

• To conserve plant parts of banana germplasm that can mostly be propagated vegetatively
• As a viable alternative to complement and reduce the large space required for field banks.
• In vitro conservation has low space requirements and minimal possibility of losses due to edaphic factors.
• To duplicate material contained in field banks.
• To replace field banks.
• To allow international germplasm exchange.
• To ensure a more secure conservation of germplasm for future generations.
• Costs (qualified labour, energy, supplies and infrastructure) are highly dependent on location and ecomomies of scale should be considered when taking in vitro conservation into consideration.

How should it be done

• It requires specialized laboratories and equipment with very skilled technicians and researchers.
• It also requires adaptive technologies for some more reluctant species.
• It requires sterile conditions and very well controlled artificial growth environments.
• It requires high initial investments but relatively low maintenance costs in a long-term perspective.
• In vitro conservation should only be considered if the laboratory forms part of a conservation strategy involving also other crops.

References and further reading

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

Benson E, Harding K, Debouck D, Dumet D, Escobar R, Mafla G, Panis B, Panta A, Tay D, Van denhouwe I, Roux N 2011. Refinement and standardization of storage procedures for clonal crops - Global Public Goods Phase 2: Part II. Status of in vitro conservation technologies for: Andean root and tuber crops, cassava, Musa, potato, sweetpotato and yam. Rome, Italy: System-wide Genetic Resources Programme. Available here.

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

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

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, 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.

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.

background docs banana

ActaHort_1998_490

Conservation of Plant Genetic_

INIBAP International Network for the improvement of Banana

Management of Banana_2006_

Minimum set of photos for accession identification

Mussaspp

Mussa Germplasm Distribution

Organicmanuel04_es

PlantCellTissueOrganCulture

tg8_en