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.
- 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
Back to top
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.
- 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.
- 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.
- 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%.
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.
Back to top
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.
Back to top
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:
- Necrosis (of the leaves and apex).
- Blackening of tissue / medium discoloration.
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.
Back to top
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.
Back to top