Establishment of aseptic cultures of banana

Mother plant and young shoots – source of material (photo: IITA)


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

New accessions can be received as small samples of in vitro cultures from other collections, seeds of wild types or as classical vegetative propagation materials (apex-containing parts of the banana plant). When materials come from the field, a series of procedures are required and are described below:

Initiation of shoot tip cultures from suckers

In Musa, explants can be obtained from all plant parts that contain a shoot meristem: the parental pseudo stem, its suckers, peepers, sword suckers, lateral buds or very small eyes.

Usually only the apical meristem is available from a trimmed sucker. Eventually, smaller buds (sleeping eyes) on the corm can also be used as explants for tissue culture initiation.

Explant isolation and disinfection

Surface sterilization of explants

Excision of explants

Shoot apices of bananas are enclosed in many tightly overlapping leaf initials. The shoot tip is about 2-5 mm in size and consists of the meristem covered with several leaf primordia supported on a small base of corm tissue.

In case of high risk of bacterial contamination, i.e. when suckers are harvested during the wet season, it is recommended to excise a smaller explant consisting of the meristem covered with 1-2 leaf primordia (1 mm).

Tissue culture initiation

The size of the explant is an important factor in the successful culturing of shoot tips of bananas. Very small explants increase the chance of producing bacteria-free and virus-free cultures, but the mortality rate is high. Intermediate sized explants produce clean vigorous cultures that multiply rapidly whereas very large explants tend to show more blackening and contamination, and thus lower rates of successful tissue culturing.

Initiation of tissue cultures from seeds

For wild banana species, seeds can also be used to initiate tissue cultures, if no vegetative propagation material is available. However germination of banana seeds is known to be low and erratic, therefore preference is given to direct culturing of the axenic embryo isolated from the seed (= embryo rescue).

Surface disinfection of banana seeds

Embryo isolation

Germination can start about two days after inoculation, accompanied by a colour change from white to yellow and abundant formation of root hairs. Once cultures are successfully initiated, regular testing of the plant material for asepsis should start.

Contamination indexing (bacteria)

A primary requirement in the establishment and permanent maintenance of an in vitro collection is the asepsis of the tissue cultures. Sterility of the cultures is also of paramount importance for their intercontinental exchange and whenever cultures are used for various research purposes.

To safeguard the in vitro cultures against microbial contamination, and to prevent the spread of contaminants in the tissue culture system, monitoring of the material for asepsis should be performed at crucial steps (see Tissue culture testing below) and at regular intervals during the processing of material in the genebank.

Visual examination

Most of the time however the contaminants remain deep seated inside the plant tissue and only appear in the growth medium under favourable conditions (increased sucrose concentration, change of pH of the medium, increase of the ambient temperature, deterioration of the plant tissue etc.).

Tissue culture testing

Tissue culture testing should usually be done upon introduction of new material, during storage and at annual subculturing. The screening test is simple and non-destructive. Testing involves streaking of the basal part of the shoot tip onto a semi-solid bacteriological medium.

Decontamination treatment

Contaminated cultures should be eliminated by autoclaving. However, when all replicates of one accession at initiation or in storage are tested positive, contaminated material cannot simply be discarded as the accessions may be unique and difficult to replace. They should then be subjected to a decontamination treatment.

Elimination of bacteria through meristem culture
This technique involves the isolation of aseptic meristems from the contaminated in vitro cultures. Depending on the type of contaminating bacteria, the eradication method is further refined by subjecting either contaminated in vitro or greenhouse grown contaminated plants to meristem culture.

Elimination of bacteria from greenhouse plants
In some cases, for instance when the tissue cultures are contaminated with very small sized bacteria, the method described above is less effective. For a successful elimination of these bacteria, the explant size might need to be further reduced. These smaller sized explants, however, often fail to establish in vitro. Therefore, an alternative approach to eliminate bacteria is to isolate explants from greenhouse plants, acclimatizing the in vitro plants.

Antibiotic treatments
An antibiotic treatment involves exposure of the contaminated shoot tip material to a bactericidal concentration of an antibiotic agent without affecting the growth of the shoot tip.

The antibiotic most often used is rifampicin. This antibiotic is able to kill Gram+ and Gram- bacteria without affecting the growth of the banana shoot tip (the MBC of rifampicin does not exceed the phytotoxic concentration). Other antibiotic agents tested seemed to be ineffective on most of the bacterial isolates and/or had a toxic effect on the plant tissue.

References and further reading

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, USA. pp. 225-257.

Van den Houwe I, Guns J, Swennen R. 1998. Bacterial contamination in Musa shoot tip cultures. Acta Horticulturae 490: 485-492.

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The Genebanks

The 11 CGIAR genebanks currently conserve 730,000 of cereals and grain legumes, forage crops, tree species, root and tuber crops, bananas and crop wild relatives.