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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 Y
Y 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:

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In 
, 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.

á Explants should preferably be isolated from young, vigorous and healthy looking suckers of 40-
100 cm in height with a corm diameter of about 10 cm.
á These should be collected from a flowering mother plant to guarantee trueness-to-type.

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.
Mother plant and young shoots ± source of
material (photo: Bioversity)
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á Excise from the sucker a cubical block of tissue (2x2x2 cm) containing the apical meristem.
á If there are buds on the corm surface present, excise the buds surrounded by 0.5 cm of corm
tissue.

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á Transfer each cube of tissue to a different Erlenmeyer flask.

á Soak each individual cube of tissue in ethanol (96%) and shake gently.
á Immediately decant and rinse for 30 seconds in sterile deionised water.
á Transfer the Erlenmeyer flask to a laminar air flow cabinet.
á Emerse each plant tissue cube for 20 minutes in a 2% hypochlorite solution or diluted commercial bleach solution supplemented with a few
drops of Tween 20 (surfactans). Liquid dish-washing detergent can be used if no Tween is available.
á Cover the Erlenmeyer flasks with a glass petri dish lid and swirl the solution frequently.
á Decant the hypochlorite solution.
á Then rinse three times in the space of ten minutes with sterile deionised water and decant.

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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.

á Remove the outer tissue that was exposed to the disinfectant solution from all sides of the cube.
á Flame dissection instruments repeatedly during manipulation.
á Remove stepwise the covering leaf primordia, cut away as much corm tissue as possible, as this causes blackening of the culture during
initiation.
 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).

á To excise the meristem tip, place the excised shoot tip under a dissecting microscope (working in a laminar flow cabinet) and further reduce
the size by removing leaf primordia and some corm tissue at the base of the explant.

 


 



á Immediately place the excised explant onto culture initiation medium.

á Push the explant a short distance into the culture medium to ensure good contact.
á Keep the cultures in complete darkness for one week to reduce blackening (oxidation of polyphenols).
á Growth of the tissue should start about two weeks after inoculation, accompanied by a swelling of the leaf sheet tissue and corm tissue, a
change of colour from white to green, and abundant oxidation of polyphenolic compounds that are released from the corm tissue
(blackening of the tissue and surrounding growth medium).
á Transfer the explants to fresh medium every two weeks during the first eight weeks in culture.
á Trim the blackened and/or swollen corm tissue, if necessary, leaving the viable leaf primordia tissue of the explant intact.
á After 10-12 weeks the explant will develop into a shoot.
á Record the survival rate and visible contamination at each transfer for each replicate.

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.

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For wild banana species, seeds can also be used to initiate tissue cultures, if no vegetative propagation material is available. 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.

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á Soak the seeds for 2-3 days in tap water, without stirring, in an Erlenmeyer flask. Refresh the water daily in order to avoid fungal
contamination or growth.
á Soak the seeds for two minutes in EtOH (95 % v/v) in an Erlenmeyer flask and cover the flasks with a lid.
á Decant and transfer the flasks to a laminar air flow cabinet. Disinfect the seeds in a 1.5% hypochlorite solution and 1-2 drops of Tween for
20 minutes and shake gently. Wash the sterilized seeds with sterile deionised water rinsing twice in 15 minutes.

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á Use forceps to fix the seed on a sterile cutting pad.

á Cut the seed lengthwise next to the micropyle.
á Remove the plug and pick up the embryo.
á Place the embryos on embryo germination medium in glass test tubes.
á Incubate the embryos under normal conditions for banana shoot tip cultures.

Germination starts 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.

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 A primary requirement in the establishment and permanent maintenance of an Y
Y
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 Y
Y
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.

 
 


á Bacterial contamination can be detected as faint µclouds¶ in the medium around the basal part of the explants or culture. Therefore, the use
of a clear gelling agent (Gelrite) in tissue culture will facilitate the detection of contamination.
á Sometimes even colony growth is detected around the tissue on the surface of the growth medium.

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 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.

á The test should be done at subculturing just before transferring the explant to the growth medium.
á A single shoot tip should be isolated from the culture and trimmed to a suitable size for transfer to fresh culture medium.
á The corm basis of this shoot tip should be streaked three times onto bacteriological medium and immediately placed onto plant growth
medium.
á The petri dishes should be sealed with parafilm and incubated at 28°C.
á The standard testing medium is a broad spectrum bacteriological medium, which allows growth of a wide range of bacteria. The medium
includes nutrient agar (8%), enriched with yeast extract (5 g/l) and glucose (10 g/l).
á Most bacteria are fast growing and will appear on the medium after two or three days, but slow growing contaminants may require up to
eight weeks to appear on the medium. It is thus important to observe the medium for at least two months after testing.
á In case of inconclusive results, alternative and complementary detection methods can be applied:
› Using specific/selective growth medium.
› Macerating the tissue and incubating in liquid the bacterial growth medium.
› Using polymerase chain reaction (PCR) methods.

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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.

á Before applying a decontamination treatment it is recommended to characterize the isolated bacteria in order to determine the most
effective method of eradication.
á For routine application, meristem culture, for which only a rough characterization of the contaminant is required, is preferred over treatments
with antibiotics of which the therapeutic success depends on the identification of the contaminant, bacterial sensitivity and phytotoxicity.
Moreover, the application of antibiotic agents may involve the risk of developing resistant bacterial strains.

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

á Culturing 1 mm sized meristems, directly isolated from contaminated Y
Y
plants, is found most effective to eliminate bacteria
likeY
sp.
á The shoot tip must be isolated from the contaminated culture and further reduced to 1 mm in length under a dissecting microscope. Young
leaves must be removed until the dome covered by two to three leaf primordial remains.
á It is important that the corm tissue is carefully and maximally removed, as bacteria often reside in this tissue.
á The excision of the meristem tip should be done under stringent aseptic conditions in order to minimize the risk of transferring bacteria to
the aseptic inner meristematic zone of the explant.
á It is essential that the explant material only comes into contact with sterile instruments and surfaces. Between use, forceps and scalpels
should be sterilized (in a glass bead sterilizer or flame) and the cutting pad should be sterilized before the surface comes into contact with a
freshly cut tip.

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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 Y
Y. Therefore, an alternative approach to eliminate bacteria is to isolate explants from greenhouse plants,
acclimatizing the Y
Y
plants.

á The contaminated Y
Y
plants should be transferred to potting soil in PET pots in a greenhouse and grown for a period of 3-6 months or
until the stems reach a diameter of at least 5 cm.
á The plants should be kept dry for 2-3 weeks and then explants of 1-3 mm tips can be isolated to re-initiate Y
Y
cultures.
á The newly established cultures should be re-tested for at least five subculture cycles in order to confirm their
bacteria-free status.

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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.

á Prepare liquid MS-based regeneration medium containing rifampicin HCl (100 mg/l) in Erlenmeyer flasks. Place one contaminated tip of 0.5
mm in 10 ml medium in an Erlenmeyer flask and incubate for 30 days at 28°C in light on a rotary shaker (60 rpm) and light intensity of at
least 25 m m² s-1.
á After one month, the treated plant material should be taken out of culture. The aseptic shoot tip should be excised and cultured on a fresh
antibiotic-free medium.
á The newly established cultures should be re-indexed for at least 5 subculture cycles in order to confirm their bacteria-free status.

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Van den Houwe I, Panis B, 2000.

Y conservation of banana: medium term storage and prospects for cryopreservation. Razdan MK,
Cocking E, editors. Conservation of Plant Genetic Resources Y
Y. Vol. 2. M/S Science Publishers, USA. pp. 225-257.
„