An effective indexing method for banana tissue culture provides long-term freedom from bacterial contamination

The global banana industry relies on tissue culture for uniform plants for better farm management, improved production and for prevention of pests and diseases otherwise transferred in soil or plant material. Banana tissue culture is also required by quarantine authorities to import/export banana plants and used in banana cultivar collections to safely maintain plantlets free of disease in the long term and protected against impacts of weather events or disease incursion. Production needs to be efficient to minimise costs and to deliver large quantities of plants on schedule. Both commercial tissue culture production and research applications involving long-term storage of banana tissue culture and cryopreservation are negatively impacted by bacterial contamination. Endophytic bacteria in tissue cultured plantlets that build up over time and adapt to culture conditions are a problem in banana tissue culture. While fungal contamination is relatively easy to identify and remove from the system, bacteria in tissue culture plants is not always apparent and can rapidly spread during plant micropropagation. Commonly bacteria may only become obvious after an extended culture time such as towards the end of a mass production cycle or in long term storage in a cultivar collection. The widespread bacteria contamination towards the end of a production cycle causes significant loss of plants and unreliable supply. Use of antibiotics rarely eliminates bacteria and can disadvantage plant growth and does not provide a contamination solution. This paper describes an effective assay allowing identification of explants used in banana tissue culture that contain culturable bacteria. Banana cultures initiated via meristem culture were indexed using a combination of four methods using modified nutrient agar that includes: streaking explant, plating macerated tissue taken adjacent explant into agar and broth, and observation of initiated cultures. Cultures initiated and indexed using this multi-culture assay have remained completely free from culturable bacteria for more than 12 years. This paper describes the bacterial screening assay and demonstrates its effectiveness by producing long-term bacterial free banana tissue cultures within the Australian banana germplasm collection.