Reversion of inflorescence development in Euphorbia millii and its application to large-scale micropropagation in an air-lift bioreactor

An efficient, simple micropropagation protocol was developed for Euphorbia millii Des Moulines using inflorescence explants and an automated, low cost, air-lift bioreactor system. Inflorescence meristems of the main axis usually reverted to vegetative meristems (a meristernatic dome with leaf primordia) 1 week after inoculation on MS medium supplemented with benzyladenine (BA) and indole-3-butyric acid (IBA). Of the various plant growth regulator combinations tested, MS medium with 1 mg l(-1) BA and 0.3 mg l(-1) IBA proved to be most effective. On this medium the percentage reversion was maximal, and the number of explants producing multiple shoots was the highest. The highest percentage of inflorescence reversion and the highest number of multiple shoots induced per explant were observed when inflorescences from the first stage of development were used. The lowest response was observed on third stage inflorescences. Vegetative shoots obtained from reverted explants of inflorescences were used as inocula for multiplication studies. Comparisons between solid and bioreactor cultures (i.e., continuous immersion with or without a net, or temporary immersion in liquid media using ebb and flood) revealed that shoot multiplication and growth were more efficient using a continuous immersion bioreactor (with a net). In vitro regenerated shoots were cultured hydroponically for 6 weeks; 100% of plants rooted and were acclimatised successfully in growing media containing peat moss:vermiculite:perlite (1:1:1). RAPD DNA marker analysis of micropropagated plants and their mother plant confirmed the genetic uniformity of the regenerants.