Plant Cell, Tissue and Organ Culture 77: 283–285, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 283 Research note Colchicine induced embryogenesis in maize Bohuš Obert 1,∗ & Be´ ata Barnab´ as 2 1 Institute of Plant Genetics and Biotechnology, Slovak Academy of Science, Akademick´ a 2, P.O. Box 39/A, 95007 Nitra, Slovak Republic; 2 Agricultural Research Institute of the Hungarian Academy of Sciences, Brunsvik utca 2, 2462 Martonv´ as´ ar, Hungary ( ∗ requests for offprints: E-mail: bobert@scri.sari.ac.uk) Received 31 March 2003; accepted in revised form 9 October 2003 Key words: anther culture, colchicine, embryogenesis, microspore, Zea mays L. Abstract The present study involves in vitro androgenesis of Zea mays L. using anther culture. We tested combinations of single factors and their influence on microspore induction. Embryogenic induction of microspores within anthers in in vitro conditions was the best when combination of cold treatment, TIBA (0.1 mg l -1 ) in media and colchicine (0.02% during first 3 days of culture) was applied, but colchicine alone can be factor, which can stimulate or initiate embryogenesis in anther culture of maize. Abbreviations: ELS – embryo like structure, NAA – napthalene acetic acid, TIBA – 2,3,5-triiodobenzoic acid In cereal anther cultures, chromosome doubling of haploid cells (or tissue) in certain stage is a critical step in producing doubled haploid plants. Several in- vestigations were undertaken to study the effect of incorporation of colchicine in the induction medium for anther culture, since first report on ‘direct genome doubling’ was published in wheat (Barnabas et al., 1991). The effect of colchicine on anther culture de- rived plants was investigated as well in several cereals: wheat (Szakacs and Barnabas, 1995; Redha et al., 1998; Zamani et al., 2000), maize (Saisingtong et al., 1996; Barnabas et al., 1999), rice (Chen et al., 2001), or triticale (Arzani and Darvey, 2001). A number of abiotic stresses can elicit microspores to irreversibly alter their development to androgenic pathway. In our experiments, temperature and colchicine were used as abiotic stresses capable of inducing androgenesis (Touraev et al., 1997). A responsive maize genotype A 18 was used as anther donor plants. The plants were raised in climate chambers using the ‘Bk’ programme for phytotronic cultivation of maize (Tischner et al., 1997). Tas- sels were collected prior to emergence of the leaf sheath, when the microspores were judged to be at the mid-uninucleate stage of development. After collection, if cold pretreatment was applied the excised tassels were covered by aluminium foil and then stored at 7 ◦ C. After 7 days of cold pretreatment a few anthers from different parts of the tassel were removed, and examined microscopically to verify the developmental stage. Parts of the tassels containing mid- or late-uninucleate microspores were surface sterilised with 20% sodium hypochlorite for 20 min and then washed three times with sterile distilled water. Anthers were inoculated onto the surface of induction media (Genovesi and Collins, 1982) supple- mented with 5 g l -1 charcoal, 500 mg l -1 casein hy- drolysate, 120 g l -1 sucrose, alternatively 0.1 mg l -1 2,3,5-TIBA and/or 0.02% colchicines for 3 days was added (Barnabas et al., 1999). pH of all media was adjusted to 5.8 and 2.5 g l -1 gelling agent gelrite was used. The induction media was autoclaved using a general protocol. The cultures were incubated in the dark at 29 ◦ C for 28 days. After this period, the number of responding anthers and number of induced struc- tures (ELS and calluses) were harvested and counted. After the induction period the ELS were trans- ferred to a regeneration medium: MS macro and micro nutrients (Murashige and Skoog, 1962), RM vitamins (Barnabas et al., 1999), supplemented with 1 mg l -1