Optimisation of growing conditions for the apple rootstock M26 grown in RITA containers using temporary immersion principle Li-Hua Zhu*, Xue-Yuan Li & Margareta Welander Swedish University of Agricultural Sciences, Department of Crop Science, P.O. Box 44, SE-230 53 Alnarp, Sweden (*requests for offprints; Fax: +46-40-460442; E-mail: Li-Hua.Zhu@vv.slu.se) Key words: apple, bioreactor, micropropagation, RITA, temporary immersion Abstract The use of bioreactors may provide an efficient and economic tool for mass clonal propagation of plants if technical problems can be solved. In this paper, we report the results of experiments aimed at optimising conditions for apple rootstock M26 grown in RITA containers using the temporary immersion principle. We tested different types and sizes of explants, different concentrations of plant growth regulators (BAP, kinetin and IBA) in the multiplication and elongation phases, and medium exchange during the shoot elongation period. The results show that the higher concentrations of cytokinins were required during the shoot multiplication phase, while the lower concentrations were better during the shoot elongation phase. Hyperhydricity was increased with increasing concentration in of cytokinins during both shoot multipli- cation and shoot elongation phases. The best shoot production in terms of shoot number and shoot quality was obtained using 4.4 lmol BAP and 0.5 lmol IBA during the shoot multiplication phase and 1.1 lmol BAP and 0.25 lmol IBA during the shoot elongation phase. Medium exchange twice during the shoot elongation phase resulted in higher shoot production compared with no exchange of the medium. However, it also resulted in increased hyperhydricity. Immersion frequency of 16 times per day gave a higher mul- tiplication rate and longer shoots than 8 times per day. The explant size of 0.5 cm or 1 cm resulted in a significantly higher shoot production rate compared with that of 1.5 cm, but shoot length and hyper- hydricity were not affected by the explant size. Shoot cultures from the liquid media rooted normally in the RITA containers with more than 90% rooting and the rooted plantlets acclimatised well in the greenhouse. Abbreviations: BAP – benzylaminopurine; IBA – indole butyric acid; MS – Murashige and Skoog medium (1962) Introduction Clonal propagation by conventional tissue culture techniques is limited in commercial production due to high input of manual labour and a low degree of automation (Maene and Debergh, 1985; Sluis and Walker, 1985; Simonton et al., 1991; Chu, 1995). In contrast to conventional tissue culture techniques, the use of bioreactors may provide a promising tool for mass clonal propa- gation. Due to the use of liquid media, bioreactors have the following advantages compared with agar media: – a large number of plantlets can be more easily produced due to more uniform culture condi- tions and the ease with which, the explants can take up the nutrients; – time- and labour-saving in the handling of cul- tures because of the semi-automatic operation; – better growth and biomass production because of good aeration by forced oxygen supply; – the decrease of apical dominance and the stim- ulation of lateral bud growth, which is probably due to the loss of culture orientation. However, bioreactors developed in the past were mainly for bacterial cultures and not suitable for Plant Cell, Tissue and Organ Culture (2005) 81: 313–318 Ó Springer 2005 DOI 10.1007/s11240-004-6659-9