ORIGINAL PAPER The effect of thidiazuron level on in vitro regeneration type and peroxidase profile in Eucalyptus microtheca F. Muell Morteza Shabannejad Mamaghani Æ Mohammad Hassan Assareh Æ Mansoor Omidi Æ Mohammad Matinizadeh Æ Abbas Ghamari-Zare Æ Shokofeh Shahrzad Æ Massih Forootan Received: 30 April 2009 / Accepted: 3 August 2009 / Published online: 19 August 2009 Ó Springer Science+Business Media B.V. 2009 Abstract The green twigs of 1-year-old Eucalyptus mi- crotheca F. Muell seedlings were cultured on modified MS medium, supplemented with a-naphthalene acetic acid (NAA) and kinetin (Kin) hormones at 12 different concentrations. After 4 weeks, the combination of 1 mg l -1 NAA ? 1 mg l -1 Kin induced the highest number of axillary shoots. Mean- while, embryogenic calli were observed in media containing 4 mg l -1 NAA ? 0.5 mg l -1 Kin, without any regenera- tion. The hormone treatments were followed by subculturing the twigs in different levels of thidiazuron (TDZ). The com- bination of 1 mg l -1 NAA ? 1 mg l -1 Kin together with 0.01 mg l -1 TDZ resulted in an increase of direct shoot, while higher amounts of TDZ led to adventitious shoot induction. Somatic embryogenesis was observed in the treatment con- taining 0.01 mg l -1 TDZ ? 4 mg l -1 NAA ? 0.5 mg l -1 Kin. The peroxidase (POD) band patterns in regenerated plantlets were investigated in order to determine the effect of different levels of TDZ on loci synthesis. A dimer locus, a tetramer locus and two epigenetic bands (a new band for NAA ? Kin and the other for TDZ) were observed in the POD profiles. In case of low (0.01 mg l -1 and 0.1 mg l -1 ) levels of TDZ, one heterozygote allele was disappeared from dimer locus, while at higher TDZ levels, the dimer locus lost its stability and tetramer locus showed a high activity. Thus, POD allele patterns seems to be a feasible marker for different types of regeneration. Keywords Adventitious shoot Á Direct shoot Á Eucalyptus Á Peroxidase Á Somatic embryogenesis Á Thidiazuron Abbreviations Kin Kinetin NAA a-Naphthalene acetic acid POD Peroxidase TDZ Thidiazuron Introduction Approximately 16 million ha of lands in Iran are located in the desert marginal area. Eucalyptus spp. is found to be an adaptable species which can be utilized in stabilizing the eroding lands (Assareh 1998). Over 700 species, subspe- cies and natural hybrids from Eucalyptus genus have been identified (Watt et al. 1999; Pinto et al. 2002). Among those, Eucalyptus microtheca F. Muell is native to Aus- tralia and has been introduced to the southern subtropical M. Shabannejad Mamaghani (&) Department of Plant Breeding, Faculty of Agriculture and Natural Resources, Islamic Azad University, Karaj Branch, P.O. Box 31485–313, Karaj, Tehran, Iran e-mail: m.shabannejad@gmail.com M. H. Assareh Á A. Ghamari-Zare Á S. Shahrzad Á M. Forootan Department of Biotechnology, Research Institute of Forests and Rangelands, P.O. Box 13185–116, Tehran, Iran M. Omidi Department of Plant Breeding, Faculty of Agriculture, University of Tehran, P.O. Box 31587–11167, Karaj, Tehran, Iran M. Matinizadeh Forest Research Division, Research Institute of Forests and Rangelands, P.O. Box 13185–116, Tehran, Iran 123 Plant Growth Regul (2009) 59:199–205 DOI 10.1007/s10725-009-9404-x