Microshoot culture is a significant method for proliferation of woody plants. Walnut (Juglans sp.) is an important economical plant originated from Iran. One of the main aspects in walnut micropropagation is the induction of rooting in microshoots. This work analyzed morphological and biochemical changes during in vitro adventitious rooting of five Persian walnut (Juglans regia L.) cultivars in order to knowing how this ability can be increased for better breeding. Morphological and biochemical parameters such as rooting percentage, root number, length of root and changes in peroxidase activity and its isoenzymatic pattern in Electrophoresis technique were monitored to characterize the rooting response. The best rooting performance was in microcuttings of ‘Sunland’ cultivar, followed by ‘Chandler’, ‘Z63’, ‘Kerman’ and ‘Howard’, respectively. Protein content showed the same changes pattern in all cultivars that confirms its metabolism as crucial steps for root formation. Variations in rooting response were markedly indicated with peroxidase isoenzymes indicators. Maximum increase of peroxidase activity, observed at day 35– initiative phase of rooting- was associated with enhanced rooting ability in ‘Sunland’ and ‘Chandler’ cultivars. Having the special activity pattern during root development in all walnut cultivars, peroxidase could be a reliable and successive marker for this physiological process.

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