&p.1:Abstract The potential use of the rol genes from Agro- bacterium rhizogenes to improve the root system horti- cultural characteristics was evaluated in transgenic aspen (Populus tremula) plants, harboring the rol genes under their native promoters. Southern blot and RT-PCR ana- lyses confirmed the presence and expression of A. rhizo- genes rolC and rolB genes in four different phenotypical- ly selected transgenic clones. Several of the observed phenotypic modifications were related to rol-gene ex- pression and included, in particular, modified root sys- tems. All in vitro-cultured rol-transgenic plants exhibited extensive root formation in a hormone-free medium, as well as a larger root surface area and mass, as compared to a uidA (β-glucuronidase-encoding)-transgenic aspen line and control (non-transformed) plants. Adventitious root formation in stem segments of rol-transgenic plants exhibited very rapid kinetics, resulting in a much shorter rooting time for rol-transgenic stem segments (e.g. 10 days for 80% rooting in rol-transgenic lines T-26 and T- 27, as compared to more than 18 days for control non- transformed or uidA-transgenic aspen plants). rol-Trans- genic plants maintained the capacity for 100% rooting throughout the year, versus 70–80% rooting in non-trans- formed plants during the winter. The four rol-transgenic lines exhibited differences in root development; in two of them enhanced root development was accompanied by increased shoot fresh weight. The root:shoot fresh weight ratio was always higher in rol-transgenic lines than in non-transformed plants. In the T-27 rol-transgen- ic line, the propagation coefficient of shoot-bud regener- ation in liquid root culture was almost three times higher than in non-transformed plants. To the best of our knowl- edge this is the first report on quantitative phenotypic al- terations in rol-transgenic woody plants. &kwd:Key words Agrobacterium rhizogenes · Populus tremula · rol Genes · Root formation · Transgenic plants&bdy: Introduction Adventitious root formation and root system perfor- mance are important factors in propagation of cuttings from different species, as well as in their further estab- lishment. The choice of a suitable organ and tissue type (e.g. stem, leaf or root cuttings) for propagation is of prime importance for the improvement of root formation in cuttings, and depends on the tissue-specific potential for root regeneration. Pruning (Scott 1987) and etiolation of stock plants to obtain easy-to-root juvenile material (Maynard and Bassuk 1987), the use of root-inducing growth regulators (Loach 1988), and propagation under fog and mist (Harrison-Murray et al. 1988; Millbocker 1987) are some of the techniques which have been pro- posed to improve root formation from cuttings. In vitro propagation can be helpful in overcoming rooting problems because different treatments can be easily employed in tissue culture. The use of different nutrient media, accurate hormone concentrations, well- controlled environmental conditions and a suitable juve- nile plant source can improve rooting percentages and root performance. However, the high cost of tissue-cul- ture propagation (Mudge et al. 1986) has prompted a search for genotypes with better rooting performance following a shorter period of culture (Strode and Abner 1986). Abolishing the need for hormones in the medium, reducing the time required for adventitious root forma- tion and increasing rooting percentages can reduce the cost of media components and shorten culturing time, thereby increasing the propagation coefficient. The phytopathological soil bacterium Agrobacterium rhizogenes can trigger root formation in infected plants by transferring a DNA fragment (T-DNA) from the Ri (root-inducing) plasmid to wounded plant cells (Zambry- ski et al. 1989). Using molecular methods of insertion and deletion analysis, four different loci have been iden- T. Tzfira · B. Vinocur · A. Altman ( ✉ ) · A. Vainstein The Kennedy-Leigh Centre for Horticultural Research and The Otto Warburg Center for Biotechnology in Agriculture, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O.B. 12, Rehovot 76–100, Israel e-mail: altman@agri.huji.ac.il; Fax: +972-8-9468263&/fn-block: Trees (1998) 12:464–471 © Springer-Verlag 1998 ORIGINAL ARTICLE &roles:Tzvi Tzfira · Basia Vinocur · Arie Altman Alexander Vainstein rol-Transgenic Populus tremula: root development, root-borne bud regeneration and in vitro propagation efficiency &misc:Received: 1 December 1997 / Accepted: 8 March 1998