Annals of Botany

Roles of rols in root regulation

Like its close relative Agrobacterium tumefaciens, A.rhizogenes is a naturally occurring agent of plant genetic transformation; it is the causative agent of ‘hairy root’ disease in which roots proliferate at the infection site. Two groups of ‘oncogenes’ on the rootinducing plasmid, the aux genes and the rol genes, are important for this transformation. Interestingly, as pointed out by Alpizar et al. (Montpellier, France, pp. 929–940), the roots may be excised from the plant and grown readily in culture, and used for regeneration of whole plants and the study of root growth and physiology. In common with A. tumefaciens, A.rhizogenes shows differing abilities to infect different dicot plants. For both bacteria, coffee (Coffea arabica) is a difficult host (although methods for generating GM coffee have now been developed). The authors have previously achieved transformation of coffee with A. rhizogenes but the excised roots did not proliferate and soon died. In the new series of experiments, 62 different hairy root clones were grown under different conditions. All of the clones were shown by PCR to have the rolB and rolC oncogenes integrated into their genome but none of them had the bacterial aux genes. Added auxin was thus required for proliferation of all clones and was optimally supplied as 0.5 μM IBA. Sucrose was not essential but its inclusion at 2 % (w/v) was optimal for root growth with low (20 μmol m^-2 s^-1) light intensities or darkness favouring regeneration. Under optimal conditions, the 62 clones have been maintained and sub-cultured for over 3 years. Although there is some variation between clones in respect of root length and the proportion of fine roots, the majority of their phenotypes did not differ significantly from the roots of non-transformed plants. They therefore constitute a useful system for study of root characters such as resistance to nematodes.