The Horse Chestnut Lines Harboring the rol Genes

An Agrobacterium rhizogenes-mediated transformation system for Aesculus hippocastanum L. has been developed. Wounded androgenic embryos of A. hippocastanum were inoculated with bacteria containing the pRiA4 plasmid, with the uid A sequence as a reporter gene. The hairy roots emerging from the wounded sites of androgenic embryos were isolated and maintained in Murashige and Skoog's (MS) liquid hormone-free medium. Five hairy root lines have been maintained in vitro for 4 years with unchanged growth rate and might be a suitable source for secondary metabolite production. The transformation events have been confirmed by a polymerase chain reaction specific to the rol A, B, C and D genes. The absence of residual contaminating bacteria has been shown by a polymerase chain reaction specific to the vir D1 sequence.