Agrobacterium rhizogenes-mediated DNA transfer to Aesculus hippocastanum L. and the regeneration of transformed plants

Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 muM 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 muM BA. Following elongation on MS medium supplemented with 1 muM BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four.