Mycorrhizal associations between Tuber melanosporum mycelia and transformed roots of Cistus incanus

We set out to establish root cultures of a host plant with the aim of obtaining dual cultures of Tuber melanosporum mycorrhiza on transformed roots. Seedlings of Cistus incanus germinated under sterile conditions from seeds collected in the wild were treated with Agrobacterium rhizogenes. Nine hairy roots collected from different seedlings were cultured individually by repeated subculturing. The hairy root clones differed in growth rates and in morphology (branching frequency and distance between side roots). Root growth in a liquid medium exhibited a lag phase of about 2 weeks and an exponential phase lasting about 12 days before the start of the stationary phase. Hairy roots could be kept alive on medium M, a special solid minimal medium (low in Fe2+, BO43–, Ca2+, Cu2+ and Zn2+, very low in PO43– and lacking MoO42–, NH4+ and Co2+), for more than 7 months. T. melanosporum could be grown on the same medium for long periods only by subculturing the fungus with the roots. A mycorrhizal association developed between the roots and the T. melanosporum mycelium within 3 months. The association consisted of elongated roots with a mantle and a Hartig net surrounding two to three layers of cortical cells. Swollen, club-like root tips were discernible 5 months after inoculation. The mycorrhized roots could be subcultured and propagated on medium M and maintain the mycorrhizal association.