Seeds of O. ficus-indica were obtained from a plant cultivated at the Botanic Garden of Palermo, Italy. Seeds of P.
aselliformis were purchased from Koehres Kakteen (Germany). To establish the in vitro cultures, sixty seeds of O. ficus-indica were chemically scarified with concentrated H2SO4 for 15 min and rinsed three times under aseptic conditions with sterile distilled water (SDW) and germinated on MS medium (Murashige and Skoog 1962) supplemented with 146 mM sucrose and 1 µM gibberellic acid (GA3) (pH 5.7 ± 0.1). Seedlings of O. ficus-indica (Fig. 1a) were used as starting material to obtain suitable micropropagated rootstock for in vitro micrografting. Preliminary experiments showed that the best results in terms of rootstock production were obtained when explants were cultured in presence of 2 µM 6-benzylaminopurine (BA). This plant material was subcultured every 30 days and maintained as rootstock source indefinitely (Fig. 1b).
Forty-five seeds of P. aselliformis were disinfected for 1 min in 70 % ethanol, 25 min in 2 % sodium hypochlorite (w/v), rinsed four times under aseptic conditions with SDW and germinated on MS medium (Fig. 1c).
All cultures were maintained in a climate chamber at 25 ± 1 °C under a 16 h day length, and a photosynthetic photon flux of 50 µmol m−2 s−1 provided by Osram cool-white 18 W fluorescent lamps.
Micrografts were carried out using O. ficus-indica as rootstock and P. aselliformis seedlings as scion. Micrografting procedures were based upon those described by Estrada-Luna et al. (2002) with few modifications. Micrografts were performed on 10 mm long fresh cut rootstock of O. ficus-indica without roots. Shoots were cut under aseptic conditions at the base and at the top with a single stroke of a razor blade. The scion was cut into apical and sub-apical segments (slices) of 5 and 3 mm, respectively. To maximize the production of slices no areoles were left in the remaining hypocotyl. The remaining part was discarded.
The freshly cut scion was brought in contact with freshly cut rootstock tissue (Fig. 1d), the two bionts were incubated on medium to induce root formation. Periodically, axillary shoots that emerged from rootstocks were removed. The different steps of micrografting were carried out on PGR free MS medium and maintained as described above. Micrografts were done in separate batches. When sub apical explants were used, each set comprised 90 micrografts while, when apical explants were used, each set was of ten micrografts.
Well developed micrografts 25 mm long were collected and washed in SDW to remove the medium, then transferred into autoclaved Jiffy 7® peat pellets soaked in 40 ml of SDW. After transplanting, plants were maintained under 95 ± 5 % relative humidity in a growth chamber till roots grew out of the pellet, and then transferred to the greenhouse, exposed to daylight conditions at 25 ± 2 °C day, 20 ± 2 °C night. Micrografting response was quantified by the number of non-necrotic scions and the number of offsets produced by the scions 60 days after micrografting. Scion growth was evaluated by recording the mean number of new shoots and the shoot length 3 and 6 weeks after grafting.