Direct shoot regeneration from basal leaf segments of Lilium and assessment of genetic stability in regenerants by ISSR and AFLP markers

Here, we report a widely applicable procedure for direct shoot regeneration via basal leaf segments of Lilium. Leaf segments (0.8-1.0 cm long and 0.4 cm wide) were excised from leaves on shoot nodes 3 to 6 of 4-wk-old in vitro stock shoot cultures. The segments were wounded by three transverse cuts across the midvein on the abaxial side, with 1 mm between cuts, and cultured with the abaxial side in contact with a shoot regeneration medium composed of half-strength Murashige and Skoog medium supplemented with 1 mg/l naphthaleneacetic acid, 0.5 mg/l thidiazuron, 30 g/l sucrose, and 7 g/l agar (pH 5.8). The cultures were incubated for 4 wk under a 16-h photoperiod at 23 +/- 2A degrees C for adventitious shoot regeneration. With this procedure, a mean shoot regeneration frequency of 92-100% and mean number of shoots of 4.7-7.0 per segment were obtained in five Lilium species and hybrids, which represent diverse genotypes of Lilium and are commercially popular lilies. Histological studies with Lilium Oriental hybrid Siberia revealed that meristemoids initiated from subepidermal cells on the adaxial side of the explant and eventually developed into adventitious buds, without callus formation. In an assessment of genetic stability in the regenerants of Siberia, no polymorphic bands were detected by intersimple sequence repeat and only 0.73% polymorphic bands were detected by amplified fragment length polymorphism. The morphologies of the regenerants were identical to those of the control. These results demonstrated that the regenerants were genetically and morphological stable. Thus, this procedure has great potential application for micropropagation, genetic transformation, and preparation of shoot tips for cryopreservation and cryotherapy for virus eradication of Lilium.