Efficient Lilium longiflorum plantlet regeneration from transgenic callus culture lines after ten years of sub-culture

The development of a long-term callus storage method for the commercially valuable flower bulb Lilium longiflorum is important for molecular breeding. A regeneration protocol was developed for undifferentiated cell callus culture lines of 'Osnat' (five transgenic and two non-transgenic lines), established in 2003 from bulb-scale segments. The transgenic plants were designed to produce pollen-less flowers and therefore carried the rolB gene under the control of the lat52 promoter, the nptII selection gene and the GUS reporter gene uidA, the last two genes driven by the CaMV35S promoter. After ten years of sub-culture, the calli retained kanamycin resistance but could not regenerate. GUS specific reaction and the presence of all three transgenes in the cultures were confirmed by PCR and RT-PCR. A protocol was developed to restore plantlet regeneration from the callus culture lines in five stages. Initially, 96 plantlets of ca. 10 mm tall with a 2-3 mm bulblet were isolated after regenerant development and transferred to the plantlet growth stage. At the end of the plantlet growth stage, 48% of the plantlets developed normally (5-8 mm bulblet), and were successfully acclimatized in the greenhouse. During the following season, 20-75% of plants flowered for each line. Another 214 plantlets from the same seven lines were studied after regenerant development and again displayed normal phenotypes after the plantlet growth stage (with variation between lines of 28-77%). The presence and activity of the transgenes in the phenotypically normal plantlets was confirmed by PCR and RT-PCR. Phenotypic characterization of the transgenic and control plants required more than one flowering season. These results suggest that this approach may be used as a long-term callus storage method.