Interspecific lily hybrids: a promise for the future

In order to introduce new characters such as resistances, flower shape and colour, from wild species into the cultivar assortment of lily it is necessary to overcome interspecific crossing barriers.. Several techniques have been used for wide interspecific lily crosses with species and cultivars from the different sections of the genus Lilium (L. longiflorum, L. henryi, L. canadense L. concolor, L. dauricum, L. candidum, L. rubellum, L. martagon, Asiatic and Oriental hybrids). Hybrids originating from intersectional crosses (e.g. L. longiflorum x L. concolor, L. longiflorum x L. dauricum, L. longiflorum x L. henryi, L. longiflorum x L. martagon, L. longiflorum x L. candidum, L. longiflorum x Asiatic hybrids (LA), L. longiflorum x Oriental hybrids (LO), L. longiflorum x L. rubellum, L. longiflorum x L. canadense, Oriental x Asiatic hybrids (OA) and L. henryi x L. candidum) have been produced. Especially the Oriental x Asiatic hybrids are a break-through in lily breeding and a promise for the future. In general wide interspecific lily hybrids show F1-sterility. Using chromosome doubling techniques tetraploids with restored fertility are produced from these diploid hybrids. At this moment a crossing programme at polyploid level with these hybrids is being carried out

Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in total twenty lily cultivars representing Lilium longiflorum, Oriental × Trumpet and Longiflorum × Asiatic hybrids were maintained on a medium with 8.3 µM picloram (PIC). In this study, they were tested for their regeneration potential by transferring them onto a regeneration medium supplemented with 0.4 µM PIC and 0.044 µM 6-benzyladenine. Regeneration was obtained in all cultivars examined and the percentage varied from zero to 89 % in the 36 callus lines. Regeneration frequency was significantly influenced by the genotype (cultivar). Subculturing the calli every 4 weeks by refreshing the regeneration medium contributed positively to bulblet formation, when compared to an eight week subculture frequency. It was found that the regeneration ability generally decreased with an increasing age of the callus cultures for all cultivars. The origin of the callus (style or filament) did not lead to significant differences in regeneration frequency, but there was an interaction between callus origin and genotype. Calli of eight randomly chosen cultivars were co-cultivated with Agrobacterium tumefaciens strain AGL0 carrying binary vectors with the gus gene as reporter and putative transgenic plants were produced. GUS histochemical assays demonstrated transient and stable expression of the gus gene in both calli and regenerated lily plants. Transient expression frequencies ranged from 0.3 to 20.6 % while stable transformation was much lower, only 1.4 % as the maximu

Transgenic lilies via pollen mediated transformation

We have developed a procedure for the production of transgenic lilies by using the pollen grain as vector for DNA delivery. First, a particle gun was used for the introduction of the NPTII gene (for kanamycin resistance) into pollen of lily (Lilium longiflorum), cv ‘Gelria’. Subsequently the bombarded pollen was used for pollination of flowers of cv ‘Indian Summer’. A large number (approx. 400,000) of seeds were harvested, of which 65,000 were tested for in vitro germination in the presence of kanamycin. Three plants that developed well on media with 50μg kanamycin were obtained. These plants were grown-on in the greenhouse. Regeneration of bulbscale explants on kanamycin confirmed the resistance of these selected plants. Furthermore, by using PCR the presence of the transgenes in the genome of the putative transformants was established. To investigate the transfer of the transgenes to the next generation, crosses were made using the three plants as male or female parent. The offspring were tested again for germination on kanamycin-containing medium, and the presence of kanamycin-resistant as well as kanamycin-sensitive seedlings proved that transfer and segregation of the transgenes had occurred. The kanamycin-resistant seedlings of the first generation were positive in the PCR reaction. The results clearly show that transgenic lily plants have been obtained. However, segregation analysis showed that transmission of the genes to the F1 was not mendelian. This will be investigated by following the transfer of transgenes to future generations

Transgenic lilies via pollen mediated transformation

We have developed a procedure for the production of transgenic lilies by using the pollen grain as vector for DNA delivery. First, a particle gun was used for the introduction of the NPTII gene (for kanamycin resistance) into pollen of lily (Lilium longiflorum), cv ‘Gelria’. Subsequently the bombarded pollen was used for pollination of flowers of cv ‘Indian Summer’. A large number (approx. 400,000) of seeds were harvested, of which 65,000 were tested for in vitro germination in the presence of kanamycin. Three plants that developed well on media with 50μg kanamycin were obtained. These plants were grown-on in the greenhouse. Regeneration of bulbscale explants on kanamycin confirmed the resistance of these selected plants. Furthermore, by using PCR the presence of the transgenes in the genome of the putative transformants was established. To investigate the transfer of the transgenes to the next generation, crosses were made using the three plants as male or female parent. The offspring were tested again for germination on kanamycin-containing medium, and the presence of kanamycin-resistant as well as kanamycin-sensitive seedlings proved that transfer and segregation of the transgenes had occurred. The kanamycin-resistant seedlings of the first generation were positive in the PCR reaction. The results clearly show that transgenic lily plants have been obtained. However, segregation analysis showed that transmission of the genes to the F1 was not mendelian. This will be investigated by following the transfer of transgenes to future generations