Isolation of Female Gamete Cells from Unpollinated Ovules in Lilium

Ovules obtained from opened flowers of Oriental hybrid 'Casa Blanca', LA hybrid 'White Tycoon', Lilium x formolongi 'Hakuryu' and L. formosanum were wounded by a surgical blade, and then treated with an enzyme mixture. Afterwards, ovule sections were dissected using a handmade glass needle and the tip of a 30G short needle syringe under a stereomicroscope to remove the outer and/ or inner integument. The isolated embryo sac with nucellar tissue was cut off by the 30G short needle syringe, and was then pushed off by a macromanipulating system to isolate female gamete cells. The isolated gamete cells could be easily captured and handled by a microcapillary system that was connected to an electric micropump. The combination of a surgical cutting with the use of an enzymatic treatment, shortened significantly the total operating time for the isolation of female gamete. However, the optimal incubation time for the enzymatic treatment differed between cultivars and species. It varied from 1 to 1.25 and 2.5 h for L. x formolongi 'Hakuryu', L. formosanum and L. 'Casa Blanca', respectively. No isolated gamete cells could be obtained from ovules of ` White Tycoon' due to the fact that the cut flowers were stored at low temperature for several days.