Efficient genetic transformation of Lilum longiflorum and Ornithogalum dubium by particle acceleration followed by prolonged selection in liquid medium

Genetic transformation mediated by bombardment with microscopic metal particles carrying target genes is the preferred method for the introduction of foreign genes into monocotyledonous plants. The fact that most flower bulbs are monocotyle-donous and that almost all commercial cultivars are propagated vegetatively makes them good candidates for molecular breeding through microprojectile bombard-ment. We report here on a development of a reliable method for an efficient genetic transformation of both Lilium longiflorum and Ornithogalum dubium using a particle inflow gun to deliver gene constructs into the target plant tissue, followed by a prolonged selection in the dark in liquid medium supplemented with kanamycin. The system was first optimized for Lilium longiflorum 'Snow Queen'. Based on the level of transient GUS expression, liquid-grown cell clumps are more competent than leaves. Large cell clusters (2-10 mm) maintain their organogenic potential while smaller clusters (<2 mm) cease to grow and die. The liquid-grown tissue cultures have a level of competence for transformation about 50-70 times greater than that of solid-grown callus cultures, and compact cell clusters are more competent than loose clusters. The cells were bombarded with a pCAMBIA2301 vector, carrying nptII gene conferring kanamycin resistance and GUS reporter gene. Following selection for 4-6 months in a liquid medium supplemented with 80 mg l(-1) kanamycin in the dark, the cell clusters were transferred to a regeneration medium in the light where hundreds of transgenic plantlets developed. The plants retained their stable transgenic state when grown in the greenhouse for two seasons. The transformation of O. dubium was similar in principle to that of L. longiflorum with three major differences: lily liquid-grown cultures grew more rapidly and had a higher potential for somatic embryo development. Ornithogalum cultures under selection took longer to develop into semi-organized cell clumps of sufficient size to allow continued shoot regeneration, were mostly organogenic, and the regenerated plantlets had higher rate of vitrification.