Transformation of Lilium Longiflorum Plants for Cucumber Mosaic Virus Resistance by Particle Bombardment

Lilium species (Liliaceae) are a significant floriculture commodity and one of the three major bulb crops in the commercial market. Lilies are monocotyledonous plants that have been generally recalcitrant to molecular genetic manipulation because of limitations that restrict utilization of transformation technologies that are routinely applied to dicotyledonous plants. Cucumber mosaic virus (CMV) causes a serious disease of lily. It is known that transgenic plants bearing a disabled CMV replicase gene can be resistant to that virus. About 5000 pieces of morphogenic calli, 3-4 mm in diameter, derived from segments of sterile bulblet scales of L. longiflorum Thunb. cv. ‘Snow Queen’ were microprojectile bombarded, using a Finer-type of bombardment apparatus, followed by bialaphos selection. The plasmid p35SAc containing PAT selectable marker gene encoding phosphinothricin-Nacetyltransferase (that detoxifies the herbicide “Basta”) under the control of 35S promoter and the nopaline synthase (NOS) terminator; and the plasmid pSAN101 containing the CMV defective replicase gene under the control of Act promoter and NOS terminator, were used. After several rounds of callus selection, plants were regenerated in vitro. PCR analysis indicated that these plants contain both the PAT and the disabled CMV replicase transgenes. INTRODUCTION Lilium species (Liliaceae) are a significant floriculture commodity and one of the three major bulb crops in the commercial market (Robinson and Firoozabady 1993). Lilies, like other important floral bulb crops, are monocotyledonous plants, which have been generally recalcitrant to molecular genetic manipulation because of restrictions of transformation technologies that are routinely applied to dicotyledonous plants. The recent development of microprojectile bombardment systems (Klein et al., 1988; Sanford, 1988) and their demonstrated utility for DNA delivery into plant cells with high morphogenic capacity presents an opportunity for transformation of monocotyledonous floriculture crops (Sanford et al., 1993). During the last decade, particle bombardment has been used to obtain transgenic plants of floriculture crops such as tulip (Wilmink et al., 1992), orchid (Kuehnle and Sugii 1992), gladiolus (Kamo et al., 1995) and lily (Langeveld et al., 1995; Watad et al., 1998). However, there are no reports that document recovery of transgenic L. longiflorum plants bearing genes that may confer virus resistance. We report here the recovery of transgenic lily plants after microprojectile bombardment of morphogenic primary callus with plasmids containing uidA reporter and PAT selectable marker genes and the plasmid pSAN101 containing the CMV defective replicase gene. Plants were regenerated under bialaphos selection. The presence of the transgenes in regenerated plants was determined by polymerase chain reaction (PCR) amplification. Proc. 10 IS Virus Diseases Ornamentals