Transformation of carnation by microprojectile bombardment

Transgenic carnation (Dianthus caryophyllus L.) plants were produced by microprojectile bombardment of highly regenerative stem segments. A two-step regeneration procedure based on the use of two different cytokinins-6-benzylaminopurine and thidiazuron-was employed for the production of adventitious shoots from stem segments. The size of the original stem was found to affect the regeneration efficiency of stem segments: the highest efficiency of adventitious shoot regeneration was obtained with segments originating from stems with two mature leaves, as compared to those with four, six or eight mature leaves. The tissue culture procedure was shown to be suitable for a number of standard and spray cultivars. Bombardment of cultivar "White Sim" stem segments was performed with a plasmid containing uidA and bar genes encoding β-glucuronidase and phosphinothricin-acetyltransferase, respectively. Transformation frequency was determined, based on the transient expression of uidA in stem segments. Following selection in the presence of the herbicide bialaphos, about 70 plantlets per 100 stem segments were recovered. Upon analysis, about 3% of these recovered plantlets exhibited strong stable uidA expression throughout the plant. Presence of the bar gene in plants stably expressing uidA was confirmed by Southern blot analysis.