Improvement of regeneration and transformation systems for Cyclamen persicum using somatic embryo culture

Abstract

Cyclamen persicum is one of the most important pot plants in the world. We developed an effective method for somatic embryo-mediated plant regeneration and genetic transformation using Agrobacterium, with somatic embryos as the source of plant material. Numerous somatic embryos were formed from the leaf segments of Cyclamen cv. Fragrance Mini 16 weeks after initiation on a 2,4-D- and kinetin-containing medium. The maximum regeneration frequency from the somatic embryos was achieved using a regeneration medium containing 0.1 mg l � 1 benzylaminopurine, 0.01 mg l � 1 a-naphthaleneacetic acid, and 0.2 mg l � 1 gibberellin. After co-cultivating the somatic embryos with Agrobacterium strain LBA4404 (the plasmid vector pIG121Hm) that harbors genes for hygromycin phosphotransferase, neomycin phosphotransferase, and b-glucuronidase, hygromycin-resistant clones were selected on a medium containing 5 mg l � 1 hygromycin. Hygromycin-resistant plantlets were regenerated from 14 hygromycin-resistant embryos. Using this transformation system, 74 independent plants were obtained from 1-ml packed cell volume, approximately 2,000 embryos.