Plant Cell Rep (2006) 25: 513–521 DOI 10.1007/s00299-006-0114-9 CELL BIOLOGY AND MORPHOGENESIS Xiaoping Wei · Xiaoping Gou · Tong Yuan · Scott D. Russell A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica Received: 18 September 2005 / Revised: 8 December 2005 / Accepted: 14 December 2005 / Published online: 10 February 2006 C  Springer-Verlag 2006 Abstract Plumbago zeylanica is a unique model for studying flowering plant gametogenesis, heterospermy, and preferential fertilization, yet understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. We found three key factors for enhancing successful regeneration: (1) tissue source of explants, (2) combination and concentration of growth regulators, and (3) culture conditions. The highest frequency of shoot regeneration was achieved using hypocotyl segments cultured on MS basal medium supplemented with BA 2.0 mg/l, NAA 0.75 mg/l, adenine 50 mg/l and 10% (v/v) coconut milk under subdued light at 25 ± 2 ◦ C; under these conditions, each hypocotyl segment produced over 30 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Regenerated shoots rooted easily on half-strength basal MS medium and were successfully established in the greenhouse. Using this tissue culture protocol, reporter gene GUS under the constitutive CaMV 35S promoter was introduced into P. zeylanica cells of petiole, cotyledon and hypocotyl with A. tumefaciens strains AGL1 and LBA4404. Transient expression was observed in all recipient tissues. Stable transgenic calli originating from petiole were obtained. Keywords Agrobacterium tumefaciens . Gene transformation . Growth regulators . Organogenesis . Plumbago zeylanica . Regeneration Abbreviations AD: adenine free base . AS: acetosyringone . BA: 6-benzylaminopurine . CM: coconut milk . GUS: β-glucuronidase . IAA: indole-3-acetic acid . IBA: indole-3-butyric acid . Communicated by R. Reski X. Wei · X. Gou · T. Yuan · S. D. Russell () Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA e-mail: srussell@ou.edu Tel.: +1-405-325-4391 Fax: +1-405-325-7619 MS medium: Murashige and Skoog (1962) medium . NAA: 1-naphthaleneacetic acid . NPTII: neomycin phosphotransferase . PGR: plant growth regulator . SEM: scanning electron microscopy . X-Gluc: 5-bromo-4-chloro-3-indolyl-β-d-glucuronide . ZT: zeatin Introduction Plumbago zeylanica, a perennial flowering plant used as a medicinal herb for certain disease treatments in southern Asia (Chopra et al. 1996), has emerged as a model for the study of classical sexual reproduction in angiosperms. As a plant representative of both cytoplasmic heterospermy and preferential fertilization, P. zeylanica is potentially a valuable system for molecular dissection of the mecha- nisms involved in double fertilization and gamete identity. Its mature male gametophyte contains a vegetative nucleus and two strongly dimorphic sperm cells (Russell and Cass 1981; Russell 1984; Russell and Strout 2005). One sperm cell, which possesses more mitochondria and is physically associated with the vegetative nucleus, preferentially fuses with the central cell to form endosperm, whereas the other sperm cell, which contains more plastids and is unasso- ciated with the vegetative nucleus, fuses with the egg to give rise to the zygote and embryo (Russell and Cass 1981; Russell 1984, 1985). Because the simplified embryo sac of this plant lacks synergids, the observation of fertilization events is simplified, making P. zeylanica an ideal model to trace sperm cell fate during double fertilization (Russell 1980, 1982, 1983). In recent years, our group has worked toward developing tools for studying gene expression during preferential fer- tilization using cDNA libraries, EST sequences, functional classification, in situ hybridization and qRT-PCR of P. zeylanica sperm cells (Zhang et al. 1998; Gou, Yuan, Wei and Russell, unpublished data). Although common genetic elements may be activated in Arabidopsis or other easily transformable plants, divergence of such elements in distantly related species often complicate functional