Production of Transgenic Camelina sativa Plants via Agrobacterium -Mediated Transformation of Shoot Apical Meristems

Abstract

A method to produce transgenic Camelina sativa plants in cvs. PI650159 and PI650161 was developed. Micropropagated shoot meristem cultures were established from in vitro germinated seedlings and used as target tissues for Agrobacterium-mediated transformation. A plasmid harboring enhanced green fluorescent protein, β glucuronidase and neomycin phosphotransferase II genes were used to optimize parameters for transgenic plant production. Kanamycin at 40 mg∙l−1 was effective in suppression of non-transformed cells while permitting growth of transgenic tissues. Shoot apical meristems co-cultivated with Agrobacterium exhibited stable enhanced green fluorescence protein (EGFP) and β glucuronidase (GUS) expression after culture on plant regeneration medium. We observed transformation efficiencies of 53.33% in cv. PI650159 and 98.33% in cv. PI650161. The presence of transgenes in both cultivars was confirmed by PCR, while quantitative real-time PCR detected single copy integration in Pl650161 and two copy integration in Pl650159. Transgenic plants exhibited EGFP and GUS expression in all tissues including shoots, leaves, buds, floral organs, seeds, and pods. Our results demonstrate a simple and efficient technique using apical shoot meristems for production of transgenic C. sativa plants that can be used for transfer of desirable traits.