SAAT: SONICATED ASSISTED AGROBACTERIUM-MEDIATED TRANSFORMATION OF TROPICAL MAIZE EMBRYOS

Abstract

Particle bombardment and transformation mediated by Agrobacterium are the main processes to introduce gene in maize in an asexual way. Recently, a new and potentially more efficient method for plant transformation via Agrobacterium was developed to reach the proper cells in the target tissue. This technique called Sonicated Assisted Agrobacterium-mediated Transformation (SAAT) involves subjecting the plant tissue to brief periods of ultrasound in the presence of Agrobacterium. SAAT treatment produces small and uniform fissures and channels throughout the tissue, allowing the Agrobacterium easier access to internal plant tissues. The objective of this work was to transform immature embryos of tropical maize inbred lines using the SAAT technique. Immature maize embryos were cultured for five days on callus induction medium. Also, prior to SAAT treatments, embryos were inoculated with Agrobacterium LBA4404 harboring the plasmid pCAMBIA C2-208 and submitted to sonication for 30 seconds. Different transformation parameters were analyzed in transient expression assays and 37.5 to 87.5 percent of embryos showed positive histochemical GUS assay. Resistant calli, obtained from the medium containing 30 mg.L-1 hygromycin were used to regenerate plants in MS basal medium supplemented with 0.5 mg.L-1 IBA (indole butyric acid) and 1 mg.L-1 BAP (Benzylaminopurine). GUS expression was verified in the leaves of plants regenerated and resistant to hygromycin.

Keywords

• Zea mays
• genetic transformation
• immature embryos
• transient expression
• Gus assay