Use of direct somatic organogenesis for Agrobacterium-mediated transformation of onion

Abstract

An Agrobacterium tumefaciens mediated DNA delivery is being developed for onion (Allium cepa L.) using organogenic structures formed on the ovaries as target tissue. An indirect transformation procedure using A. tumefaciens strains LBA4404 and EHA105 in combination with four plasmids was used for establishing an efficient transformation protocol. Studies were focused on different protocols for co-cultivation, selection and detection of optimal reporter and selection genes in onion. A histochemical GUS test and visual detection of the GFP protein were used for optimization of transformation treatments. Several pretreatment and co­ cultivation protocols achieving transient expression were studied, and the optimal was a combination of sonication (10 s) and vacuum treatment (5min, 35 nuriHg). Among several selection media studied, optimal results were obtained using phosphinotricin (2.5 mg/1) as selection agent and timentin (150 mg/1) for good suppression of bacterial growth. The transgenic nature of individual regenerants after sixmonths on selection media was confirmed by polymerase chain reaction using primers for detection of uidA and bar genes. Using this test, three regenerants were positive for both bar and uidA genes and three regenerants were positive for the bar gene only.

Keywords

• tissue cultures
• genetic transformation
• onion
• Allium cepa
• direct somatic organogenesis
• Agrobacterium