Effective Agrobacterium–mediated transformation of pineapple with CYP1A1 by kanamycin selection technique

Abstract

Initial calli was induced from leaf base of aseptic shoots. These calli were co-cultivated with Agrobacterium tumefaciens strain LBA4404 harboring a plant expression vector pUHA1 containing a human cytochrome P4501A1 (CYP1A1) gene for 3 days. Then, the infected calli were transferred to differentiation medium. Adventitious buds were generated after about 10 day’s incubation. The generated shoots were cultured on a medium containing 30 to 50 mg L^-1 Km for screening. The selected Km-resistant shoots were subsequently transferred to rooting medium for rooting. We derived 95 Km-resistant plants from four infection groups in total. The transformation frequency was 0.12 to 2.69%. Further analysis by polymerase chain reaction (PCR) and Southern blotting confirmed that the positive frequency of Km-resistant plants was 53.58%. We also confirmed that these protocols below were essential for A. tumefaciens-mediated genetic transformation of pineapple calli: taking agar as medium gelling agent, adding AS to co-culture medium, increasing selection times and gradually increasing the concentration of Km in the selection medium.

Key words: Ananas comosus, CYP1A1 gene, genetic transformation, Agrobacterium tumefaciens.