A reproducible protocol for regeneration and transformation in canola (Brassica napus L.)

Abstract

The objective of the present study is to develop an efficient protocol for shoot and plant regeneration using five commercial canola cultivars grown under the Egyptian agricultural conditions. The regeneration efficiency from hypocotyl explants was examined. The data indicated that embryonic calli were formed within two weeks in the presence of 1 mgl-1 2,4-D. Adventitious shoots emerged from the embryonic callus in the presence of 4.5 mgl^-1 BA. The cultivars showed a varied response to shoot regeneration. Regeneration frequency was high in the cultivar Sarow-4 (68%) followed by Masrri L-16 (64%) compared with the other cultivars tested. Hypocotyl explants from the cultivars Sarow-4 and Semu-249 were inoculated and co-cultivated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBI-121 containing the neomycin phosphotransferase-II gene (NPT-II). The resulted putative transgenic plantlets were able to grow under knanamycin containing medium. The stable integration of the NPT-II gene into the plant genomes was tested by PCR using NPT-II -specific primers. The GUS gene expression can be detected only in the transgenic plants. The reported protocol in the present study is repeatable and can be used to regenerate transgenic canola plants expressing the genes present in A. tumifaciens binary vectors.

Keywords: Agrobacterium, canola, GUS assay, regeneration, fransformation, NPT II gene