Regeneration and transformation efficiencies among five Egyptian clover cultivars (Trifolium alexandrinum)

The objective of the present investigation is to develop an efficient method for shoot and plant regeneration using five commercial Egyptian clover (Trifolium alexandrinum) cultivars grown under the Egyptian agricultural conditions. The regeneration efficiency from hypocotyls and petiole explants was examined. The data indicated that embryonic calli were formed within one week in the presence of 1.5 mgl-1 2,4-D. Adventitious shoots emerged from the embryonic callus in the presence of 1.5 mgl-1 BA. The petiole explants show higher callus and shoot induction percentages compared with the hypocotyls in all cultivars tested. The cultivars showed a varied response to shoot regeneration. Regeneration frequency was high in the cultivar Serw 1 follwed by Helaly 22 and 19% respectively compared with the other cultivars tested. Petiole explants from the cultivars Serw 1 and Helaly were inoculated and co-cultivated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBI-121 containing the ?- glucurodinase (gus) gene under the genetic control of CaMV-35S promoter and nopalin- synthase terminator. The resulted putative transgenic plantlets were able to grow under knanamycin containing medium. The stable integration of the gus gene into the plant genomes was confirmed by dot blot and by PCR analysis using 35 S promoter specific primers. The transformation percentage of the clover cultivars Serw 1 and Helaly was 56.2 and 28 % respectively. The gus gene expression can be detected only in the transgenic plants. The developed protocol for regeneration of transgenic plants in the present study is repeatable and can be used to regenerate transgenic clover plants expressing the genes present in A. tumifaciens binary vectors.