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Abstract
As perquisite for the production of insect resistant clover plants, callus induction and plant regeneration capacity among five Egyptian clover cultivars were compared. The embryonic calli were formed within one week in presence of 1.5 mg / l2,4D. The embryonic calli were transferred to MS medium supplemented with 1.5 mg / lBA. The cultivar serw 1 exhibited higher regeneration frequency (22%) followed by Helaly (19%) while sakha 4 showed the least regeneration value (5%). To optimize the transformation method, petiole explants from cultivars serw 1 and helaly were transformed with gus gene using Agrobacterium-mediated gene delivery system. The stable integration of the gus gene was confirmed by PCR and dot blot analysis. Histochemical gus assay confirmed the stable expression of the gus gene. The transformation frequency was 56.2% and 28% for serw 1 and Helaly, respectively. In order to produce transgenic plants expressing cry1Ia5, serw 1, helaly and gimmeza 1 cultivars were used, the transformation percentage were 25.4%, 17.6% and 16%, respectively. PCR, southern blot analysis and RNA dot blot were used to confirm the stable integration and expression of the Cry1Ia5 gene into the plant genome. Four transgenic lines expressing the cry1Ia5 gene were bioassayed for resistance to second larval instar of spodoptera littoralis