Agrobacterium tumefaciens mediated transformation of chicory (Cichorium intybus L.) with Mytilus galloprovincialis foot protein type five (MGFP-5)

Abstract

Mytilus galloprovincialis foot protein type 5 (Mgfp-5) can be used as medical adhesive or in underwater environments due to its significant adhesive ability and biodegradable properties. To obtain sufficient Mgfp-5 for further study, genetic engineering has been abundantly used, while plant genetic engineering was rarely applied. This study reports an efficient protocol for the direct regeneration of chicory (Cichorium intybus L.), with a regeneration frequency of 79.18% in the case of inoculation on MS+1.5 mg/L 6-BA+0.2 mg/L NAA medium. The plant expression vector pRI101-Mgfp of the Mgfp-5 gene was transformed into chicory via Agrobacterium mediation. After plant selection and regeneration, regenerated plants with resistance were successfully obtained. The efficient transformation system of the Agrobacterium-mediated genetic transformation consists of leaf explants immersed in Agrobacterium at O.D.(600) of 0.6 for 10 min after being pre-cultured for 3 d, followed by co-culture for 3 d. Then, these explants were transferred to medium containing 50 mg/L kanamycin 1 selection power. As proved via PCR, RT-PCR, and Western blot analysis, the Mgfp-5 gene was integrated into the chicory genome, accompanied by normal transcript and expression, as well as stable genetic transformation and inheritance. These results underpin future research toward providing a feasible method for the production of Mgfp-5 protein.