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N.Matvieieva, A.Shachovsky, O.Kvasko, N.Kuchuk

High frequency genetic transformation of Cichorium intybus L. using nptII gene as a selective marker


TSitologiia i genetika

Cichorium intybus L. is an important vegetable crop used as salad (leaf form) and for the production of coffee substitutes (root form). At the same time these plants also can be used in biotechnologies for synthesis of pharmaceutical proteins. Here we report the possibility of high frequency genetic transformation of C. intybus L. Agrobacterium rhizogenes or A. tumefaciens- mediated transformation were used for construction of transgenic chicory hairy roots and plants. Plasmids used contained target human interferon ifn-α2b gene, Mycobacterium tuberculosis ESAT6:Ag85B antigene esxA::fbpBΔTMD fused gene and human telomerase reverse transcriptase hTert gene. Using of nptII gene as a selective one was preferable to the bar gene for chicory. In this case the frequency of transgenic plants or hairy roots formation was significantly higher. Cultivation of explants on the medium with Basta in concentration 1-2 mg/l have led to plants death or to significant reduction of number of shoots formed. Frequency of hairy roots formation varied from 5,9 to 42,3% after A. rhizogenes-mediated transformation. Frequency of regeneration of transgenic plants varied from 10 to 86% after A. tumefaciens-mediated transformation. Both A. rhizogenes and A. tumefaciens- mediated transformation frequency depended on the type of explants, roots or cotyledons, and vector used. Usage of A. tumefaciens carried pCB063 plasmid (target esxA::fbpBΔTMD fused gene and nptII selective gene results in the most effective regeneration of transgenic plants with regeneration frequency up to 86%. In the case of chicory A.rhizogenes-mediated transformation the highest regeneration frequency up to 42,3% was demonstrated using pCB161 vector with ifn-α2b target gene and nptII selective gene.

Key words: Cichorium intybus L., Agrobacterium-mediated transformation, nptII, bar, human ifn-α2b gene, esxA::fbpBΔTMD gene


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