Matvieieva N, Shakhovsky A, Kvasko O, Kuchuk N
Tsitol Genet. 2015 Jul-Aug;49(4):11-6.
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 can also be used in biotechnologies for synthesis of pharmaceutical proteins. Here we report the possibility of high frequency Agrobacterium rhizogenes- or A. tumefaciens-mediated transformation of C. intybus L. for construction of transgenic "hairy" roots and plants. The used plasmids contained target human interferonifn-α2b gene, Mycobacterium tuberculosis ESAT6:Ag85B antigene esxA::fbpB(ΔTMD) fused gene and human telomerase reverse transcriptase h Tert 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 carrying pCB064 plasmid (target esxA:fbpB(ΔTMD) fused gene and nptII selective gene) resulted 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 p CB161 vector with ifn-α2b target gene and nptII selective gene.
菊苣是一种重要的蔬菜作物，其叶片可用于制作沙拉，根可用于生产咖啡替代品。同时，这些植物还可用于生物技术领域，用于合成药用蛋白质。在此，我们报道了通过发根农杆菌或根癌农杆菌介导的高频转化菊苣，以构建转基因“毛状”根和植株的可能性。所使用的质粒包含目标人干扰素α2b基因、结核分枝杆菌ESAT6:Ag85B抗原esxA::fbpB(ΔTMD)融合基因和人端粒酶逆转录酶h Tert基因。对于菊苣而言，使用nptII基因作为选择基因比bar基因更合适。在这种情况下，转基因植株或“毛状”根的形成频率显著更高。在含有浓度为1-2 mg/l的草铵膦的培养基上培养外植体，会导致植株死亡或形成的芽数量显著减少。发根农杆菌介导的转化后，“毛状”根的形成频率在5.9%至42.3%之间。根癌农杆菌介导的转化后，转基因植株的再生频率在10%至86%之间。发根农杆菌和根癌农杆菌介导的转化频率均取决于外植体的类型（根或子叶）以及所使用的载体。使用携带pCB064质粒（目标esxA:fbpB(ΔTMD)融合基因和nptII选择基因）的根癌农杆菌，可使转基因植株的再生最为有效，再生频率高达86%。在菊苣发根农杆菌介导的转化中，使用带有ifn-α2b目标基因和nptII选择基因的p CB161载体，显示出最高再生频率，可达42.3%。
