Agrobacterium Mediated Genetic Transformation of Carrot (Daucus carota) with a Tobacco Osmotin Gene

Lack of water is a problem that affects the entire the Middle East, where most countries are unable to meet demands. And by 2050, per capita water availability is expected to fall by half. Genetic engineering could be one of many approaches to solve this problem. Carrot (Daucus carota) is a major vegetable crop and is also used as an animal feed, with a world production of 23.3 million megatons. Carrots and parsnips (Pastinaca sativa) are the only umbellifers of international repute as root crops. Enhanced carrot drought tolerance and disease resistance might have a large economic impact on the Middle East and the world in general. Osmotin and osmotin-like proteins are stress proteins belonging to the plant PR-5 group of proteins induced in several plant species in response to various types of biotic and abiotic stresses. It was named Osmotin due to its induction by low water potential of the growth medium and a correspondence between the level of osmotin protein produced and the degree of osmotic stress. Transgenic strawberry, cotton and mulberry expressing the tobacco-osmotin gene showed improved drought tolerance and disease resistance. We transformed carrot plants with a tobacco-osmotin gene under the control of CaMV 35S promoter using an Agrobacterium mediated transformation technique. Seven day-old hypocotyls were grown on MS medium for six weeks before being inoculated with Agrobacterium. Then, the transformed callus was kept on MS medium that had Kanamycin as a selectable marker and Cefotaxime to prevent bacteria overgrowth.  After three months, regenerated plants were transferred to soil under growth chamber conditions. PCR using osmotin gene-specific primers was conducted on DNA extracted from tissue of putative transgenic carrot plants.  Positive results suggest that the gene has been integrated into the carrot genome, and confirmation by southern blot hybridization is currently underway.