Efficiency of direct injection and co-cultivation methods in genetic transformation of oilseed rape (Brassica napus L.) seedlings leaves by Agrobacterium rhizogenes ATCC15834

Aims:The study was aimed to investigate the efficiency of direct and co-cultivation methods in the genetic transformation of Brassica napus at the molecular level. Methodology and results:The seedlings' leaves were prepared from explants parts of oilseed rape and inoculated with a culture of A. rhizogenes ATCC15834 using direct injection and co-cultivation methods.Cultures of hairy roots were created, then were aggregated and placed on a solid MS medium and sub-cultured by transferring them 3-4 times on an MS solid medium containing graded concentrations (100, 200 and 250 mg/L) of cefotaxime until obtaining hairy root cultures free of bacteria.Moreover, the DNA was extracted and amplified from each type of hairy root by polymerase chain reaction (PCR).The results show that the process of the inoculation of the leaves by direct injection with the Agrobacterium rhizogenes ATCC15834 suspension at the density of 1.151 10 8 cells/mL was the most efficient way for the formation of hairy roots compared with the other densities after 30 days with 80.64% ratio and of 2.72 roots/segment average.Additionally, hairy roots were also formed on oilseed rape leaves by incubating them with co-cultivation and bacterial A. rhizogenes ATCC15834 at a density of 1.151 10 8 cells/mL for 4 h, which shows their superiority from the other incubation periods, recording 44.44% and 2.55 average.Gel electrophoresis results demonstrate that one band was amplified by PCR with a molecular weight of 590 bp, identical to the estimated size of rolC gene recovered from the genomic DNA of hairy roots using specific primers.Conclusion, significance and impact of study: It is concluded that hairy roots can be produced from oilseed rape B. napus L. seedling leaves using direct injection and co-cultivation methods by A. rhizogenes ATCC15834.