Comparing the efficiency of sonication assisted Agrobacterium -mediated and particle bombardment for the production of transgenic canola plants

In order to achieve higher transformation rate in canola plants, the efficiency of sonication assisted Agrobacterium mediated and particle bombardment was compared. The hypocotyls explants isolated from 4 days old seedlings of two canola cultivars (Serw-3 and Serw-4) were transformed using both methods. The explants were treated by sonication for different time (5,10,20 and 30 sec.) before co-cultivation with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector (PBI-121-gfp) that carried genes coding for kanamycin sulphate, and green fluorescence protein (gfp) under the genetic control of 35-S CMV promoter and nos terminator. The data indicate that 5 sec. sconication treatment is enough to get higher transformation rate (55 %). Based on our data, we could draw the following conclusions: the transformation frequency of the two tested canola cultivars after biolistic gun was 25% and 16%, while the sonication assisted Agrobacterium gave 33% and 55% for the cultivars Serw-4 and Serw-3, respectively. The stable integration of the T-DNA in the T0 transgenic plant genomes was confirmed by PCR and dot blot analyses. The PCR analysis using primers specific to the 35 S-promoter showed a clear band (250 bp) corresponding to the expected size of the 35-S promoter only in transgenic plants however; no such bands were seen in the non-transformed plants. The dot blot analysis data indicate that clear dots specifically hybridized with the probe prepared from the gfp gene. The data of the present study reveal that 5 sec. sonication followed by Agrobacterium transformation resulted in high frequency of transgenic shoot production comparing to biolistic mediated transformation. The improved transformation protocol reported here is repeatable and can replace the ordinary Agrobcaterium transformation method and could be used to express the genes of interest in economically important crop plants.