Genetic Transformation Of Glyphosate Resistance Gene And The Identification Of Transgenic Plants In Brassica Napus

Glyphosate is a broad-spectrum herbicide used to control weeds, especially annual broadleaf weeds and grasses known to compete with commercial crops. It is found to be low toxic to human and animals and easy to decompose in soil. The resistance against glyphosate has become one of the breeding goals in Brassica napus.. In this study, a leading cultivar of B. napus (Zheda619) was used as an experimental material. In this investigation, we establish the efficient transformation and regeneration system using rapeseed hypocotyl and cotyledonary petioles. Tentative transgenic plants were selected by using PCR analysis. The main results are as follows:1. Based on the previous results of our lab, we further investigated the following parameters i.e. age of seedlings, glyphosate and hormones concentrations for the regeneration of shoots and roots. Results showed that6-7day-old cotyledonal petioles and8day-old hypocotyls had higher regeneration frequency. Glyphosate at13.0mg/L significantly reduced the shoot regeneration percentage and it considered to be a suitable dose for the selection of cotyledonal petioles and hypocotyls. Furthermore, at root induction stage there was no difference in the inhibition of root induction of MS (full or half strength) media supplemented with glyphosate. Higher root induction rate was observed in MS media with glyphosate compared to1/2MS media.2. The Agrobacterium-medlated transformation method required antibacterial agents to control its growth at callus induction stage. For this purpose, we used timentin (250mg/L) as a bacteriostatic agent that was also helpful to promote the shoot regeneration. In precultivation stage, the browning rate of explants significantly decreased with the extension of precultivation time, but frequency of callus and shoot regeneration was reduced. To overcome this problem we found that3days precultivation was the best precultivation time to reduce browning process and also promoted the shoot regeneration effectively.3. We also found that Agrobucterium-mediated transformation using the hypocotyl as an explant was performed better than using cotyledonary petioles. Moreover, infection time (5min) could reduce the harm of Agrobcterium and increase the differentiation of resistant shoots. The PCR analysis showed the transformed plants regenerated from the hypocotyl of rapeseed cultivar Zheda619.