Genetic Transformation Of EPSPs To Rape (Brassica Napus L.) Mediated By Agrobacterium Tumefaciens

In my thesis, restorer line of "SHUZHA No.9"which is a new sort of hybrid rape(brassica napus) is used as my experimental material .The factors which influence the effect of the transformation are discussed, including the factors like pre-culture, seed age, concentration of hormone, etc. The plants are transformed by Agrobacterium tumefaciens-mediation method, and during the induction and differentiation, some of them are proved by PCR and Southern-blotting. Some transgenic plants and their offspring show high degree of herbicide- resistance and are fertile. The details are shown as that followed:1. An optimized system of re-generation is formed, through comparing and observing the different situations of the induction and differentiation of the explant. Experimentation shows the following results: The differentiation frequency of hypocotyl is higher than that of cotyledon; explant with age of 8 days has a rather high rate of inducing Callus; pre-culture will increase the differentiation frequency of hypocotyl; the differntiation frequency of bud is the highest when MS+3mg/L6-BA; AgNO₃ can increase the differentiation frequency of hypocotyl; increasing the density of agar and decreasing that of sugar can decrease the phenomenon of vitrification.2. Agrobacterium tumefaciens which contains the vector p35S-2300-EPSPs is used to transform the plants of rape(Brassica napus). The condition of transforming is also optimized, and the best conditions of culture media are conformed as the followings:â…  medium of shooting, MS;â…¡ medium of pre-culture, MS+0.2mg/L6-BA+0.5mg/L2,4-D+6mg/LAgNO3+100mg/LAs;III medium of co-culture, MS+0.2mg/L6-BA +0.5mg/L2,4-D+6mg/L AgNO3+100mg/LAs;IV medium of inducing callus, MS+3mg/L6-BA+0.5mg/L2,4-D+6mg/L AgNO3 +600mg/L carb;V medium of differentiation, MS+3mg/L6-BA+6mg/LAgNO3+600mg/L carb+ 20mg/Lkan;VI medium of rooting, l/2MS+0.2mg/LNAA+300mg/Lcef3. Resistant seedlings are obtained after being selected by kan and glyphosate, and are elementarily proved to have EPSPs gene via PCR test.4. Three of the four positive plants proved by PCR are confirmed as transformed plants by Southern-blotting.5. In order to test the herbicide-resistance of transformed plants, the experiment has been done that comparing the leaves of the transformed plants(To) which have been daubed with Glyphosate with the non-transformed plants daubed with Glyphosate. Some transgenic plants show high herbicide-resistance. These transformed plants are planted in the experimental field after being trained and then they show fertile.6. Twenty seeds of transgenic plants(Ti) gained by self-mating are chosen randomly, are sowed into pots, and then are tested by 3g/kg glyphosate, compared with non-transgenic plants which have been sowed in the same time. The results show that non-transgenic plants become withered and then all dead; but transgenic plants still grow well.7. All the seeds gained by self-mating are sowed into experimental field. 2g/kg Glyphosate is used to test the herbicide-resistance of the plants. However, the results show that there are few plants have herbicide-resistance, and they don't match the Mendel Law.