Optimization Of Agrobacterium-mediated Transformation System Of Cotton And Defense Gene Transfer

In this study chitinase and β-1,3-glucanase were transferred into Gossypium hirszrtum CCRI 24 by Agrobacterium tumefaciens .The main results as follows:1.The time when the explant can be obtained in genetic transformation. Asepsis treated seedlings were wiped off their seed coats,and then sterilized by 0.1%Hg for 5 min . After washed 3 to 5 times by sterilized water ,they had been grown on MS1 culture medium for 7 days. Then they were easily infected by Agrobacterium and induced calli.2.Ascertain of the screening pressure of antibiotics .Through concentration grading experiment, 75mg/Lwas regarded as the screening pressure of resistance-callus.3.The aptimal concentration and infection time when Agrobacterium infects explant .When the Agrobacterium that had entered log-growth period was used to infect hypocotyl, the induction rate of resistance-callus was about 40%.4.Ascertain of the time of co-culture . Infected explant had been cultured for 48h on the MS2 culture medium that had been added selective antibiotics and antibiotic for microbe-inhibition so as to induce resistance-callus .In this way, the explant could be fully infected by Agrobacterium and also the over-growth Agrobacterium would not poison the explant and inhibit the growth of calli .5.Optimum of culture medium.① Callus-inducing medium: MS1+IAA,Ktand 2,4-D each 0.1 mg/L,pH=6.0； ② Embryo-callus inducing medium: MS1+ IAA and KT,IAA/KT=1/4,pH=6.5；③ Embryogenic bud and regenerated seeding inducing medium: MS1+IAA,6-BA each 0.5mg/L, Asp,Gln each 1g/L ,active carbon 1.5g/L,pH=6.2 .6.A totel of 9 transgenic plant had been gaind. By PCR detection ,the foreign genes had been integrated in the genome of Gossypium hirsutum CCRI 24.7. Using this optimized transformation system, we obtained four transgenic plants into which the sigle GO gene was transferred.and the detection by PCR method showed that the target gene has been inserted into the genome of CCRI 394.