Lab 07 - tobacco transformation

Lab 07 - tobacco transformation - At this point you should...

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Lab 7 Tobacco Transformation 1. Start Agrobacterium culture to be used from a glycerol stock. Grow up overnight at 28 ° C, under the proper selection. We will do this for you. 2. Tobacco has been grown aseptically. On the next day cut the leaves off the tobacco plantlets as in the previous lab. In general, larger leaf pieces regenerate more readily than small ones. Explants will regenerate from cut edges or wounded areas. Place the leaf pieces onto DBI medium. Put 6 pieces per dish, right side up. 3. Spin down the Agrobacterium culture. Resuspend the pellet in ½ volume of liquid MS medium (hormone-free). 4. Use a pipette to gently inoculate the tobacco tissue – place one drop of the culture at the base of each leaf piece so that capillary action will draw it between tissue and medium. 5. Incubate in low light for 24-48 hours.
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Unformatted text preview: At this point you should see a little cloudy bacteria around the leaf edges. Bacteria should not be growing over the top of the tissue; if it is you used too much inoculum. 6. Transfer tissue to selective medium (DBI with 200mg/l cefotaxime and 50mg/L kanamycin) in 20x100 mm plates. Put plates under lights in growth room. (We will do this for you.) 7. Expect to see shoots after about 10 days. Excise shoots when they are large enough to handle. 8. Test shoots for GUS activity: slice a thin section off the bottom of the stem and submerge the section in GUS (x-gluc) stain at 37 C for 4-5 hour or overnight. 9. Transfer shoots positive for GUS activity to rooting medium. DBI media (per Liter) MS salts 4.3g Thiamine 4mg Myo-inositol 100mg Sucrose 30g IAA 1mg Kinetin 2mg pH 5.6 Agar 8g 30...
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