Floral dip and fluoresent maker, very simple transformation Print
Written by nguyen   
Friday, 01 May 2009 17:16

Floral dip of Agrobacterium mediated transformation combine with fluorescent marker gene, a very simple method which any lab can do for plant transformation and it is easy than ever. Below is step by step:

1. Growth plant for transformation, 4 plants per pot and two pots are enough to get 50-100 transgenic seeds

 

 

2. As plant have flower cut main first flower make plant have more flower and waiting until ready for transformation

 

 

3. Depend your pot size, use the plastic or paper cup to hold Argrobacterium solution for dip flower in

 

 

4. Dip all flower in the Argrobacterium solution in few seconds

 

 

A kid at 8 years old can do that too

 

 

5. Lay down pots in the tissue paper to absorb transformation solution and put a glass cover tray to keep humidity

 

 

6. After overnight lay down put the pots stand up in the tray

 

 

7. Repeat step 4-6 after 4-5 days of previous transformation, do two or three transformations

 

 

8. Water plant until the seed almost matured and let them dry

 

 

9. Use tray with net that small enough for seed go through

 

 

10. Depending the fluorescent of gene use filter and light correspond with it, for DsRed gene use red filter and green light to collect the fluorescent seed

 

 

11. Because the Arabidopsis is too small, can use microscope and attached filter and flash light as the picture to get the transgenic seed easily.

 

 

12. Through microscope you can see the red seed as below

 

 

13. Three different genes yellow, green and red shown as picture below

 

 

14. At T2 plants you can see a segregation of red seed and WT in the silique with ratio 3 to 1 if one copy of gene inserted or multi copy inserted but at same loci

 

 

See more picture here

 

References:

Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.. Plant J. 1998 Dec;16(6):735-43.

 

Last Updated on Wednesday, 24 February 2010 21:31