Generating single insertion homozygous tDNA lines in Arabidopsis.

When making transformants it is generally desirable to select for single insertion line and eventually homozygous line. You can get single insertion homozygous line from the T2.

 

1)      Perform your floral dipping

2)      Select your transformant (T1) on plate (kan or hygro) or soil if your are using Basta.

3)      Collect seeds (T2) from individual plants (do not pool, label well)

4)      Plate roughly 30 seeds (T2) from individual plants on selection media (MS with antibio or basta).

5)      Count the number of resistant seedlings. If using KAN do not count seeds that do not germinate count the green healthy seedling versus yellowish sick looking ones. Since your marker is dominant you should have ¾ resistant for a single insertion. Anything above that should be considered as multiple insertion and rejected.

6)      Transfer 8 resistant plants in soil, collect seeds (T3) from individual plants (do not pool, label your envelopes well to identify each plants).

7)      Plates 10 seeds from each plants on plates. The population that is 100% resistant comes from a homozygous parent. Bulk up seeds from this one (T3), your can also transfer your 100% resistant progeny to soil. You can pool the seeds from that generation (T4).