Integration of transgene is  random and all T0 plants regenerated from independent event will probably have gene integration at different locus. The T-DNA integrated in plants genome will be same but due to integration at different locus, all plants will not have same Southern blot profile. In T0,  transgene remain in the hemizygous condition and upon selfing T0 plants, the transgene will segregate and T1 plants exhibit phenotypic ratio of 3:1 (3 with transgene phenotype and 1 without transgene phenotype) and genotypic ratio 1:2:1 (1 transgene in homozygous, 2 transgene in hemizygous condition and 1 without transgene). Expression level of transgene will vary and again it depends on locus where gene is integrated (transcriptionally active or inactive domain of genome).

In general:

1. If you have a single-copy transgene insertion for a T0 line, the hemizygous T0 line is A/- (for example), then T1 progeny can have 3 kinds of genotypes: A/A, A/- and -/-.

2. The Southern analysis band pattern from A/A and A/- plants should be the same.

3. If the A allele is a selectable marker gene (such as kan selectable marker gene), and if no gene silencing occurs, A/A and A/- plants should survive the selection of antibiotic kanamycin.

4. Due to 'dosage effect', A/A (homozygous) transgenic line can have a stronger expression level of the gene than A/- (heterozygous) transgenic line does. This has been well documented.

5. If your T0 line has = or > 2 copies of transgenes integrating in different chromosomes, your Southern analysis patterns for T1 progeny may be different depending on the segregation results of the transgenic loci.

The progeny of the single copy independent T0 transgenic event will give the same southern pattern after selfing in T1, which will be genotypically identical in terms of the transgene integration event but there will be segregation of 3:1. One T0 plant is the independent event of integration which will differ from the other T0 plants. Selfing of one particular event will give same pattern if stable transformation and integration of the single copy transgene is there.

Thanks for the valuable inputs. So if I understand correctly, my T1 progeny lines should have a similar southern pattern if my T0 was a single copy insertion? Also, can the difference between homozygous and heterozygous lines show up in a Southern?

Hi Madhurima,

To your zygosity/Southern question:

1. If you have a single-copy transgene insertion for a T0 line (A/-, hemizygous), then T1 progeny derived from this T0 line will have 3 kinds of genotypes: A/A (homozygous), A/- (heterozygous) and -/-.

2. The Southern analysis should show: 1 band for A/A lines, 1 band for A/- line, 0 band for -/- lines. The sizes of the bands from A/A and A/- lines should be the same on the x-ray film.

3. The intensity of the band from A/A lines should be higher ('darker') than that from A/- lines, because the homologous lines have 2 copies of the 'A' allele. However, sometime it is hard to distinguish them from the radioautography. You may need to do a 'progeny test' to see the segregation ratio to determine which one is homozygous or heterozygous. Alternatively, you can assess transgene zygosity using quantitative PCR (qPCR ).

>>So if I understand correctly, my T1 progeny lines should have a similar southern pattern if my T0 was a single copy insertion?

Here if your T1 lines originating from different T0 (single copy) lines, the pattern will be different between different lines. For example, T0-line 1, T0-line2, and T0-line(n) will show different patterns in T1-line 1, T1-line2, and T1-line(n) because they are 3 independent lines.  But 3 different clones within T0-line1 will show same pattern.

YOUR understanding that : T1 progeny lines should have a similar southern pattern if my T0 was a single copy insertion.

the pattern (size) of the signals observed in southern blots generally differs between two independent events, as DNA integration is random. This difference can only be observed depending on the restriction digestion profile you used. For suppose, if you digest the DNA with an enzyme that releases the gene of your interest, then the signal pattern will be same in all the different independent transformants.

if you digest the genomic DNA with an enzyme that linearizes (for this you need to have the restriction map of your plant expression unit), then you will get signals of different sizes... So, called independent transformants.

Hope you understood

Regards