3 replies to this topic

[theen]

Hi all,

I transformed Arabidopsis with a binary construct via agrobacterium mediated in planta method. I collected the seeds and grown on Kanamycin and selected the transgeic plants (T1). I n order to make homozygous plants , I selected seeds again on Kanamycin from T1 plants. 2 lines are showing both resistant and susceptible plants, indicating presence of homozygous/heterozygous and escapes. But the remaining 2 lines, all the plants are kanamycin resistant. How could this happen? is there any possibility of getting homozygous in T2 itself?? Any help or suggestion in this regard will be highly appreciated

Edited by theen, 23 March 2009 - 03:54 AM.

[theen]

theen, on Mar 23 2009, 04:54 AM, said:

Hi all,

I transformed Arabidopsis with a binary construct via agrobacterium mediated in planta method. I collected the seeds and grown on Kanamycin and selected the transgeic plants (T1). I n order to make homozygous plants , I selected seeds again on Kanamycin from T1 plants. 2 lines are showing both resistant and susceptible plants, indicating presence of homozygous/heterozygous and escapes. But the remaining 2 lines, all the plants are kanamycin resistant. How could this happen? is there any possibility of getting homozygous in T2 itself?? Any help or suggestion in this regard will be highly appreciated

Is this possible if the the transgene has multiple insertions in the plant genome??

[bob1]

Insertion in such a way that there is complete penetrance (i.e dominance).

[suhasbn]

theen, on Mar 23 2009, 04:54 AM, said:

Hi all,

I transformed Arabidopsis with a binary construct via agrobacterium mediated in planta method. I collected the seeds and grown on Kanamycin and selected the transgeic plants (T1). I n order to make homozygous plants , I selected seeds again on Kanamycin from T1 plants. 2 lines are showing both resistant and susceptible plants, indicating presence of homozygous/heterozygous and escapes. But the remaining 2 lines, all the plants are kanamycin resistant. How could this happen? is there any possibility of getting homozygous in T2 itself?? Any help or suggestion in this regard will be highly appreciated

Hi Theen,

You are right. There is possibility of having multiple inserts. Although the Agro-mediated transformation is belived to yield transformants with single inserts, its been observed that on an average the insertion copy number could be 1.5 to 3. In such cases pick up the transgenic lines which showed you both resistant and susceptible phenotypes (3 resistant : 1 Susceptible) as their genetic constitution could be (1RR: 2Rr: 1rr), and an indication that these might have single inserts. But again you need to confirm them by further experiments.

regards