How to select homozygous transformants? - Transgenic Arabidopsis (Sep/08/2008 )
Hi!
Recently I transformed Arabidopsis with GFP fusion protein construct. I am selecting the transformants from T1 seeds. They are supposed to be heterozygous. I can use PCR or GFP phenotype to select them. But how can I get homozygous seeds from those heterozygous?
I do not think end-point PCR works.
Is there a easy way to identify the HM seeds?
Thank you!
Recently I transformed Arabidopsis with GFP fusion protein construct. I am selecting the transformants from T1 seeds. They are supposed to be heterozygous. I can use PCR or GFP phenotype to select them. But how can I get homozygous seeds from those heterozygous?
I do not think end-point PCR works.
Is there a easy way to identify the HM seeds?
Thank you!
typically I use the resistance marker in selecting for homozygous seeds - you have to grow the seeds two more generations until all of the seeds on a plate are resistant for your marker. You can speed up the process by picking individual brown siliques off the plants before the entire plant is completely dried.
Even though smu2 is correct, you can also try semi quantitative western blots. Use leaves from 10d old plants and do blots using a-GFP antibodies. You MUST do an equal loading control. After the blot, you will find that some lanes have a stronger signal (two fold), those are your homozygous plants. The others are heterozygous. By doing this, you'll probably save two months!
Perhaps you can try iducing haploids in anther culture (MS without any hormones).
Or, better, treatment of anthers with 0,5% colchicine for 24 hours and then you can transfer them to MS to get dihaploids. After that you can test them with PCR, and positive plants should have two copies of your gene. I don't know much about A. thaliana, but it works this way with N. tabacum.
Another way - FISH. Yust check chromosomes under fluorescent microscope, and if you see two signals on two chromosomes - you have homozygous plant.
Third option - if you know where has your insert been... ummm... inserted 
(do you?) you can try PCR with primers specific for that region od A. thaliana genome, not GFP. If you ger one heavy (long, high Mr) band - you have homozygous plant, if you have one heavy and one light band, you have heterozygous plant. If you get only one light band, you don't have your gene in this plant. But I'm not sure if that would work... It should in theory 
. M.
Or, better, treatment of anthers with 0,5% colchicine for 24 hours and then you can transfer them to MS to get dihaploids. After that you can test them with PCR, and positive plants should have two copies of your gene. I don't know much about A. thaliana, but it works this way with N. tabacum.
Another way - FISH. Yust check chromosomes under fluorescent microscope, and if you see two signals on two chromosomes - you have homozygous plant.
Third option - if you know where has your insert been... ummm... inserted
(do you?) you can try PCR with primers specific for that region od A. thaliana genome, not GFP. If you ger one heavy (long, high Mr) band - you have homozygous plant, if you have one heavy and one light band, you have heterozygous plant. If you get only one light band, you don't have your gene in this plant. But I'm not sure if that would work... It should in theory . M.Thanks man!
But it seems to be random insertion. So I may not use the PCR method. Anyway, thank you guys a lot. and I am still working on the selection of heterozygous plants. Plants grow slowly after hygromycin treatment. I also posted a new topic about fast selection of transgenic plants. If interested, write a line on the topic.
