how to identify a hemizygous indiviual?

Delta-Notch

wrote...

13 years ago

After I integrated a transgen into Arabidopsis I would get a hemizygous plant, right?

Now I want to have a homozygous plant.
Therefore I have to cross the plant with itself?
The result might be: 25% no transgen
50% hemizygous
25% homozygous for the transgene

What experiment would you prefer to differ between homozygous and hemizygous plant?
- RFLP?
-TILLING?
- PCR melting curve?

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duddy

wrote...

Staff Member

13 years ago

hmm, lemmi think...

Okay, arabidopsis thaliana is a diploid with a small number of chromosomes (five). If you transform a transgene into it, why would you get a hemizygous plant? Hemizygous zygous means when you're neither heterozygous or homozyous... for example, a male with a single allele on his X chromosome is hemizygous for that gene because there isn't a second allele to determine whether he his homo or heterozyous. Where are you interesting this gene, which chromosome?

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duddy

wrote...

Staff Member

13 years ago

In addition, is this the protocol you are using.. I've personally never transformed a gene to a plant (only bacteria) so im not 100% sure...

http://entomology.wisc.edu/~afb/protocol.html

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Delta-Notch Author

wrote...

13 years ago

I mean "hemizygous" referring to the transgene. If there is a transgene integrated into 1 of the homologous chromosome, the plant will be hemizygous to that new "(trans)gene". Did I understand correctly?

This could be a protocol, but I haven't done such an experiment, yet. I am just interested in the theory.

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duddy

wrote...

Staff Member

13 years ago

Quote from: Delta-Notch (13 years ago)

This could be a protocol, but I haven't done such an experiment, yet. I am just interested in the theory.

Oh I see. Well, after you insert the gene, the organism becomes hemizygous for the allele. But if you cross the plant with itself, that gene won't be passed on to the progeny because in actuality, you are inserting the gene into an autosomal cell (not a sex cell). If you insert it into a sex cell, then it will get passed on. Remember, we are dealing with a multicellular organism and not a single-celled bacterium.

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Delta-Notch Author

wrote...

13 years ago

Sorry, I didn't pay attention to this detail in the protocol.

The experiment is usually done y ti-plasmid transformation. Therefore you transform only one single
cell from the plant. Afterwards you grow a new (now transgenic) plant out of that cell.

http://www.shikshasetu.in/engineering/biotech/articles/images/image1.jpg

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