co-cultivation period - (May/07/2007 )
hi
im using agrobacterium transformation.
wondering if duration of co-cultivation period would affect my plantlet (leaf and immature embryo)/
thanks in advance
Dear Heidi,
Even if extending your co-cultivation time seems a simple solution to obtain more transformants, I'm afraid it would effect your explants viability.
Keep in mind that you are forcing your explants to be contaminated, a condition that they naturally tent to avoid.
Co-colture affects differently explants depending on their sensitivity to that specifical strain of the pathogene.
Moreover, this is also a tissue-specific and species-specific response.
If you use co-cultures that lasts too long, you can also have problems in eliminating bacteria from further cultures.
Anyway, as you want your explants to be transformed by Agrobacterium, you have to reach a balance between your transformation efficiency and explants viability.
Try to expose your cultures to agrobacteria with different times of incubation.
I suggest you also to wash your cultures after co-culture with sterile water containing different concentrations of antibiotics to help agrobacteria elimination.
Good luck!
ILA
thanks you so much ila! i wasn aware of those points u mentioned.
btw I know that transformation efficiency is affected by species of plants transformed.
however does types of gene of interest transformed into plant cells affect transformation efficiency?
thanks again
I don't think that the type of gene would affect transformation efficiency...but let's start a step behind.
On one hand you have the efficiency of transformation
on the other hand you have the efficiency of interest gene expression.
These two effects are due to different genetic mechanisms.
In the first case, the efficiency of transformation depends from the response and susceptibility of your species/tissue to the Agrobacterium strain. A side consideration would be that - obviously - it's esasier that a small construct would integrate completely into the host genome, but we are not talking of the single gene of interest but of all your T-DNA.
In the second case, I believe that it depends mainly from the possibility of the promoter you choose for your gene to express in the host cells.
I hope I hit the point...
Cheers!
ILA
jus to clarify if i hav got u.....so what matters is the compatibility of gene insert n promoter rite?
and if the gene of interest is bigger, transformation might be lower cos its harder to get a bigger plasmid (T-DNA & vector) over the host.
thanks
referring to the duration of co-cultivation, will types of explant (eg leave, immature ,embryo flower) react differently under shorter or longer co-cultivation.
thanks!
and if the gene of interest is bigger, transformation might be lower cos its harder to get a bigger plasmid (T-DNA & vector) over the host.
thanks
The rest of the plasmid is designed to be expressed in the Agrobacterium. It includes genes implied in the infection mechanism, T-DNA targeting (vir genes, for example...), antibiotic resistance genes used to selct the bacterial strain and so on...
My consideration about T-DNA length was quite naive and sounded like this: a long sequence is subjected to more troubles than a short one during its transfer to host cells.
Yes, indeed!
The reaction of each explant is different, because it is composed of different cell types at different developmental stages. They have different features, so we can say that each one is characterized by its own susceptibility to a certain Agrobacterium strain.
Moreover they have different attitudes toward regeneration...
More-moreover, they are also more or less sensitive to antibiotics...
and so on...
Hope I was clear enough!
ILA
thanks ILA!