Agrobacterium tumefaciens 33970 mediated transformation of medicinal plant - Agrobacterium tumefaciens 33970 (Nov/14/2006 )

Res sir,
i have done genetic transformation of some medicinal plant with wild strain of Agrobacterium tumefaciens 33970.i have got the crown gall growth.
PCR confirmation of the insert
RT-PCR confirmation of the insert
DNA fingerprinting using AFLP technique
DNA fingerprinting with RAPD and development of SCAR markers
TAIL PCR for confirmation of the introduced DNA fragment
RACE PCR for identification
please suggest the most suitable molecular techniques for the charecterization of transformed tissue.
please write in detail bcoz i am a very new researcher of this field
regards
rinki

I didnt get the idea of what u mean by characterization of the tissue
did u mean that u have to confirm it is transformed ?
then that will depend on the insert gene and its product

for example if its a protease enzyme u can do assay tests , along with controls of course


what si the plasmid that u used ? didnt it have any reporter gene ? or selectable marker?
u can use that to select the transformed tissue and confirm it

also doing pcr studies only confirms the gene presence, not the quanitity.
u will have to se how much ur transgene is epxressed too

try doing southern for dna--
northern for RNA --if ur looking for protein expression after transformation
and western for protein ,

hope it has helped u a bit
regards

dear sir
thanks for replying my e mail request .
i am getting the tumrous growth.
so i think there should be the integration of following genes
tms1
tms2
tmr
nos nopaline synthase
how should i design the primers .
what universal primers i can use.
i want send u some papers
can i send them in the attachments.
please let me know
regards
rinki

Dear rinki

if ur checking for the genes in the tumour tissue
u can check either by pcr after DNA isolation or do southern
for pcr u can design primers accroding to the gene sequence( more specific ) do u have the gene sequences? if u have the gene sequence of the Ti plasmid u can get the coding regions and then design from them both forward and reverse.

for southern , u can isolate the plasmid , try to find out a restriction site that cuts the genes and elute that region from the gel and use as probe , u can also use random oligo primers . u can try and search if they are available commerically , try for plant tissues , but the result will be more specific if u use homologous probes.

for nopaline synthase u can also try RNA isolation and RT PCR or northern , so that u can see the level of the RNA and u can say that gene is there and functioning .

also if u have antibody aganist nopaline synthase u can get total protein extracted from the tissue and try western , if protein is there and detected by western u can say ur gene is there and functioning properly , do western first then if nothing happens do RNA or if u dont have antibody do RNA

are u only studying the tumour cells or are u putting any other insert gene? if so finding its presence will be better

send the papers in my PM or attachments here

glad i could help somewhat though whati am doing is very different from ur doing , hence dont know how much i can help you

regards
laxmi

What you need might be confirmation of successful transformation I think, so you might use following:

Classic confirmation of transformation: Southern hybridization
Rapid identification of transformation: PCR
Or nopaline synthase assay (transgenic product)

Other might not necessary

dear rinki

both methods are good pcr and non-pcr based
pcr method is for quick confirmation
and southern is for quantifying how much of ur gene is there in the transformed tissue

if u have time and the resources do both

i would suggest southern since as rye said its classic confirmation experiment
if ur submitting this data for publishing or to others , southern result would be acceptable

regards

laxmi

res mam , there is some problem regarding uploading the file through bioforum .
please can u send ur e mail id .
regards
rinki