To study the function of my gene. I have cloned my gene in PBi vector and then i have moved the vector to Agrobacterium tumefaciens strain EHA 105. now I am thinking to proceed for tobacco transformation to study the function of gene.
Please guide me.
Thanks and regards
Hi Harshraj,
Depending on your research goal, there are 2 choices for doing tobacco transformation.
1. Transient assay by using Agroinfiltration. You need tobacco plants, but they don't need to be sterilized. You can just inject the Agrobacterium (contains your binary vector) solution into plants' leaves, and after 2-3 days, you can isolate recombinant proteins (just one example) from the injected areas for assay.
2. Stable transformation, by dipping sterile leaf discs (from tissue-cultured plants) into Agrobacterium solution for transformation, and then select these infected leaf discs on medium with proper antibiotics. Transgenic shoots surviving selection will come out from the leaf discs. Cut those shoots and stick them into 'rooting' medium. Then you will have those 'transgenic' plants. They are called 'putative' transgenic, because you still need to confirm it using assays such as Southern analysis.
Hi Harshraj,
Depending on your research goal, there are 2 choices for doing tobacco transformation.
1. Transient assay by using Agroinfiltration. You need tobacco plants, but they don't need to be sterilized. You can just inject the Agrobacterium (contains your binary vector) solution into plants' leaves, and after 2-3 days, you can isolate recombinant proteins (just one example) from the injected areas for assay.
2. Stable transformation, by dipping sterile leaf discs (from tissue-cultured plants) into Agrobacterium solution for transformation, and then select these infected leaf discs on medium with proper antibiotics. Transgenic shoots surviving selection will come out from the leaf discs. Cut those shoots and stick them into 'rooting' medium. Then you will have those 'transgenic' plants. They are called 'putative' transgenic, because you still need to confirm it using assays such as Southern analysis.
hello guys,
I am also interested in the transient expression of the protein but little bit confused in the choice of experimental material like what will be more good Onion (epidermal cells) or Tobacco plants?
Please Guide me also.
Thank you
Hi Dear
Very recently I did transient expression of a promoter using agroinfiltration (using needleless syringe). To get quick expression of your gene, you can use tobacco especially Nicotiana benthamiana because N. benthaminana is more prominent to agroinfiltration. As a refernec you can read SPARKES, 2006 (Nat Protoc. 2006;1(4):2019-25. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants.
Sparkes IA1, Runions J, Kearns A, Hawes C.)
aLL THE BEST
Thank you Yuan-Yeu Yau and Chet Ram for your answer. I want to study both stable and transient expression.
@ Ambika and Harshraj,
1. Onion (epidermal cells) is usually used for bombardment. Besides, onion is monocot, may not efficient for agro-transformation. People just bombard naked DNA (plasmids) into onion epidermal cells. Depending on your research goals, you can study localization of your gene-of-interest in the cells by seeing GUS or GFP expression when you fuse your gene with GUS or GFP (see attachments #1 and #2). This is just one example. There are different applications.
2. In the attached picture #2, the legend is:
"Mitochondrial localization of OsPHB1 protein in onion epidermal cells. Constructs carrying 35S::GFP (A–C), 35S::OsPHB1-GFP (D–F), or 35S::GFP-OsPHB1 (G–I) were bombarded into onion skin epidermal cells. Expression and localization of GFP signal was observed 12 h after bombardment. Mitochondria were specifically stained with MitoTracker Red (A, D, and G). C, F, and I, The merged images of A and B, D and E, and G and H, respectively."
3. You can use "tobacco plants" for Agroinfiltration, but again, it depends on your research goals (see attachment #3).
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