Wild type Agrobacterium Ti plasmid cannot be used as gene cloning vectors because of
1) Large size of Ti plasmid; difficult to handle
2) presence of oncogenes or tumor causing genes (auxin and cytokinin)
3) lack of unique restriction sites and marker site within T-DNA
There ate two genetically engineered Ti plasmid based vectors. They are Co-integrate vectors and binary vectors.
Making of Co-integrate vectors
In this strategy, both the T-DNA with our gene of interest and vir region are present in the same vector used for transformation.
At first; an intermediate vector is made using E.coli plasmid + vir region + T-DNA borders + origin of replication+pBR 322 sequences.
Second vector is a disarmed pTi vector = gene of interest+ some markers+pBR 322 sequences.
Both intermediate vector and disarmed pTi has some sequences in common (pBR 322 sequences).
Therefore by homologous recombination, co-integration of two plasmids will take place within Agrobacterium.
Now we have a cointegrate vector that has both T-DNA with our gene of interest with in the T-DNA borders and vir region. This complete vector is used for transformation eg:pGV2260.
|Transformation of tobacco plant using Cointergrate vector|
Binary vector strategy: Two vector strategy
Here two vectors are used. This vector was devised based on the knowledge that vir region need not be in the same plasmid along with T-DNA for T DNA transfer.
|Mini-Ti or micro Ti plasmid|
Binary vector consists of a pair of plasmids
1) A disarmed Ti plsmid: This plasmid has T-DNA with gene of interest + ori for both E.coli and Agrobacterium. Also called as mini-Ti or micro Ti plasmid eg: Bin 19
2) Helper Ti plasmid has virulence region that mediates transfer of T-DNA in micro Ti plasmid to the plant.