1. PLANT VECTORS REKHA PULICHERLA

2. Plasmids
A circular piece of DNA that is not a part of the bacteria’s genome. It is ‘extra-chromosomal.’
Analogy: The Plasmid is the little dog, and the Chromosomal DNA is the big dog. (The cell cannot live without the big dog however…)

3. Carrier for DNA during the recombinant DNA process.
Plasmid-piece of free-floating DNA in the cytoplasm of bacteria.
Double-stranded, circular molecules that replicate independently of the chromosome.

4. Common features of eukaryotic vectors include
Most vectors carry marker genes that allow the recognition of transformed cells
Others die due to the action of an antibiotic
They are called as selectable markers
Kanamycin resistance used for selection of transformants

5. The other features of vectors include
Multiple unique restriction sites
Bacterial origin of replication
Agrobacterium Ti plasmid is the most preferred as it has a wide range of host range and capacity to transfer genes due to T-DNA border sequences

6. Ti &Ri Plasmids Most commonly used vectors in plant systems
Ti plasmid is found in tumor inducing strains of A.tumefaciens
Ri plasmid is found in root inducing (hairy root disease) strains of A.rhizogenes
T-DNA is a part of both the plasmids

7. Ti Plasmids Ti plasmids have 4 regions in common
Region A comprising the T (transferred)-DNA responsible for tumor induction
Region B responsible for replication
Region C responsible for conjugation
Region D responsible for virulence called as vir (virulence) region

8. T-DNA Regions An onc (oncogenecity) region consisting of 3 genes-
tms1, tms2 (shooty locus)
tms2 (rooty locus)
This region is responsible for synthesis of 2 phytohormones namely
IAA (indole acetic acid-auxin)
IPA (isopentyladenosine monophosphate-cytokinin)

9. These genes encode enzymes for synthesis of the hormones
Their incorporation in the plant genomes lead to synthesis in host plant
This in turn alter the host development leading to formation of crown gall

10. An os region responsible for synthesis of unusual amino acids or sugar derivatives called as opines
Opines are derived from variety of compounds found in plant cells
E.g. – octopine and nopaline
The synthesis of these two enzymes are coded by T-DNA
Depending whether Ti plasmid encodes octopine or nopaline it is described as octopine type Ti plasmid or nopaline type Ti plasmid

11. Ti plasmid carry genes that can catabolize opines which are utilized as source of carbon and nitrogen
The T-DNA region is flanked by 25 bp sequences which is essential for its transfer
Vir region is also essential for transfer of T-DNA

12. vir region The vir region (35 kbp) is organized into 6 operons
vir A, vir B, vir C, vir D , vir E and vir G
Genes A,B,D and G essential for virulence
vir C and E are required for tumor induction
Vir A sense the phenolic compounds like acetosyringone and hydroxyacetosyringone

15. Vectors based on Ti plasmids &Ri plasmids

16. The following properties of Ti plasmids did not allow them to be used directly as vectors
Large size( kb)-usually small version is preferred
Absence of unique restriction enzyme sites
Tumor induction property

17. Auxin and cytokinin genes should be removed as it leads to production of these hormones in plants which can prevent or hinder plant growth
Opine synthesis gene as it is not useful to transgenic plant and it also lowers yield

18. Recombinant DNA technology has overcome these constraints and a number of Ti plasmid vectors have been created
Two plasmid derived cloning vector systems
The binary cloning vector
The co integrate cloning vector

19. The Binary vector system contains
Origin of replication for both E.coli &A.tumefaciens
A selectable marker gene – to check the transformation efficiency
A target gene – both of these inserted between T-DNA left and right border

21. A binary T-DNA plasmid for delivering the Bt gene to plants (not a cointegrate vector)
(NPT or kanr)
(35S-Bt gene-tNOS)
(Spcr)

22. Transfer the Promoter into the T-DNA Shuttle Vector
Use Shuttle vector and Helper Ti plasmid instead of Ti plasmid

23. The Cointegrate vector contains
Origin of replication for E.coli
A T-DNA right border
A plant selectable marker gene
A target gene
A sequence of Ti plasmid DNA that is homologous to a segment on the disarmed Ti plasmid

24. The disarmed Ti plasmid contains
The T-DNA left border
The vir gene cluster
An A.tumefaciens origin of replication (ori)
The recombination between the cointegrate vector and disarmed Ti plasmid results in the final recombinant Ti plasmid

25. Strategies to avoid Bt resistant insects
Use of inducible promoters (that can be turned on only when there is an insect problem)
Construction of hybrid Bt toxins
Introduction of the Bt gene in combination with another insecticidal gene
Spraying low levels of insecticide on Bt plants
Use of spatial refuge strategies