This document discusses genetic engineering techniques using Agrobacterium tumefaciens and its tumor-inducing (Ti) plasmid. The Ti plasmid contains a segment of transferred DNA (T-DNA) that is integrated into the host plant genome and causes tumor formation. Genes of interest can be inserted into the T-DNA, which is then transferred to the plant cell. However, wild-type Ti plasmids also contain oncogenes, so alternative vectors like plant viruses have been developed that can introduce foreign DNA into plant cells without causing tumors. Cauliflower mosaic virus is one such virus vector that has been used, as its small, 8kb double-stranded DNA genome can be easily manipulated.

1. GENETIC ENGINEERING AND
MOLECULAR TECHNIQUES
Karthi.M
MSc Project Student-Cancer Biology Lab
Dept. of Biochemistry

2. Ti-Plasmid
*Tumor-inducing principle – Transferred from the bacterium to the plant at the wound
site.
*Zaenen at al. (1974) – virulent strains of A.tumefaciens harbor large plasmids (140-235
kbp)
*double stranded circular DNA - Agrobacterium tumefaciens
*Agrobacterium is a gram negative soil bacterium - infects over 3000 dicots - causes
crown gall disease
*This plasmid is denatured at higher temperatures and loses tumorgenic properties.

3. It encode for enzymes for catabolism of opines such as permease and oxidase

4. Ti-Plasmid Gene Maps

5. *Octopine is formed with two amino acids; Arginine and Alanine.
*Nopaline is made up of Arginine and Glutamine.
*Octopine and nopaline are not found in healthy plant tissues
*The opines are catabolized and used as the energy source by the bacterium.
*During the infection through a wound, the plant cells begin to proliferate and form tumors
and the plant tissues begin to synthesize opines.
*It has T DNA which is of 20kb
*In addition it has several genes such as vir genes for virulence
*ori gene for origin of replication, tra genes for transfer and genes for opine synthesis.

6. *Virulence gene Responsible for the transfer of T DNA into the host cell and
integration of T DNA with host genome
*Tra genes encode proteins necessary for transfer of T DNA into the host.
*A small, specific segment of the plasmid, about 23kbp in size, is found integrated in
the plant nuclear DNA at an apparently random site  this DNA segment T-DNA
(transferred DNA)
*It carries genes that confer both unregulated growth and the ability to synthesize
opines upon the transformed plant tissue.
*These genes are non-essential for transfer and can be replaced with foreign DNA

7. *Structure and organization of nopaline plasmid T-DNA sequences are usually simple
i.e. there is a single integrated segment
*Octopine T-DNA TL (carries the gene required for tumor formation)
 TR (carries the gene for opine synthesis)
*The two segments are transferred to the plant genome independently and may be
present as multiple copies
*In Ti-plasmid itself  T-DNA is flanked by 25 bp imperfect direct repeats  border
sequences
*It is not transferred intact to the plant genome, but they are involved in the transfer
process

8. *Genes in the virulence region are grouped into the virABCDEFG
*virA codes for a receptor which reacts to the presence of phenolic compounds such as
acetosyringone, which leak out of damaged plant tissues
*virB encodes proteins which produce a pore/pilus-like structure
*virC binds the overdrive sequence
*virD1 and virD2 produce endonucleases which target the direct repeat borders of the
T-DNA segment
*virE binds to T-strand protecting it from nuclease attack, and intercalates with lipids to
form channels in the plant membranes
*virG activates vir-gene expression after binding to a consensus sequenceonce it has
been phosphorylated by virA

9. Genetic transformation Process

10. *In transferring gene of interest to Ti plasmid, an intermediate vector is used such as
pBR 322.
*T DNA portion of the Ti plasmid is separated. T DNA is inserted into the pBR 322
vector which results in formation of a shuttle vector.
*This shuttle vector can replicate in E. coli and in Agrobacterium.

11. Drawback
*Ti-plasmid  natural vector for genetically engineering plant cells
*Because it can transfer its T-DNA from the bacterium to the plant genome
*However, wild type Ti-plasmids are not suitable as general gene vectors because the
T-DNA contains oncogenes that cause disorganized growth of the recipient plant cells
* Deleting all of its oncogene

12. Plant viruses can be used as episomal
expression vectors
*Alternative to stable transformation using Agrobac. or direct DNA transfer, plant viruses
can be employed as gene transfer and expression vectors

13. Advantages to use of Viruses
1able to adsorb to and introduce their nucleic acid into intact plant cells.
However for many viruses, naked DNA or RNA is also infectious, allowing r.combt vec
to be introduced directly into plants by methods such as leaf rubbing
2infected cells yield large amt of virus, so r.combt viral vectors have the potential for
high-level transgene expression.
3viral infections are often systemic
4viral infections are rapid
5 all known plant viruses replicate episomally

14. The first plant viral vector were based on
DNA viruses
Vast majority of plant viruses have RNA genomes.
However, two group of DNA viruses that are known to infect plants
 The caulimoviruses and geminiviruses – 1st to be developed as vectors because of
the ease with which their small, DNA genomes  manipulated in plasmid vectors
 Caulimoviruses – the type member is cauliflower mosaic viruses (CaMV).
 8 kb dsDNA genome of several isolates has been completely sequenced

15. Map of the cauliflower mosaic virus
genome
8 coding regions
Different reading frames
DNA strands with the 3 discontinuities
Major transcripts – 19s & 35s

16. Revealing an unusual structure characterised by  3 discontinuities .
There are eight tightly packed genes, expressed as two major transcripts 35s RNA
(which essentially represents the entire genome),
 19s RNA (which contains the coding region for gene VI )
 Promoter and terminator sequences for both transcripts have been utilized in plant
expression vectors
 35s promoter is particularly widely used
 Only two of the genes in the CaMV genome for non-essential ( gene II & gene VII)

17. CaMV is icosahedral capsid
Max capacity of the CaMV is 8.3kb
Removal of all non-essential genes, represents max insert size of less than 1kb
This restriction in the capacity for foreign DNA represents a major limitation of CaMV
vectors