This is the genetic engineering information..

1. Vectors e.g.
plasmid, cosmid,
phage, BAC, YAC
etc
sample/source
PCR product with
sp. Restriction
ends,
Bacterial
Transformation:
Selection step 1
Selection step 2
AmpR/TetR or LacZ (blue-
white screening)
• Recombinant
bacteria
• Bacteria with re-
circular plasmid
• Bacteria without
plasmid
Transformation
with competent
bacterial cell

3. Plant Transformation:

4. Gene transfer:
The process that moves a specific piece of DNA into cell.
To introduce genetic diversity into plant population.
To select superior plants carrying genes for desired traits.
Maintained the range of plant varieties.
Genetic transformation:
The desirable gene transfer from one organism to another
The subsequent stable integration
The expression of a foreign gene into the genome.
Transgene: The transferred gene
Transgenics : The organism that develop after a successful gene transfer
Transformed plants: The plants that carry the stably integrated foreign genes.
Gene Transfer in Plants

5. Transient and stable gene expression
Transient expression:
The transferred DNA may be expressed for only a short period of time
following the DNA transfer process.
Transient assay:
Analysis of gene expression
Rapid monitoring of gene transfer
Stable expression
DNA is integrated into the plant nuclear or plasmid genome
Expression occurs in regenerated plants
DNA is inherited in subsequent generation
The developmental potential is important, transformed cell should be
capable of regenerate into fertile plants.

6. Marker genes
A set of genes are used for monitoring and detection of plant transformation
system in order to know whether the DNA has been successfully transferred
into recipient cells.
The marker gene is induced along with the target gene.
Marker genes are two types-
1. Reporter or scoreable genes (quantitative)
2. Selectable marker genes (qualitative)
Easy assay technique, requires no DNA extraction, electrophoresis, or
autoradiography.

7. Expression vector

8. Reporter genes
Screenable, scoreable, quantitative reporter genes
Show immediate expression in cells
Analysis of plant gene expression and standardization of parameters for
successful gene transfer in a particular technique
Should have the following features
1. Detection with high sensitivity
2. Low endogenous background
3. There should be a quantitative assay
4. Assay should be non destructive
5. Assay should be require a minimal amount of effort and expense

9. Opine synthase (ocs, nos genes):
Reporter genes
Enolpyruvyl Shikimate Phosphate Synthase (EPSP):
Bar Gene:
2. Herbicide Resistance Genes:
1. Antibiotic Resistance Genes :
Neomycin Phosphotransferase II (NPT II):
Hygromycin Phosphotransferase (hpt):
Selectable Marker Genes
Chloramphenicol acetyl transferase (cat gene):
Bacterial luciferase (lux F2 genes):
Firefly luciferase (luc gene):
Green fluorescent protein (gfp gene):
β-Glucuronidase (GUS) (uidA gene):

10. Opine synthase (ocs, nos genes):
octopine and nopaline are common opines in Agrobacterium, respectively
produced by the synthase genes ocs and nos.
octopine synthase and nopaline synthase genes present in T-DNA of Ti (tumor
inducing) or Ri (root inducing) plasmids of Agrobacterium.
The transformed status of the plant cells can be easily detected by the presence
of these opines.
Opines can be separated by
electrophoresis and identified.
The enzyme activities
responsible for the production
of opines can also be assayed,
enzymes are stable, the
enzymatic assay is inexpensive
and easy to perform.

11. Chloramphenicol acetyl transferase (cat gene):
The gene for chloramphenicol acetyl transferase (CAT) is a widely used
reporter gene in eukaryotic organism.
This gene carries out acetylation of chloramphenicol.
The enzymatic assay is very sensitive and relatively simple.

12. β-Glucuronidase (GUS) (uidA gene):
β-Glucuronidase (GUS) producing from bacterial gene (uidA) is the most
commonly used reporter gene in assessing plant transformation.
β-Glucuronidase assays are highly sensitive and nonradioactive assay.
Quantitative estimation by fluorometric method and qualitative estimation by
histochemical assay.
enzyme localization can be detected by fluorogenic substrate 4-MUGluc, or
chromogenic substrate X-gluc.
Quantitative
Fluorometric
Qualitative
histochemical

13. Green fluorescent protein (gfp gene):
Green fluorescent protein (GFP), coded by gfp gene, is being widely used .
Assays of GFP are easier and non-destructive and early screening of even the
primary transplants can be done by GFP.
Gene for GFP has been isolated from jelly fish Aequorea victoria which is a
luminescent organism.
GFP emits fluorescence which can be detected under a fluorescent
microscope.

14. Firefly luciferase (luc gene):
The enzyme firefly luciferase, encoded by the gene luc, isolated from north
American firefly photinus pyralis.
the enzyme catalyses the oxidative decarboxylation of D-luciferin (ATP
dependent) which results in the emission of light that can be detected by
sensitive luminometers.
Bacterial luciferase (lux F2 genes):
The bacterial luciferase genes (luxA and luxB) have originated from Vibrio
harveyi and V. fischerii.

15. Selection of transformed cells is a key factor in successful genetic
transformation.
Selectable marker genes enable the transformed cells to survive on media
containing toxic level of selection agent whereas nontransformed cells get
killed.
Selectable marker genes such as antibiotic resistance genes, herbicide
tolerance genes and several antimetabolite genes are in common practice.
The usefulness of a particular resistance marker depends on
the characteristics of selection agent
the resistance gene
the plant material
Selectable Marker Genes

16. 1. Antibiotic Resistance Genes as Markers:
Antibiotic resistance genes used as selectable markers are basically microbial
in origin from E.coli .
Antibiotics are toxic to plants by inhibiting protein synthesis particularly in the
cell organelle like chloroplast.
Neomycin Phosphotransferase II (NPT II):
NPT II or amino glycoside 3’ –phosphotransferase II enzyme is the most
widely used antibiotic resistance gene in plant transformation strategy.
The enzyme is encoded by the nptII gene which is derived from Tn5
transposon.
It inactivates a number of aminoglycoside antibiotics (protein synthesis
inhibitor) such as including kanamycin, neomycin and puromycin.
Kanamycin is the most commonly used selective agent.

17. Hygromycin Phosphotransferase:
The hygromycin phosphotransferase gene (hpt) is derived from E.coli.
Hygromycin B is an aminocyclitol antibiotic that kill non transformed cells
by blocking protein synthesis during selection process.
It is more toxic than kanamycin.

18. 2. Herbicide Resistance Marker Genes:
Bar Gene:
Bar gene encodes phosphinothricin acetyl transferase (PAT), which
inactivates herbicides like biolophos, phosphinothricin. This gene has
been cloned from two different strains of Streptomyces hygroscopicus and
S. viridochromogenes.
This is based on damaging of non-transformed tissues by inactivating
glutamine synthase (GS) which is required for the assimilation of NH3 and
in the regulation of nitrogen metabolism in plants.
Inhibition of glutamine synthase results in the accumulation of NH3 ,
highly toxic to the cell which causes death of plant cells.
Bar gene encodes PAT which converts herbicides into a nonherbicidal
acetylated form and confers resistance to the cell.

19. Enolpyruvyl Shikimate Phosphate Synthase (EPSP):
In plants, the enzyme 5-enolpyruvyl-shikimate -phosphate synthase is
essensial for biosynthesis of aromatic aminoacids like tyrosine,
tryptophan, etc.
Glyphosate (N-phosphonomethyl- glycine) is a potential herbicide, is
widely used to kill weed plants by competitive inhibitor of enzyme
EPSP synthase, and thereby blocking aromatic aminoacid production.
In transformation selection technique, mutant EPSP synthase gene is
used as herbicide resistant marker. Expression of mutant EPSP synthase
gene in transformed tissues is able to survive against glyphosate
herbicide included in the culture medium. In contrast, non-transformed
cells get killed by inhibition of EPSP synthase enzyme by herbicide.

20. Cloning vector

21. pCAMBIA1305.1
11846 bp
Lac Z alpha
kanamycin (R)
hygromycin (R)
GUSPlus
Catalase intron
T-BORDER (R)
pVS1 Sta
T BORDER (L)
pBR322 bom
NOS polyA
CaMV35S polyA
2x CaMV35S promoter
CaMV35S promoter
pBR322 ori
pVS1 rep
BamH I
Bgl II
Bst EII
Bst XI
EcoR I
Hind III
Kpn I
Nco I
Pml I
Pst I
Sac I
Sal I
Spe I
Xba I
Nhe I (2026)
Nhe I (5467)
Xho I (8910)
Xho I (10004)
Sma I (11052)
Sph I (11083)
Binary vector

26. Transfer of T-DNA to plant cells.

27. The induction of vir genes and T-DNA transfer.