Genetic tool

1. Genetically engineered Microbe
Agrobacterium Tumifaciens

2. Agrobacterium
• Agrobacterium is a genus of Gram-negative bacteria established by H. J.
Conn that uses horizontal gene transfer to cause tumors in
plants. Agrobacterium tumefaciens is the most commonly studied species
in this genus.
• It is famous for taking advantage of its host by injecting DNA derived from
itsTi plasmid into the host causing the plant to create galls which excrete
opines .
• Agrobacterium tumefaciens (updated scientific name Rhizobium
radiobacter, synonym Agrobacterium radiobacter) is the
causal agent of crown gall in over 140 species of eudicots.
• Agrobacterium tumefaciens was first isolated from grapevine galls in 1897
• Symptoms are caused by the insertion of a small segment of DNA from a
plasmid, into the plant cell,[5] which is incorporated at a random location
into the plant genome.
• Economically, A. tumefaciens is a serious pathogen of walnuts, grape
vines, stone fruits, nut trees, sugar beets, horse radish,

4.  Size of the plasmid: ~250 kbp and 206,479 nucleotides long, the GC content is 56%
and 81% of the material is coding genes.
 The Ti plasmid exist as independent replicating circular DNA molecules within the
Agrobacterium cells.
 The T-DNA is variable in length in the range of 12 to 24 kb.
 The Ti plasmid has three important region:-
1. T-DNA region: This region has the genes for the biosynthesis of auxin (aux),
cytokinin (cyt) and opine (ocs), and is flanked by left and right
borders.
• T-DNA borders- A set of 24 kb sequences present on either side (right & left) of T-
DNA are also transferred to the plant cells.
• It is clearly established that the right border is more critical for T-DNA transfer.
2. Virulence region: The genes responsible for the transfer of T-DNA into host plant
are located outside T-DNA and the region is reffered to as vir or virulence region .
• At least nine vir-gene operons have been identified. These include vir A, vir G, vir
B1, vir C1, vir D1, D2, vir D4 and vir E1,E2.
3. Opine catabolism region: This region codes for proteins involved in the uptake and
metabolisms of opines
4
Ti Plasmid

6. Infection And Pathogenesis
1. Signal induction to Agro bacterium: The wounded plant cells
release certain chemicals-phenolic compounds and sugars
which are recognized as signals by Agro bacterium. The signals
induced result in a sequence of biochemical events in Agro
bacterium that ultimately helps in the transfer of T-DNA of T-
plasmid.
2. Attachment of Agro bacterium to plant cells: The Agro
bacterium attaches to plant cells through
polysaccharides, particularly cellulose fibres produced
by the Bacterium. Several chromosomal virulence (chv)
genes responsible for the attachment of bacterial cells to
plant cells have been identified.

7. T – DNA Transfer And Integration
3. Production of virulence proteins: As the signal induction
occurs in the Agro bacterium cells attach to plant cell, a
series of events take place that result in the production
of virulence proteins.
• To start with, signal induction by phenolics stimulates vir
A which in turn activates (by phosphorylation) vir G. This
induces expression of virulence gene of Ti-plasmid to
produce the corresponding virulence proteins
(D1,D2,E2,B etc.).

8. T – DNA Transfer And Integration
4. Production of T-DNA strand:-
The right and left borders of T-DNA are recognized by
vir D1/vir D2 proteins. These proteins are involved in the
production single-stranded T-DNA (ss DNA), its protection and
export to plant cells. The ss T-DNA gets attached to vir D2.
5. Transfer of T-DNA out of Agro bacterium:-
The ss T-DNA –vir D2 complex in association with
vir G is exported from the bacterial cell. Vir B products form
the transport apparatus.

9. T– DNA Transfer And Integration
6. Transfer of T-DNA into plant cells and integration:-
The T-DNA –vir D2 complex crosses the plant plasma
membrane.In the plant cells, T-DNA gets covered with vir E2.
This covering protects the T-DNA from degradation by
nucleases. Vir D2 and vir E2 interact with a variety of plant
proteins which influences T-DNA transport and integration.
• The T-DNA – vir D2, vir E2- plant protein complex enters the
nucleus through nuclear pore complex. Within the nucleus,
the T-DNA gets integrated into the plant chromosome
through a process referred to illegitimate recombination.

11. Overview of the Infection Process

12. Construction of vector
with desired genes

13. Transformation – the process of obtaining transgenic plants
Transgenic plant – a plant with a foreign gene (or genes) from
another plant/animal that is incorporated into its chromosome
Marc Van Montagu and Jeff Schell, discovered the gene transfer
mechanism between Agrobacterium and plants,which resulted in
the development of methods to alter the bacterium into an
efficient delivery system for genetic engineering in plants.
Most common genes (and traits) in transgenic or biotech
crops
herbicide resistance
Insecticide resistance
Bt genes in field corn (maize)
virus-resistance (coat-protein) genes
Transgenic plants – An introduction

15. Example
IMAGE: Mol bio of the cell by Albert (pg no:599)

16. Genetically modified soybean
• Roundup Ready
Soybeans are glyphosateresistant soybeans produced by Monsanto.
• Glyphosate kills plants by interfering with the synthesis of
the essential amino acids phenylalanine, tyrosine and tryptophane
• Roundup Ready Soybeans express a version of EPSPS from the CP4
strain of the bacteria, Agrobacterium tumefaciens, expression of
which is regulated by an enhanced 35S promoter (E35S) from
cauliflower mosaic virus (CaMV), a chloroplast transit peptide (CTP4)
coding sequence from Petunia hybrida.
• The plasmid with EPSPS and the other genetic elements mentioned
above was inserted into soybean germplasm with a gene gun

17. Golden rice contains increased levels of pro-vitamin A .
Traditional rice is white (a).
The prototype of golden rice was developed in 2000 and is a light yellow
color (b). It contains 1.6 mg/g of carotenoid.
In 2005, new transgenic lines were developed that dramatically increased the amount of
carotenoid synthesized, making the rice a deep golden color (c).
This latest form contains 37 mg/g of carotenoid
karthikumarbt@kcetvnr.org 17

18. Miraculin - taste-modifying protein – miracle fruit, the red berries of
Richadella dulcifica - shrub native to West Africa
Active principle - protein miraculin - not sweet
Unusual property - turn a sour taste into a sweet taste
Sour foods - lemons, limes & grapefruit, taste sweet when tasted
together with this protein
karthikumarbt@kcetvnr.org 18

19. • Dr Eady Crop & Food Research in New Zealand and his
collaborators in Japan produces Tearless Onion
• As onions are sliced, cells are broken, generate sulphenic acids -
unstable rearrange into a volatile gas then diffuses by air –
reaches the eye - reacts with the water to form a diluted solution
of sulphuric acid
Tear glands produce tears to dilute and flush out the irritant

20. Approved Transgenic plants.....
• Soybean
• Corn
• Cotton
• Oil Seed rape
• Sugarbeet
• Squash
• Tomato
• Tobacco
• Potato
• Papaya
• Rice

21. COLORED FRIUTS/FLOWERS/VEGETABLES
The-orange-purple-green-cauliflowers
karthikumarbt@kcetvnr.org 21

22. World's First Blue Roses On Display In Japan
Tokyo, Japan –
World's first blue roses have been unveiled to the public
for the first time at an international flower fair in Japan,
following nearly two decades of scientific research.
The blue-hued blooms are genetically modified and have been
implanted with a gene that simulates the synthesis of
blue pigment in pansies.
Its scientists successfully pioneered implanting into the
flowers the gene that produces Delphinidin,
the primary plant pigment that produces a blue hue
but is not found naturally in roses.
The Blue Rose was
developed by Suntory
Flowers
karthikumarbt@kcetvnr.org 22

23. Biodegradation of explosives (TNT, RDX)
Aresa – Danish biotech company planting tabacco plant to detect
TNT and Enzymatic detection & destruction
19 strains of Rhodoccus – use RDX as N2 source
Cytochrome p450 system - breakdown
karthikumarbt@kcetvnr.org 23