BT Cotton FINALS RECOMBINANT COTTONS.pdf

BIOTECHNOLOGY

1. - Soumya Halder (341)
- Biswarup Ghosh (352)

2. Biswarup Ghosh
Bt cotton

3. Definition:
•Bt cotton is a genetically
modified (GM) variety of
cotton that has been
engineered to produce a
natural insecticide to kill
bollworms and other
caterpillar pests. These
bollworms are major
agricultural pests that can
devastate cotton crops.
BtCotton

4. What is Bt ?
Bacillus thuringiensis (Bt) is a
naturally occurring soil bacterium
that produces proteins toxic to
certain insect pests. These
proteins, known as Bt toxins, have
been used in agriculture as
biopesticides to control pests.

5. •Btis classified as a gram-positive bacterium, meaning it
has a thick cell wall composed of peptidoglycan that
retains the crystal proteins produced by the bacterium.
•Btis a naturally occurring bacterium commonly found in
soil, sediment, and plant material. It has adapted to
diverse environments and is often used as a biological
control agent in agriculture.
•Bthas the ability to form endospores, which are
dormant, tough structures that protect the bacterium
from harsh environmental conditions such as high
temperatures, UV radiation, and chemical treatments.
These spores enable Btto survive and persist in soil for
extended periods.
•Btproduces crystal proteins, known as delta-
endotoxins or Bttoxins, which are toxic to various insect
pests.
Bacillus
thuringiensis ?

6. The Cry
group of
proteins
W hy BT ?
“Cry” represents crystals

7. Cry genes
oIn 1902, an unknown bacteria was isolated for the first time by the Japanese scientist Ishiwata, who was studying the cause of mortality in
silkworm larvae which he also called Soto disease. Then the bacteria was coined Bacillus sotto.
oIn 1911, a German scientist, Ernst Berliner, isolated a bacterial strain in dead moth larvae in Mediterranean flour, located in a flour mill in the
German state of Thuringia. For this reason, Ernst named this Bacillus thuringiensis ( Bt!)
Crystals are synthesized after stage II of sporulation
and accumulate in the cell where they can represent up
to 30% of the dry weight of the sporulated cells.
oMechanism of cytotoxic action of particular Bt inclusions, called parasporal, was shown in silkworm larvae (Bombyx mori).
oChanges in the permeability of the intestinal walls of the insect were observed, consequently causing its death. These results showed that the
parasporal inclusions contain crystals of endotoxin, which was the cause of the larva deaths.
oThe successful use of Bt in agriculture lies in the production of crystal proteins called Cry, which have specific cytotoxic activity against
different insect orders (discussed in the next slide)
oDuring the sporulation phase, Bt forms a protein crystal in seven distinct stage : - (a) phase I: axial filament formation
(b) phase II: formation of the forespore septum
(c) phase III: parasporal crystals and pre spore formation
(d) phases IV–VI: exospore formation, primordial cell wall
development, and the transformation of spore nucleoids
(e) phase VII: spore maturation and cell lysis
oSeveral authors have described the presence of different forms of Bt crystals, such as dipyramidal, pyramidal, cuboidal, flat rhomboid, spherical, and
rectangular. The most commonly found shape is the dipyramidal crystal. These crystals may include one or more-endotoxins, also known as Cry proteins

8. Cry proteins
Peptide sequence of Cry 5Aa gene
responsible for toxicity
Cysteine
Asparagine
Proline
Glutamine
Lysine
Aspartic Acid
Leucine
Arginine
Valine
CNPNQPCKDDLDRV
oProduce δ-endotoxins, which are 80,000X more toxic than organo-phosphate insecticide(but specific to certain pests only)
oAre inactive precursors in bacterium & human & other mammals. But activated after solubilizing in the alkaline environment of the larval midgut.
oThis secreted as protoxin is cleaved by protease in insect gut cells, which bind inside gut. This triggers pore formation in cell
membrane, disrupting ion balance and an ultimatum of cell lysis. This results in surface epithelium damage of gut. So osmotic
imbalance causes cell lysis. Hence, the insect gets unfit to feed, an starves to death !
oAre synthesized from cry genes during the sporulation phase of the bacterium's life cycle and are released into the surrounding
environment. (later discussed in detail)
oHave different types :-
TYPES EFFECTS ON : -
Cry I (130-140 kDa)Lepidoptera (moth & butterfly)
larvae
Cry II (65 kDa) Lepidoptera & Diptera larvae
Cry III-A & Cry III-B Coleoptera (beetles)
Cry IV Diptera Larvae
Cry V Nematode larvae
Cry VI No data yet
Did you know that are
more than 150 different
Cry toxins !
Sporulation in
Bacillus Thuringiensis
Cry protein crystals !
Mesosome
Cell Wall
Plasma Membrane
Axial Filament
Ovoid Inclusion
Exosporium
BipyramidalParasporal Crystal
Spore
C-terminal extension in long
protoxins is dispensable for toxicity
and induce formation of crystal
inclusion bodies within the bacteria.

9. •Cry proteins are classified into three groups through a phylogenetic approach
•Tertiary structure of the N-terminal domain, called domain I, is a set of seven α-helices,
among which the central α-helix is hydrophobic and surrounded by six amphipathic helices; this
helical domain is responsible for membrane insertion and pore formation.
•Domain-II consists of three antiparallel β sheets with exposed loop regions, and
Domain-III is a β sheet. The most exposed regions in the tertiary structure of the
protein are domains II and III, which are involved in receptor binding. And the
process of receptor binding is mainly contributed to the three surface exposed
loops.
•Domain-III is still unknown. Domain III can also possibly function for receptor binding
and insect specificity
•Domain I result in low or no toxicity on the tested insects
•The Cry protein, because of its essential function(s) in the biological system, retains its
function intact throughout evolution of organisms despite allowing diversification
elsewhere in its structure. It is conserved.
Cry 1Aa

10. These crystals are solubilized in alkaline insect
midgut environment.
1
Cry proteins are released in the form of protoxins (inactive,
active), which still lack toxic biological activity.
Alkaline pH conditions ranging from 8 to 11 are found in lepidopteran
and dipteran insects; some Cry proteins require neutral or slightly
acidic pH conditions, which are present in coleopteran insects. Thus,
Cry proteins are specific
2
3This soluble cry proteins, are processed by intestinal
proteases & their peptides from both N - & C-termini are
cleaved
4they bind to various membrane receptors of the cells of the
insect’s intestine
5They form oligomers
6Mainly bound to proteins like cadherins, they induce
pore formation
7
Anionic imbalance(release of H+, K+, Na+, and Ca2+
ions) in the cell, causes an increase in cAMP and
activate the apoptotic process known as cytolysis.
The activation is mediated by the action of proteolytic
enzymes, such as cathepsin-G and chymotrypsin, which are
located in the digestive system of the insect larva
Proteins such as cadherins, amino
peptidases, and alkaline peptidases are
recognized by the domains of endotoxin
and, recently, the binding of-endotoxin to
other proteins, such as ATP binding
transporter proteins(ABC), has been
demonstrated
Another model suggests that the toxin
monomer can bind to a cadherin receptor
and activate Mg2+- dependent signal
transduction, a pathway that leads to
cell death

12. oTo date, 731 genes encoding Cry proteins have been found with 272 holotypes.
o These proteins have molecular weights between 30 kDa and 140 kDa
o Crystal proteins have been widely used in genetically modified cultures; these transgenic cultures can produce Bt crystals, making them
insect resistant
o There are vegetative insecticidal proteins (Vip) including Vip1, Vip2 and Vip3. Vip1 and vip2 proteins constitute binary toxins mainly active
against Coleoptera whereas Vip3 proteins are active against Lepidoptera
BBMV- brush border membrane vesicle

13. oThe Vip3 MOA, although remaining poorly
understood, resembles the MOA of Cry
toxins but differs in the cell receptor
used for toxin binding.
oCry and Cyt proteins are produced during
sporulation as parasporal inclusions and
are typically found in this bacterium.
This feature has allowed the production
of sprayable Bt-based biopesticides
wherein the protein crystals constitute
the active ingredient of the
bioinsecticide.
oSecretable proteins are produced during
the vegetative growth and are later
secreted (and diluted) into the culture
medium. This behaviour has prevented
their commercial production as
formulated biopesticides relegating
their use to the construction of
transgenic crops exhibiting resistance
to susceptible insect pests (e.g., Vip
cotton, etc.)
oVip proteins are further not as specific
as Cry protein.

14. Multiple
alignment of
Domains II
and III of 18
Cry proteins.
The shading
reflects the
conservation
profile at
60%
consensus
of amino
acids

15. Phylogenetic tree of the 18 sequences constructed
by the ME method. Numbers under the nodes
indicate bootstrap values.
Scale bars represent level of amino acid sequence
divergence.
The numbers above each arrow refers to bootstrap
value.
You can too
see these
18 cry
protein
sequences
from
GenBank
using these
accession
numbers

17. Intermediate vector virus with
the GOI
Helper vector virus with the vir
gene

19. WATCH THIS VEIDEO TO INTERPRETE GENE TRANSFER IN PLANT VIA COINTEGRATE VECTORS

20. G ene structure

21. Plasmid construction of Cry5Aa
“PRI101-An-Cry5Aa”
constructed by Institution of Cotton Science
sequence (L07026.1) published in GenBank by Sangon Biotech (Shanghai) Co., Ltd
✓Previously Cry1Aa gene was used. But with time insect resistance lead
to usage of Cry5Aa gene
✓The cry gene here is of 697 bp & the plasmid is 10,417 bp
✓Npt II inactivates kanamycin, catalyzing transfer of terminal phosphate
of ATP to kanamycin. (selection marker for plant) (/proof)
✓Npt (neomycin Phosphotransferase) also confers resistance to
aminoglycoside antibiotics like neomycin too. (/proof)
✓Primers were designed by Primer 5.0, just as follow:
✓5′-GCCATCCAGGTTGCTATCT-3′
✓5′-GCTGACGAAAGAATGCTGA-3′
✓ColE1 ori-site for E.coli Cloning
✓Ri ori-site for Cloning in Agrobacterium
✓The GOI forged by Hind-III & Sph-I
Sph I Hind III
Cry5Aa is a novel Bt gene,
there was no any ideal detection
method to be applied

22. Kanamycin test and PCR analysis of
Cry5Aa transgenic line
PCR analysis of Cry5Aa transgenic line.
M: DNA Mark;
1: Non-transformed Control
9: positive control
2–8: Cry5Aa transgenic lines
T0 generation were harvested in 2010. Total 1829 seeds of T1 generation germinated normally while
the other seeds failed to survive. The expanded leaves were sprayed with kanamycin solution at
seedling stage for preliminary identification. Total 142 plants without yellow plaque indicated that the
positive rate of Cry5Aa transgenic line by kanamycin test was 7.76%. Meanwhile, PCR analysis was
performed after genomic DNA extraction from transgenic plants. Specific band were identified in
140 plants showed that the positive rate was 7.4% by PCR analysis.

The seeds of T2 generation were harvested from positive T1 transgenic plants. A total of 845 T2
plants were obtained at cotton base of Hunan Agricultural University in Changsha, Hunan in the same
year. There were 618 positive plants without yellow spots after spraying with kanamycin solution.
While the positive rate of transgenic line was 73.1% by kanamycin test and the positive ratio
detected by PCR was 73.45%.
Gene flowing monitoring of Cry5Aa gene

23. Pollen Tube Pathway
✓The petals were stripped off and the plasmid was pushed into fertilized
ovary with a 50 μL medical-micro injector and each ovary was injected
with 5 μL of diluted plasmid.
✓So the ovaries, as they grow, will tend to be carrying the Cry5Aa gene
transformed by our agrobacterium vector
✓So they will grow to a transgenic plant, having innate resistance to
certain pests, without any use of pesticides.

24. Effect on targeted pests
➢Adjusted death rate of fed cotton bollworm of four Cry5Aa transgenic
lines including JX0010, JX011, JX012 and JX0020 was 82.00%, 100%,
69.50% and 76.50%,
➢Jin 1 and GK19 were used as negative and positive control,
respectively. Jin1 is the receptor and GK19 is a Cry1Ac Bt transgenic
cotton variety widely planted in China
➢The result showed that the maximum pollen spreading distant of
Cry5Aa transgenic line JX0010 was 30 m, which indicated gene flow
of the novel Bt gene is limited.

25. Effect on non-targeted pests
✓The experimental results illustrated the
populations of all four tested non-target pests
including cotton aphid, miridae, two-spotted
spider mite and cotton changed among
different growth and development stages.
✓ However, there was no significant difference
between Cry5Aa transgenic line JX0010 and
the non-transgenic control Xiangmian10,
which maybe indicated that the novel Bt gene
had less influence on these non-target pests.

26. Advantage
•It helps in improving the crop yield, thereby, raising the farmer’s income.
This results in increased farm production.
•They help in controlling soil pollution as the use of synthetic pesticides is
reduced.
•Bt crops help in protecting beneficial insects.
•It can easily feed an increasing population due to increased yields in a
short time.
•It leads to the production of disease-free crops owing to the reduction of
pesticides.
•It leads to more productivity in a small area of land.

27. Disadvantage
•Bt crops are costlier than naturally grown crops.
•It can disrupt the natural process of gene flow.
•The pests might become resistant to the toxins produced by these crops
and the crop production might decline.

29. Problems of Bt-cotton farming in India
•High Cost: Bt cotton seeds are significantly
more expensive than traditional varieties,
which can lead to debt for farmers, especially
smallholders who may lack access to credit.
•Loss of Seed Saving:Bt cotton seeds are often
hybrids that don't produce viable seeds for
the next planting season. This forces farmers
to buy new seeds each year, increasing their
dependence on seed companies.
•Knowledge Gap:Some farmers lack sufficient
knowledge about using Bt cotton effectively,
including proper pest management
techniques and responsible use of
insecticides.

30. Where are we on Bt cotton in INDIA
Basic Reasearch :
•National Botanical Research Institute (NBRI), Lucknow
•National Research Centre on Plant Biotechnology (NRCPB), New Delhi.
•International Centre for Genetic Engineering & Biotechnology (ICGEB, New
Delhi).
•Central Institute for Cotton Research, Nagpur.
•National Chemical Laboratory (NCL), Pune
•Bhabha Atomic Research Centre (BARC), Mumbai,
•University of Agricultural Sciences, Dharwad.

31. •"Btcotton has made India a cotton
exporting country. We thought of
ourselves as exporters of wheat
and rice, but today we import
wheat. No country as large as India
can survive on imports for its food
needs,”…”…"The success
achieved in cotton must be used to
make the country self sufficient in
rice, wheat, pulse and oil seed
production".
The former Home Minister
P. Chidambaram on Bt
crops

32. Let’s make Our INDIA
"Biotechnology is the future. It is the only way we are going to be able to feed and fuel the world's growing population."
-Craig Venter

33. References -
o”Principles of gene manipulation & genomics”-Primrose
o“Transformation and functional verification of Cry5Aa in cotton”-
(2021) 11:2805
o“Pollen grains as a target for introduction of foreign genes into
plants: an assessment”- 8 January 2011
o Review-”Importance of Cry Proteins in Biotechnology: Initially a
Bioinsecticide, Now a Vaccine Adjuvant”-23 sept 2021
o“Adaptive Evolution of cry Genes in Bacillus thuringiensis:
Implications for Their Specificity Determination”-2007
o“Plant Transformation via Pollen Tube-Mediated Gene Transfer”
December 2014, 07 July 2016
o“Vip3A, a novel Bacillus thuringiensis vegetative insecticidal
protein with a wide spectrum of activities against lepidopteran
insects”- may 1996
o Letter-”Is the Insect World Overcoming the Efficacy of Bacillus
thuringiensis?”- may 2017
o United States Patent -Peffley et al. /Patent No.: US 6,583,335
B1/ Date of Patent: Jun. 24, 2003
o“Interaction of Bacillus thuringiensis Cry1 and Vip3A Proteins
with Spodoptera frugiperda Midgut Binding Sites”-Apr 2009
oGoogle Bard
oOpen AI
HUMBLE THANKS TO - (for their work and imageries has been used)
o Shihao Zhao
o Feng Wang
o Qiuping Zhang
o Jiayi Zou1
o Zhangshu Xie
o Kan Li1
o Jingyi Li
o Bo Li1
o Wen He1
o Jinxiang Chen
o Yunxin He
o Zhonghua Zhou
o Jin-Yu Wu
o Fang-Qing Zhao
o JieBai1
o Gang Deng
o Song Qin
o Qi-Yu Bao
o Ellen B. Peffley
o Randy Allen
o Ping Song
o all of Lubbock Xiaomin
Shang
o Plano,
o all of TX (US)
THANK YOU