Bt Cotton - (CUSAT) – Plant Biotechnology laboratory

Bt Cotton
Presented By :- Raji R. Nair
MSc Biotechnology
CUSAT

Bt cotton :- transgenic cotton with insect killer gene that is
transferred to it from soil bacteria, Bacillus Thuringiensis (Bt).

Bacillus thuringiensis
• a Gram-positive, soil dwelling bacterium.
• a genome size of 2.4 to 5.7 million base pairs.
• Aerobe, capable of producing endospores.
• Discovered by Ishiwaki (1901) in diseased silkworms.
• Isolated from gut of diseased flour moth larvae in
Thuringberg, by Ernst Berliner.
• Produces Cry toxin ---- extracted and used as pesticide.

• Cry toxin – Insecticidal Crystal Protein (ICP)
• ICPs are one of the several classes of endotoxins produced by the
sporulating bacteria.
• Classified as δ- endotoxins
• Cry toxins have specific activities against insect species of the
orders:
◦ Lepidoptera (moths and butterflies)
◦ Diptera (flies and mosquitoes)
◦ Coleoptera (beetles)
◦ hymenoptera (wasps, bees, ants and sawflies)
◦ and nematodes.

• The family of genes coding for cry toxin is the Cry gene family.
• A common characteristic of the cry genes is their expression during the
stationary phase
• Upon sporulation, B. thuringiensis forms crystals of proteinaceous
insecticidal δ-endotoxins – ‗ crystal proteins‘ or Cry proteins -- encoded
by cry genes.
• In most strains of B. Thuringiensis, the cry genes are located on the
plasmid.

Action of Cry toxin !!
• 1. Insect eats Bt crystals and spores.
• 2. The toxin binds to specific receptors in
the gut
• 3. The crystals cause the gut wall to
break down, allowing spores and normal
gut bacteria to enter the body.
• 4. The insect dies as spores and gut
bacteria proliferate in the body.

• More than 170 toxin-encoding genes have been isolated form Bt
collections
• Bt action is very specific.
• Different strains of Bt are specific to different receptors in insect
gut wall.
• Bt toxicity depends on recognizing receptors, damage to the gut
by the toxin occurs upon binding to a receptor.
• Each insect species possesses different types of receptors that will
match only certain toxin proteins, like a lock to a key.

• Pre-requisites for the functioning of Bt toxins:
• The pest must take a few bites of the plant tissue;
{ Bt transgenics are not effective against sucking pests (Homoptera,
with wings without scales), as they do not ingest plant tissue.}
• An alkaline environment (pH 9.5 and above) in the gut of the
insect pest is essential for the Cry proteins to dissolve in the gut
fluids and to be converted into an active molecule to function as an
insecticidal compound.
• The lining of the mid-gut (brush border) of the pest must have an
appropriate receptor for a particular toxin to bind to. The pest
specificity of different Bt toxins depends upon the presence of
appropriate receptors

Cotton !
• Cotton is a member of the genus Gossypium and belongs to
the Malvaceae family
• More than 95% of commercial cotton is Upland cotton –
Gossypium hirsutum
• The fruit of the cotton plant is more familiarly known as the
boll, which contains approximately ten cotton seeds that are
surrounded by the fibers (lint) which grow from the coats of
the seed.

Cotton – Indian perspective !!
• Cotton is a leading commercial crop grown for its valuable fibre
• India ranks number one in the world accounting for 20% of the total area
planted under cotton
• Highly susceptible to insects
• Fifty percent of the total insecticides consumed in the country are used
only for cotton crop.
• The total loss due to damage to cotton crop is estimated to be more than
Rs.1200 crores.
• India has the lowest yield of the top 10 producers
• Export is also less !!

Bt cotton ?
• All Bt cotton plants contain one or more foreign genes derived
from the soil-dwelling bacterium, Bacillus thuringiensis; thus, they
are transgenic plants.
• The insertion of the genes from B. Thuringiensis causes cotton
plant cells to produce the crystal insecticidal proteins ; Cry
proteins.
• These insecticidal proteins are effective in killing some of the most
injurious caterpillar pests of cotton, such as the larvae of tobacco
budworms and bollworms.

Why Bt cotton ??
• The chemical control to suppress insect pest are proving to be ineffective
due to development of high level of resistance.
• High level of resistance require repeated application of insecticides
leading to heavy expenditure, crop failures, and viscous cycle of debt for
farmers.
• The cotton hybrids containing Bt gene produces its own toxin for
bollworm attack thus significantly reducing chemical insecticide use and
providing a major benefit to cotton growers and the environment.

Cotton

The erratic performance of Bt insecticides in cotton is attributed to
four reasons:
• The toxin is rapidly degraded by ultraviolet light, heat, high leaf
pH, or desiccation.
• Caterpillars must eat enough treated plant tissue to get a lethal
dose of the toxin, since the toxin has no contact effect.
• The sites where tobacco budworms and bollworms feed are
difficult to cover with the foliar-applied sprays.
• Bt Cry-proteins are less toxic to older larvae

Bt cotton has a built-in system that efficiently and
consistently delivers Cry-toxins to the target pests from the
time a newly hatched larva takes its first bite ....

Bollworm !!!

Bt Cotton !!!
• Bt Cotton is produced by inserting a synthetic version of a gene
from the naturally occurring soil bacterium Bacillus thuringiensis,
into cotton.
• The primary reason this is done is to induce the plant to produce its
own Bt toxin to destroy the bollworm, a major cotton pest.
• The gene causes the production of Bt toxin in all parts of the cotton
plant throughout its entire life span.
• When the bollworm ingests any part of the plant, the Bt cotton
toxin pierces its small intestine and kills the insect.

Gene Construct !!
• A gene construct (or a cassette) consisting of the chosen Bt gene
is made, along with other molecular components needed for its
expression in the transgenic crop variety.
• The construct consists of sequences of nucleotides (the building
blocks of DNA, the genetic material) –
◦ to initiate the expression of the selected gene,
◦ to promote such expression,
◦ the actual sequence for the gene
◦ a nucleotide sequence to signal the termination of the process of
expression.
• This construct is then incorporated into the tissue of a (chosen
primary) variety of the crop, and this is called an event

• Protein gene.
• The Bt gene, modified for improved expression in cotton, enables
the cotton plant to produce Cry-protein.
• The first varieties of Bt cotton produced in the United States
contained one Cry-protein gene—Cry1Ac.
• Other varieties contain a ―stacked‖ gene complex, for example—
one gene for insect control (Cry1Ac) and one gene to protect the
cotton from application of the herbicide glyphosate.

• Cotton varieties containing the Cry1Ac Bt protein provide protection
against three major U.S. Cotton pests—
• tobacco budworms, bollworms, and pink bollworms.
Bt cotton also reduces survival of other caterpillar pests such as beet
armyworms, cabbage loopers, cotton leafperforators, fall armyworms,
southern armyworms, and soybean loopers.

• Promoter. is a DNA segment that controls the amount of Cryprotein
produced and the plant parts where it is produced.
• Those used in Bt cotton and certain Bt corn varieties, cause the
plant to produce Cry-protein throughout the plant.
• Example CaMV 35 S promoter
• Genetic marker. allows researchers to identify successful
insertion of a gene into the plant‘s DNA. It also assists plant
breeders in identifying and developing new cotton lines with the
Bt gene.
• A common marker is an herbicide tolerance gene linked to the
Bt gene.

• Bt cotton contains the following three genes inserted via genetic
engineering techniques:
• The Cry1Ac gene, which encodes for an insecticidal protein,
Cry1Ac, derived from the common soil microbe Bacillus thuringiensis
subsp. kurstaki (B.t.k.).
• The nptII gene, which encodes the selectable marker enzyme
neomycin phosphotransferase II (NPTII), was used to identify
transformed cells that contained the Cry1Ac protein. It served no
other purpose and has no pesticide properties. The nptII gene is
derived from the prokaryotic transposon Tn5.
• The aad gene which encodes the bacterial selectable marker enzyme
3”(9)-O- aminoglycoside adenyltransferase (AAD) allowed for
the selection of bacteria containing the PV-GHBK04 plasmid on media
containing spectinomycin or streptomycin. The aad gene was isolated
from transposon Tn7.

• NPTII and AAD proteins are used as a selectable marker and have no
pesticidal activity and are not known to be toxic to any species.

• Gene construct MODIFICATIONS needed !!!
• Because attempts to express Cry proteins under CaMV 35S or
Agrobacterium T-DNA promoters Low Expression Level !
• Heterologous genes from non plant species need to be modified !
• Genes from different organisms have different G+C contents
• In bt cotton transgene…. The codon usage and overall G+C content
were altered
• Also several potential plant polyadenylation signals and ATTA
sequences were removed !

• The inclusion of plant- specific sequences upstream of the
translation initiation codon improves translatability.
Wild – type
gene
Modified
Gene
No : of altered bases (%) 0 390 (21%)
No: of modified codons (%) 0 356 (60%)
% G+C content 37 49
No: of potential polyadenylation sites 18 1
No: of ATTTA sequences 13 0

• Quantification of gene expression:
• It is necessary to know how a Bt gene is expressing in a
transgenic variety, in order to evaluate its effectiveness against the
targeted pest.
• All transgenic Bt cotton varieties contain some quantity of Bt
protein, though the actual quantities of the protein may vary from
one variety to the other, as well as within each variety.
• The commercial Bt cotton available today contains genes from
the isolate B. thuringiensis, ssp kurstaki that produces Cry1Aa,
Cry1Ab, Cry1Ac, Cry2A

• The choice of Bt genes depends upon the crop and the
targeted pest, as most of the Bt toxins are insect group
specific.
• For example, the proteins encoded by the genes:-
• Cry1Ac and Cry2Ab control the cotton bollworms,
• Cry1Ab controls corn borer,
• Cry3Ab controls Colarado potato beetle
• Cry3Bb controls corn rootworm.

• The advantages of the Cry1Ac in Bollgard®, over the Bt
cotton spray
• Active protein provides moderate to high dose control that
allows fair to excellent control of selected important
lepidopteran pests
• Active protein expressed in all plant parts
• Active protein expressed throughout the season
• Wash off of insecticide during rain, and degradation in
sunlight are not issues as they are with spray formulations
• Less farmer exposure to insecticide
• Labour saving technology, due to reduction of insecticide
sprays

Gene Stacking !!
• In order to compound the benefits, more than one gene is used in the
development of a transgenic, by gene stacking or pyramiding.
• Bollgard I, the predominantly cultivated pest tolerant cotton, contains
only one gene, the Cry 1Ac, from Bacillus thuringiensis.
• Bollgard II contains the Cry 2 Ab gene, in addition to Cry 1 Ac.
• The Bollgard II event developed by Mahyco-Monsanto Biotech Ltd,
(MMBL) is technically designated as MON 15985.
• Bollgard I offers protection against only the major cotton pest, the
American bollworm (Helicoverpa armigera),
• Bollgard II provides season long control of key pests of cotton
including Spodoptera and Heliothis pests.

Bt cotton … implementation !
• Transgenic Bt cotton containing Cry1Ac was originally
developed using the American cotton variety Cocker
312, and this variety is not suitable for cultivation
outside America.
• Different local varieties of cotton are chosen for
developing transgenic Bt cotton varieties, incorporating
the same Cry1Ac event, for cultivation in different agroclimatic
zones in different countries.

• Bt cotton was first adopted commercially in 1996.
• In 2002 it was deployed commercially in nine countries,
seven of which were developing countries (China, India,
Indonesia, Argentina, Mexico, South Africa, and Colombia
[pre-commercial]), and two industrial countries (USA and
Australia).
Bt cotton was actually already on the Indian market as
early as 1998, well before it was approved for commercial
introduction in March 2002 !!!!!!

Bt cotton – in India !!
• Bt cotton, which confers resistance to important insect pests of cotton,
was first adopted in India as hybrids in 2002.

• The deployment of Bt cotton over the last eight years has resulted in
India becoming the number one exporter of cotton globally as well as
the second largest cotton producer in the world
• Almost a doubling of yield from 308 kg per hectare in 2001 to 568
kg/ha in 2009
• India is the only country to grow all four species of cultivated cotton
Gossypium arboreum and G. herbaceum (Asian cottons), G.
barbadense (Egyptian cotton) and G. hirsutum (American upland
cotton).
• Gossypium hirsutum represents more than 90% of the hybrid cotton
production in India and all the current Bt cotton hybrids are G.
hirsutum

• The first two-gene event MON15985, commonly known as
Bollgard®II (BG®II) was developed by Mahyco and sourced from
Monsanto, featured the two genes cry1Ac and cry2Ab, and was
approved for sale for the first time in 2006 – four years after the
approval of the single gene event MON531 Bt cotton hybrids in
2002-03.
• Farmers prefer multiple genes over a single gene Bt cotton hybrids
because multiple gene Bt cotton hybrids provide additional
protection to Spodopetra (a leaf eating tobacco caterpillar) while it
also increases efficacy of protection to both American bollworm,
Pink bollworm and Spotted bollworm.

• The first indigenous, publicly-bred Bt variety Bikaneri
Nerma (BN) and hybrid NHH-44Bt (expressing event BNLA-
601) were commercialized for the first time in 2009.
• They are unique because they are the first Bt cotton hybrid
and variety to be bred by a group of Indian public sector
institutes which include the Central Institute for Cotton
Research (CICR), Nagpur and National Research Centre for
Plant Biotechnology (NRCPB), New Delhi of the Indian
Council of Agricultural Research (ICAR) in partnership with
the University of Agricultural Sciences (UAS), Dharwad.

Benefits from Bt cotton in India
• India enhanced farm income from Bt cotton by US$5.1 billion in
the period 2002 to 2008 and US$1.8 billion in 2008 alone.
• A 2007 study ―Socioeconomic impact of Bt cotton‖, conducted by
the Centre for Economic and Social Studies (CESS), Hyderabad
concluded that the Bt cotton technology was superior to the
conventional cotton hybrids in terms of yield and net returns.
• Farmers noted that Bt cotton allowed earlier picking due to less
pest susceptibility, and the boll colour was superior.

PROBLEMS !!!
• Examples !!
• In Gujarat, home of the N-151 variety, Mahyco–Monsanto‘s
Bt varieties performed miserably during the first year of
planting.
• An official monitoring committee set up by the state
government reported that farmers in Gujarat ―suffered a
huge economic loss in the cultivation of Bt cotton‖ during
due to crop‘s susceptibility to wilt and sucking pests.
• In March 2010, Monsanto admitted that insects have
developed resistance to Bt cotton plant in Gujarat !

Environmental Impacts !!!
• Target pests, cotton bollworm (Helicoverpa armigera) and pink
bollworm (Pectinophora gossypiella), were effectively controlled
while the mirids (Hemiptera: Miridae) evolved to be key pests in
the cotton system.
• Changes in the populations of secondary pests of target insects
such as aphids, spider mites and mirids, leading to new problems
in controlling cotton pests.
• Occurrence of resistant target pests lead to ineffectiveness of Bt
cotton
• Several strains of cotton bollworm resistant to Bt cotton have
been obtained

Concerns !!!!
• Increased Pest Resistivity
• Gene flow to wild relatives
• Inability to address complexity of pest attack
• The total input cost is higher with no increase in yield

• India is not the first country to experience problems with Bt
cotton.
• In Indonesia, during an initial planting in 2001, crops of
Monsanto‘s Bollgard cotton were devastated by pests, while
other cotton crops suffered insignificant damage.
• Angry Indonesian cotton farmers, who had paid big money for
the Bt seeds, burned their fields in protest and forced
Monsanto to withdraw Bt cotton from the country after only
two seasons on the market

•Send
Solution ??
• a refuge of common cotton is employed to manage the resistance risk
in target pests
• The GE technology of Bt Cotton cannot directly increase yields
• It also does not affect a whole class of secondary pests, which are
immune to the Bt toxin.
Out of the frying
pan...?
The Indian farmer
seems to be
caught between
the green
revolution and the
new gene
revolution.

Wont affect soil !! And No harm to beneficial insects…
• Measurement of the amount of the protein CrylAc from the soil in
the continuous Bt cotton-growing fields from Henan, Jiangsu,
Hubei and Shandong provinces showed that multi-year planting of
Bt cotton would not lead to the accumulation of the Bt toxic
protein in the soil and thus would not affect biodiversity (Li et al.,
2005)
• Bt cotton has minimal or no effect on beneficial insects, including
honey bees, lady beetles, spiders, big eyed bugs, pirate bugs, and
parasitic wasps
• Theoretically, Bt cotton may indirectly lower the general
abundance of some beneficial insects, since it causes caterpillar
populations to decline !!!!

Supplemental insecticide treatment needed !!
• In Bt cotton, all insect pests not affected by the Bt toxin should be
managed as they would be in non-Bt cotton.
• It may be necessary to apply insecticide to control thrips, aphids,
boll weevils, tarnished plant bugs, stink bugs, and a few tolerant
caterpillars such as beet armyworms and fall armyworms !!!!
• To prevent resistance --- refugium !!
• 80:20 external sprayed refuge
• 95:5 external structured unsprayed refuge
• 95:5 embedded refuge

Problems remain unsolved !
• For the Bt technology to be successful, Monsanto stipulates that
the farmer has to set aside about 20 % of his acreage for non-Bt
cotton.
• This is essential so that the bollworm can feed partly on non
poisonous, normal cotton and remain susceptible to the Bt toxin.
• In the US with 10,000 to 30,000 acre holdings, wasting 20 % of
the acreage, even more if needed is not an issue.
• In India, with its small land holdings, the economics of Bt cotton
cannot work after setting aside 20% as an insect refuge.

• There are many kinds of cotton pests in India apart from the
bollworm.
• The use of pesticides will have to continue because
spraying will be needed to kill these other pests.
• Pesticide use will also continue because as in all tropical
countries, pest attack is far more intense and the number of
insects per acre will be far higher than in colder countries.
• It is unlikely the Bt strategy alone will be effective in
controlling the intense pest attacks common in the
tropics.

Against Bt Cotton ??
Current Science article !!

The story of the first four years of Bt cotton farming in India was
neatly summarised by P.V. Satheesh, Convenor of the Andhra
Pradesh Coalition in Defence of Diversity:
“In the first year (2002), Bt cotton was a disaster, yielding 35 per
cent less than the non-Bt cotton, even while costing four times
more than the non Bt cotton. In the third year, new diseases spread
through the soils and the plant. Cattle which grazed Bt cotton
plants started dying. And this year [2006], Bt plants have started
wilting, forcing farmers to harden their hearts and uproot them. In
the village of Mustyalapally, in the Bhongir mandal of Nalgonda,
farmers have uprooted Bt cotton from 41 out of the 51 acres
planted. The disease has spread to nearby villages, spreading
panic among farmers. Farmers complain that the plants are slowly
dying one after another because the root system is severely
decomposed, without any secondary and tertiary roots on the main
root system. Even the bolls formed on these wilted plants did not
bear any seeds.”

• For Monsanto and other transnational pesticide companies, Bt
crops are essentially an ingenious way to expand their profits in
the face of increasing competition from generic producers of
off-patent insecticides.
• Instead of selling a chemical pesticide that farmers spray,
Monsanto sells the pesticide by way of the seeds.
• And there is another advantage for the companies: farmers
growing Bt crops still rely on pesticides, and, when the costs of
the Bt technology fees are factored in, they generally end up
spending more overall to manage pests – which is good for the
pesticide makers‘ bottom line.

Conclusion…..
• The tragic fact is that the real farmers, the mass of
India‘s farming community has no idea what Bt cotton is
nor what GM crops are !!!
• Monitoring and regulation of a GM crop has been
entrusted to the very same company that is producing
and selling the GM variety!
• Mahyco- Monsanto will be monitoring the performance
of Mahyco– Monsanto !!!

• Resistance in the pest is bound to come, it is natural.
• Of course it is! But not in a few years.
• If resistance is going to come in just two to three years, does
the variety have any relevance for the farmers??
• Because when the variety fails that fast, the farmers are even
worse off than they were.....

For ?!?
• The causes for Bt cotton – problems lie not in the Bt technology
per se, but in management.
• All the important players such as the Government (not controlling
illegal and spurious seed and no seed certification policy), Bt
event developers (not choosing appropriate varieties for specific
regions), the seed dealers (insufficient post-sale guidance and
crop monitoring), and the farmers (cutting edges and not adopting
appropriate cultivation practices), have contributed to certain
deficiencies in the crop outcome.

enefits of Bt cotton ??
• Good control of bollworm species
• Significantly higher boll retention and more yield than the
control
• Reduction in chemical sprays for bollworm control (50% less
than that required for conventional commercial hybrids.
• Substantial increase in net income to farmers.
• No adverse impact on non-target organisms and the adjacent
non Bt cotton or other crops.

Summary !!
• Bacillus thuringiensis ...
• Cry proteins..
• Bt cotton … gene construct.
• Benefits
• Problems…
• Importance of proper management

THANK YOU !!!