The development and commercialization of insect-resistant transgenic Bt crops expressing Cry toxins revolutionized the history of agriculture. At the end of 2010, an estimated 26.3 million hectares of land were planted with crops containing the Bt gene (James 2011). Bt cotton has reduced the use of traditional insecticides by 207,900,000 lbs of active ingredient of insecticide (Brookes and Barfoot, 2006). Resistance is a genetic change in the insect pest — that allows it to avoid harm from Bt toxins. The high and consistent levels of ICP production in the Bt plants make them much less favorable for the development of resistance. Insect Resistance Management is of great importance because of the threat insect resistance poses to the future use of Bt plant-incorporated protectants and is said to be the key to sustainable use of the genetically modified Bt crops. The US EPA usually requires a “buffer zone,” or a structured refuge of 20% non-Bt crops that is planted in close proximity to the Bt crops. First documented case of insect resistance to Bt cotton came in 2008, when Tabashnik and coworkers found field-evolved Bt toxin resistance in bollworm, Helicoverpa zea (Boddie), in the United States. Field-Evolved Resistance to Bt Maize by Western Corn Rootworm (Gassmann, 2011) displayed significantly higher survival on Cry3Bb1 maize in laboratory bioassays. Expanded use of transgenic crops for insect control will likely include more varieties with combinations of two or more Bt toxins (pyramiding), novel Bt toxins such as VIP, modified Bt toxins that have been genetically engineered to kill insects resistant to standard Bt toxins. Transgenic plants that control insects via RNA interference are also under development. Increasing use of transgenic crops in developing nations is likely, with a broadening range of genetically modified crops and target insect pests .Incorporating enhanced understanding of observed patterns of field-evolved resistance into future resistance management strategies can help to minimize the drawbacks and maximize the benefits of current and future generations of transgenic crops.

1. Surender yadav
2009BS41D

2. Introduction to Bt toxins
 Bacillus thuringiensis (Bt) is a common gram positive, spore-forming,




soil bacterium.
When resources are limited, vegetative Bt cells undergo sporulation,
synthesizing a protein crystal, the insecticidal crystal proteins (ICPs) or
Cry Proteins.
For over 50 years, Bt has been applied to crops in spray form as an
insecticide, containing a mixture of spores and the associated protein
crystals.
The development and commercialization of insect-resistant transgenic
Bt crops expressing Cry toxins revolutionized the history of
agriculture.
Benefits
 High specificity and potency,
 Reduction in chemical pesticide applications,
 Increased crop yield.

3. Structure of Bt toxin
Cry protein domains
•Domain A - Pore formation
6α helices (250 aas)
•Domain B -receptor binding domain,
β-sheets (200 aas)
•Domain C - β sandwich (150 aas)
protects the toxin from protease

4. Bt mode of action

5. Bt Toxin Nomenclature:
Each Bt toxin will be assigned a unique name incorporating four ranks
e.g. Cry 1Aa3
•Primary rank - order of insect;
•Secondary and tertiary ranks - potency and targeting within an order
•Quaternary rank- alleles of genes coding for toxins
•Classification based on their sequence homology and specificities
•CryI genes encoded proteins toxic to lepidopterans;
•CryII genes encoded proteins toxic to both lepidopterans and dipterans;
•CryIII genes encoded proteins toxic to coleopterans;
•CryIV genes encoded proteins toxic to dipterans alone.
Crickmore , 1998

6. Introduction to Bt plants
 Bt plants have genes for the Bt toxins engineered to produce ICP toxic
to the pest species of concern.
 As the insect feeds on the Bt plant, it ingests the ICP and suffers the
same fate as if it ingested leaf tissue sprayed with Bt.
 At the end of 2010, an estimated 26.3 million hectares of land were
planted with crops containing the Bt gene globally(James 2011).
The chief advantages to Bt plants:
 The pests hiding inside plant parts controlled effectively;
 Multiple sprays are not needed;
 The dose of Bt can be more effectively regulated.
 A disadvantage of Bt plants is that insect-specific ICPs cannot be
changed during a growing season.

9. Insect resistance
 Resistance is a genetic change in the insect pest — that allows it to
avoid harm from Bt toxins.
 Only two insect species that have developed resistance to Bt foliar
sprays under commercial situations — the diamondback moth and the
cabbage looper.
 The high and consistent levels of ICP production in the Bt plant make
them much less favorable for the development of resistance,
 enough to kill the SS and RS insect genotypes, and such a dose is
impossible to maintain with foliar sprays.
 In the years prior to the development of resistance- substantial
environmental and human health benefits.
 Bt cotton has reduced the use of traditional insecticides.

10. Insect Resistance Management
 The practices aimed at reducing the potential for insect pests to
become resistant to a pesticide.
 Bt IRM is of great importance because of the threat insect resistance
poses to the future use of Bt plant-incorporated protectants.
Risk factors for pest populations evolving Bt resistance:
 Great genetic diversity in pest populations
 Sexual recombination
 Constitutive production of toxins
 Intense selection pressure on pest population
 IRM is said to be the key to sustainable use of the genetically modified
Bt crops.

11. Managing Bt resistance
 The US EPA usually requires a “buffer zone,” or a structured refuge of
20% non-Bt crops that is planted in close proximity to the Bt crops.
 “High dose plus refugia”
 Plants express enough Bt protein to kill all except rare homozygous
recessives (RR)
 Heterozygous offspring, produced when homozygous resistant
insects mate with susceptible insects, are killed
 Refugia “dilute out” heterozygous resistant individuals (RS)
 Assumption: initially, resistant RS mutants are very rare
 As for insects with recessive alleles for such genes, they are
thought to be “diluted out” by susceptible insects from the
refugia.

12. Why does adding susceptible plants
(refuges) slow evolution of Bt resistance?
 Bt resistance as recessive:
need to lose/mutate both copies of the
receptor gene to become resistant.
 Refuge is more effective the less dominant that Bt resistance is because
the RS genotypes don’t survive well.
 The development of resistance is driven by the initially very rare RR
genotypes, but for a long time they only have the RS types to mate with.
 Planting refuges minimize the differential in fitness between the more
and less resistant genotypes will slow evolution of resistance.

13. Seed mixtures strategies
 Seed mixture strategy involves random mixing of 20% non-Bt plants
among Bt plants.
 Poor compliance of the Indian farmers to grow refuge crop in Bt cotton
fields.
 Commercial packets of Bt seeds will also contain non-transgenic seeds
premixed.
 However, the strategy of seed mixtures can become ineffective and
unproductive if the frequency of resistant insect pests has already
become unmanageable.

14. Methods of growing Bt and non-Bt plants
for Bt resistance management.
Structured refugia contain two rows
of non-Bt plants (bold dotted lines)
for every eight
rows of Bt plants
Seed mixture strategy involves
random mixing of 20% non-Bt
plants (bold dots) among Bt plants.
Vageeshbabu ,2011

15. Evolution of resistance to Bt toxin
 Although there were no cases of insects developing resistance to Bt
transgenic plants in the field,
 laboratory populations of Cry1A-resistant DBM have been shown to be
able to survive on high levels of Cry1Ac
 In cases where resistance to Bt crops has evolved quickly, one or more
conditions of the refuge strategy have not been met.

16. First documented case of pest resistance to
Bt cotton
 Tabashnik, (2008) observed that the frequency of resistant alleles has
increased substantially and that there is field-evolved Bt toxin
resistance in bollworm, Helicoverpa zea (Boddie), in the United States.
 The concentration of Cry1Ac in Bt cotton was not high enough to kill
the hybrid offspring produced by matings between susceptible and
resistant H. zea.
 Thus, the so-called “high dose” requirement was not met .
 In a related case, failure to provide adequate refuges of non-Bt cotton
allowed the pink bollworm to evolve resistance to Bt cotton in India
(Bagla 2010).

17. Field-Evolved Resistance to Bt Maize by
Western Corn Rootworm (Gassmann,2011)
 Fields experiencing severe rootworm feeding contained Cry3Bb1 maize.
 These displayed significantly higher survival on Cry3Bb1 maize in
laboratory bioassays.
 A significant positive correlation between the number of years Cry3Bb1
maize had been grown in a field and the survival of rootworm
populations on Cry3Bb1 maize in bioassays.
 However, there was no significant correlation among populations for
survival on Cry34/35Ab1 maize and Cry3Bb1 maize, suggesting a lack of
cross resistance between these Bt toxins.
 Insufficient planting of refuges and non-recessive inheritance of
resistance may have contributed to resistance.

18.  To engineer crops that express at least two toxic compounds that act





independently, so that resistance to one does not confer resistance to
the other.
This approach, called gene pyramiding, became a commercial reality in
2003 with the introduction of Bollgard II,
A transgenic cotton plant that expresses the original Bt protein, Cry1Ac,
and a second Bt protein, Cry2Ab.
The two proteins act independently in that they bind to different
receptors in the insect’s midgut.
Insects homozygous for one resistance gene are rare, insects
homozygous for multiple resistance genes are extremely rare (Karim
et al. 2000)
A species cannot easily evolve resistance to both toxins because that
would require two simultaneous, independent mutations in genes
encoding the receptors (Jackson et al., 2003).

19. SmartStax corn
 The multitoxin Bt crops are designed to help delay resistance and to kill
a broader spectrum of insect pests.
 SmartStax corn has eight GE traits ‘stacked’ together – 6 for insect
resistance (Bt) and 2 for herbicide tolerance.
 Tolerance to aerial pests (three Bt genes): Cry 1A.105 (Monsanto), Cry
2Ab2 (Monsanto) and Cry 1F (Dow).
 Tolerance to subsoil pests (three Bt genes): Cry 3Bb1 (Monsanto), Cry
34Ab1 (Dow) and Cry 35Ab1 (Dow).
 Tolerance to herbicides (two genes): Glyphosate (Monsanto) and
Glufosinate (Dow).

20. Limitations of Gene Pyramid
 Greater the number of genes, more plant protein will be diverted
away from creating useful yield.
 This scenario sets the risk of significant agronomic and yield
penalties which may make the variety unattractive to the grower.
 One toxin can bind to several sites. Such a scenario can lead to
the development of cross resistance or multiple resistance of an insect
in cases where it was never exposed to the original toxin.

21. Mechanisms of Bt toxin resistance
 Bt works by binding to toxin receptor (cadherin),
which triggers cleavage of Bt protein
 Bt-resistant insects express mutated cadherin
proteins that do not bind toxins.
 Modified toxins can make resistant cadherinmutated insects susceptible again (Soberon et
al, Science, 2007)
 Toxins with independent actions bind to different
sites
 Multiple resistance: one toxin can bind to several
sites (e.g., insect develops resistance to multiple
Bt toxins after repeated exposure to one)

22.  Cadherin gene silencing with RNAi in tobacco hornworm resulted in






reduced susceptibility to the Bt toxin Cry1Ab, confirming cadherin’s role in
Bt toxicity.
The binding of protease-activated toxin to cadherin is essential for the
removal of helix α-1, which in turn promotes oligomerization.
Modified Cry1Ab and Cry1Ac toxins lacking helix α-1 (referred to as
Cry1AbMod and Cry1AcMod) could form oligomers without cadherin.
The modified toxins killed cadherin-silenced hornworm and Bt-resistant
pink bollworm that had cadherin deletion mutations.
Conversely, against susceptible larvae, the native toxins were more potent
than the modified toxins.
This implies that modified toxins had lower stability in the midgut,
reduced oligomer-forming ability, or reduced ability of oligomers to
ultimately cause mortality.
These findings demonstrate that the modified Bt toxins may be useful
against pests resistant to standard Bt toxins.

23. Effect of RNAi on cadherin protein expression

24. Responses of susceptible (APHIS-S) and resistant (AZP-R)
pink bollworm larvae

25. Efficacy of genetically modified Bt toxins against insects
with different genetic mechanisms of resistance.
 Relative to native toxins, the potency of modified toxins was >350-fold
higher against resistant strains of DBM(Px) and European corn borer
(On) in which resistance was not linked with cadherin mutations.
 Conversely, the modified toxins provided little or no advantage against
some resistant strains of three other pests with altered cadherin.
 Independent of the presence of cadherin mutations, the relative
potency of the modified toxins was generally higher against the most
resistant strains.
Tabashnik (2011)

26. Resistance to six species of insect pests

27. Potency of modified Bt toxins relative to
native Bt toxins
 The reductions in resistance ratio for modified toxins relative to native




toxins occurred because modified toxins were more potent than native
toxins against resistant strains in four of six cases
and less potent than native toxins against susceptible strains in all cases
For example, against the resistant strain of DBM, potency was >350fold higher for Cry1AbMod than for Cry1Ab, and >540-fold higher for
Cry1AcMod than for Cry1Ac.
However, against the susceptible strain of DBM, each modified toxin
was less potent than the corresponding native toxin.
Cry1AcMod was less potent than Cry1Ac against resistant strains of
Bollworm and Sugar cane borer.

28. Suppressing resistance to Bt cotton with sterile
insect releases
 An alternative strategy for delaying pest resistance to Bt crops where
sterile insects are released to mate with resistant insects and refuges are
scarce or absent.
 Unlike the refuge strategy, this approach does not require maintenance
of pest populations and thus compatible with eradication efforts.
 During a large scale, four-year field deployment of this strategy in
Arizona, resistance of pink bollworm to Bt cotton did not increase.
 A multitactic eradication program that included the release of sterile
moths reduced pink bollworm abundance by >99%, while eliminating
insecticide sprays against this key invasive pest.
Tabashnik (2010)

29.  Many plant secondary metabolites are toxic to or repel insects,
enabling host plants to escape from insect herbivores (Gatehouse
2002).
 To counteract plant defenses, insects have developed adaptive
mechanisms, which often involve a set of genes whose products
metabolize the chemicals from plants (Wittstock et al. 2004).
 Most cotton cultivars accumulate gossypol in both aerial tissues and
roots, and these phytoalexins form a chemical arsenal against
herbivorous.

30.  They
isolated a P450 monooxygenase gene, CYP6AE14, from
Helicoverpa armigera
 Expression of CYP6AE14 was induced by gossypol, and its expression
level was correlated with larval growth when gossypol was present in
the diet.
 When bollworms were fed on transgenic Arabidopsis plants producing
dsRNA against CYP6AE14 (dsCYP6AE14), expression of CYP6AE14 was
suppressed;
 After transferring to a gossypol-containing diet, the larvae showed
decreased tolerance to gossypol .
 Therefore, cotton plants are engineered to express dsCYP6AE14, which
indeed acquired enhanced resistance to cotton bollworms.

31. Effect of T1 transgenic cotton on larvae growth

32. qRT-PCR analysis of CYP6AE14 transcripts in
midgut of second-instar larvae

33. Effect of ds6-3 T2 plants on larvae growth

34. Transgenic cotton plants were less damaged by
bollworms than the control

35.  The dsCYP6AE14 cotton plant did have deleterious effects on
bollworms, but was not lethal.
 If multiple genes involved in the P450 complex were targeted by RNAi,
the deleterious effects would be magnified.

36. VipCot cotton
 EPA has conditionally registered a new cotton plant-incorporated
protectant, VipCot, of Syngenta Seeds Inc.
 VipCot produces the modified Cry1Ab and Vip3Aa19 proteins derived
from Bacillus thuringiensis (Bt) to control lepidopteran pests.
 The Vip3Aa19 protein expressed in VipCot cotton provides a unique
mode of action.
 When coupled with modified Cry1Ab in VipCot, the proteins have the
potential to provide benefits for IRM including:
 High-dose (for both proteins expressed together) against the major target
pests,
 Lack of cross-resistance (Vip3Aa19),
 The potential to delay development of resistance in other cotton varieties
expressing Cry toxins.
 VipCot (COT102 x COT67B) was developed by conventional breeding
of COT102 (Vip3Aa19) plants with COT67B (modified Cry1Ab) plants.

37.  The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses
midgut epithelium cells of susceptible insects.
 Vip3Aa19 protein is intended to control several lepidopteran pests of
cotton including tobacco budworm, cotton bollworm, fall armyworm,
beet armyworm and cabbage looper.
 There is no evidence of either a synergistic or antagonistic interaction
between Vip3Aa19 and modified Cry1Ab in cotton bollworm or tobacco
budworm.
 It demonstrate that data on the individual events and individual
proteins can be used to support the safety of the COT102 x COT67B
(VipCot) combined product.

38. Future of Bt crops in INDIA
 Whether GM Food is required or not for the country?
 if the perception is not clear; it is going to affect ongoing research.
 Need for additional biosafety studies to assess the safety of Bt protein?
 Need for setting up an independent GMO testing facility devoid of conflict
of interest?
 Limited release of Bt seeds to identified farmers under strict expert
supervision should be undertaken to evaluate its performance in public
space?
 The Biotechnology Regulatory Authority of India Bill is approved by the
Government which will replace GEAC.
 The Bill seeks to create a new body to regulate research, manufacture,
import and use of products of modern biotechnology.

39.  The adoption of biotech crops in the next five years period will be
dependent mainly on three factors:
 the timely implementation of appropriate, responsible and cost/time-
effective regulatory systems;
 strong political will and support;
 a continuing wave of improved biotech crops that will meet the priorities of
industrial and developing countries in Asia, Latin America and Africa.

40. Conclusion
 Together with the reduction of pesticide application and cost
reduction, Bt crops have brought tremendous benefit to both the
environment and farmers .
 Expanded use of transgenic crops for insect control will likely include
more varieties with combinations of two or more Bt toxins, novel Bt
toxins such as VIP
 Modified Bt toxins that have been genetically engineered to kill insects
resistant to standard Bt toxins.
 Transgenic plants that control insects via RNA interference are also
under development.

41.  Increasing use of transgenic crops in developing nations is likely, with a
broadening range of genetically modified crops and target insect pests .
 Incorporating enhanced understanding of observed patterns of field-
evolved resistance into future resistance management strategies can
help to minimize the drawbacks and maximize the benefits of current
and future generations of transgenic crops.