Plant Biotechnology Journal (2011) 9, pp. 283–300 doi: 10.1111/j.1467-7652.2011.00595.

Review article
Bacillus thuringiensis: a century of research,
development and commercial applications
Georgina Sanahuja1,†, Raviraj Banakar1,†, Richard M. Twyman2, Teresa Capell1 and Paul Christou1,3,*
1
Department of Plant Production and Forestry Science, ETSEA, University of Lleida, Lleida, Spain
2
Department of Biological Sciences, University of Warwick, Coventry, UK
3
Institució Catalana de Reserca i Estudis Avançats, Passeig Lluı´s Companys 23, Barcelona, Spain

Received 20 November 2010; Summary

revised 28 December 2010; Bacillus thuringiensis (Bt) is a soil bacterium that forms spores during the stationary
accepted 5 January 2011.
*Correspondence (fax +34 973 70 29 24;
phase of its growth cycle. The spores contain crystals, predominantly comprising one
email [email protected]) or more Cry and ⁄ or Cyt proteins (also known as d-endotoxins) that have potent and
†
These authors contributed equally to this
specific insecticidal activity. Different strains of Bt produce different types of toxin,
article.
each of which affects a narrow taxonomic group of insects. Therefore, Bt toxins
have been used as topical pesticides to protect crops, and more recently the proteins
have been expressed in transgenic plants to confer inherent pest resistance. Bt trans-
genic crops have been overwhelmingly successful and beneficial, leading to higher
yields and reducing the use of chemical pesticides and fossil fuels. However, their
deployment has attracted some criticism particularly with regard to the potential
Keywords: Bacillus thuringiensis, evolution of pest-resistant insect strains. Here, we review recent progress in the
topical pesticide, transgenic plants, development of Bt technology and the countermeasures that have been introduced
toxin, selection pressure, resistance. to prevent the evolution of resistant insect populations.

formation is conferred by genes carried on a plasmid. The

Introduction
genes, which encode Cry ⁄ Cyt proteins, become active dur-
The insecticidal properties of Bt were recognized many ing sporulation because they are controlled by a dedicated
years before the bacterium was identified, with some RNA polymerase that is also synthesized specifically while
accounts suggesting that Bt spores may have already been spores are forming. Up to 20% of the spore protein con-
in use in ancient Egypt. In the modern era, the bacterium tent is represented by these Cry ⁄ Cyt toxins (Aronson,
was isolated in 1901 by the Japanese biologist Shigetane 2002).
Ishiwatari during an investigation into wilt disease in silk The insecticidal properties of the crystals were discov-
worms, and he named it Bacillus sotto. Ten years later, the ered when dead flour moth caterpillars were found to be
same bacterium was isolated by Ernst Berliner from a dis- loaded with spores and crystals. Direct contact between
eased Mediterranean flour moth (Ephestia kuehniella) in the spores ⁄ crystals and healthy caterpillars had no effect,
the German province of Thuringia, and it was named Bacil- but when the spores and crystals were coated onto leaves,
lus thuringiensis (Siegel, 2000). The defining feature of Bt is the caterpillars stopped feeding and died. After recogniz-
its ability to produce proteinaceous crystals during sporula- ing the potential of Bt as an insecticide, Mattes (1927) iso-
tion. Bt is a member of the Bacillus cereus group of Gram- lated the Bt strain discovered by Ernst and subsequent
positive, spore-forming soil bacteria, and occasionally the field trials against the European corn borer (Ostrinia
bacteria lose their ability to form crystals and then become nubilalis) gave promising results (Husz, 1930). This work
indistinguishable from B. cereus itself. Similarly, B. cereus eventually led to the development of Sporeine, a commer-
can be transformed into Bt, and investigations into the cial Bt insecticide, which was used for the first time in
transformation mechanism led to the discovery that crystal 1938.

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd 283
284 Georgina Sanahuja et al.

towards different invertebrates. For example, Bt strains

The biology of Bt toxins
containing only Cry5- and Cry6-type toxins are active
Sporeine targeted lepidopteran insects, and the active against nematodes.
ingredient was initially thought to be a food-borne inva- At the genus and species level, it is more useful to clas-
sive pathogen. However, this theory was discarded when sify Bt strains functionally on the basis of which toxin pro-
direct injection of crystals into caterpillars was shown to teins they produce, as this is a more logical way to define
have no effect. Careful time-course analysis of the guts of the host range. The toxins can be described in terms of
caterpillars feeding on contaminated food revealed the their amino acid sequences, protein structures and modes
disruption of cilia on the brush border of midgut epithelial of activity (Crickmore et al., 1998). Cry toxins interact with
cells shortly after ingestion of the crystals, followed by cell specific receptors located on the surface of midgut epithe-
swelling and lysis. The gut contents (including bacterial lial cells and are activated by host proteases following
spores) were thus released into the body cavity, allowing receptor binding, resulting in the formation of a pre-pore
bacteria to breed. Once the supply of nutrients was oligomeric structure that is insertion competent. In con-
exhausted, the bacteria formed spores that could spread trast, Cyt toxins directly interact with membrane lipids and
to a new host feeding on either the cadaver or the con- insert into the membrane. The known Cry and Cyt pro-
taminated vegetation. teins now fall into 32 sets including Cyt1, Cyt2 and Cry1
These early experiments showed that Bt toxins needed to Cry67 (Crickmore et al., 2010; Figure 1).
to be activated in the gut, and it was soon discovered that Despite their sequence diversity, all Cry proteins share
the critical factors were an alkaline environment and the a similar overall tertiary structure, as exemplified by the
presence of specific proteases, which cleaved the innocu- six structures solved thus far by X-ray crystallography
ous pro-toxin into its active form. Once activated by prote- (Cry1Aa, Cry2Aa, Cry3Aa, Cry3Bb, Cry4Aa and Cry4Ba)
olysis, each toxin binds to receptors in the brush border (Figure 2). The C-terminal portion is involved in crystal
membrane and causes pores to open, disrupting the formation but is not part of the mature toxin, as it is
movement of solutes across the gut epithelium and caus- cleaved off in the insect gut. The N-terminal portion is
ing the influx of water. The toxins were shown to be orally the toxin itself, and it comprises three domains. Domain I
lethal to caterpillars in pure form, and the pro-toxins could is a bundle of seven a-helices, six of which are amphi-
be converted into active toxins in vitro, using specific pro- pathic encircling the seventh hydrophobic helix, and this
teases under alkaline conditions. The requirement for alka- domain is responsible for membrane insertion and pore
line conditions, specific proteases and specific receptors formation. Domain II consists of three anti-parallel
explains why Bt is harmless to mammals (which have an b-sheets with exposed loop regions, and domain III is a
acidic gut and lack the corresponding receptors) and why b-sandwich. Both domains confer receptor binding speci-
each toxin has a narrow host range. ficity thus helping to define the host range (Boonserm
et al., 2006).
A current model suggests that domains II and III initially
Toxin structure and specificity
bind to primary receptors (cadherins) which cleave the
Many researchers have attempted to introduce taxonomic toxin within domain I and induce oligomerization, which
classification systems for Bt, using various criteria such as in turn promotes binding to high-affinity secondary recep-
serotyping, phage susceptibility and plasmid profiles, and tors tethered to the membrane via C-terminal glycosyl-
this has resulted in the classification of approximately 100 phosphatidylinositol anchors (Soberón et al., 2009). The
subspecies. Although there is a good correlation between requirement for oligomerization has recently been con-
Bt subspecies and insect host range at the family level, the firmed through the isolation of dominant-negative muta-
relationship tends to break down at the genus and species tions of Cry1Ab (Rodrı́guez-Almazán et al., 2009). An
levels because most Bt strains can synthesize more than alternative model (Zhang et al., 2006) suggests that initial
one toxin, resulting in complex and overlapping host pro- binding triggers a Mg2+-dependent signalling cascade that
files. For example, most Bt kurstaki strains are specific for causes G-protein dependent cAMP accumulation and the
lepidopteran insects (butterflies and moths), whereas isra- activation of protein kinase A. Phylogenetic analysis has
elensis strains are specific for dipterans (flies) and morri- established that the diversity of the Cry family evolved by
soni strains are specific for coleopterans (beetles). Other the independent evolution of the three domains and by
strains are not active against insects at all, but are toxic swapping of domain III among toxins.

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 283–300
 A century of Bacillus thuringiensis 285

Figure 1 Phylogenetic trees representing (a) three-domain Cry proteins, and (b) related proteins (Cyt, Bin and Mtx).
Source: http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/. The phylogenetic trees are modified from a TREEVIEW visualization of NEIGHBOR treat-
ment of a CLUSTALW multiple alignment and distance matrix of the full-length toxin sequences, as described by Crickmore et al. (2010). The grey
vertical bars demarcate the four nomenclature ranks.

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 283–300
286 Georgina Sanahuja et al.

Figure 1 Continued

Process development began with improved fermentation

Development of Bt topical pesticides
and harvesting procedures, but perhaps the most impor-
Although Sporeine was used from 1938 in France, it was tant aspect was formulation. Liquid suspensions were the
not registered as a pesticide in the United States until most convenient to handle, but tended to deteriorate in
1961. By this time, other Bt products such as Thuricide storage, while powders were easier to store and transport
were already on the market, most based on kurstaki strain but drying was expensive and reformulation more compli-
HD1 and active against lepidopteran pests (Baum et al., cated for the end user. These challenges were addressed
1999). Despite their beneficial properties, these early Bt by the addition of excipients such as suspension agents to
products did not compete well against chemical pesticides prevent suspensions settling and preservatives to increase
because of their poor performance. Commercial activities shelf life, and in the case of powders by adding chemicals
concentrated on two strategies to overcome these chal- to improve pouring and wetting. UV screening agents
lenges, namely process development to increase the effi- were also used, to prevent rapid photolysis after spraying
ciency of the Bt products, and strain improvement to (Burges and Jones, 1998a). These improvements, in addi-
increase the intrinsic toxicity of the bacteria. tion to more rigorous quality control and the standardiza-

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 283–300
 A century of Bacillus thuringiensis 287

Figure 2 Structure of three-domain Cry proteins. (a) Primary structure, showing domain organization of representative members of each Cry
family. (b) Conserved tertiary structure, showing the positions of the three domains. Source: de Maagd et al. (2001).

tion of potency testing, led to a sixfold or more increase Table 1 Bt topical products based on natural strains (Kaur et al.,
in efficacy in the field (Burges and Jones, 1998b). 2000). Bt kurstaki HD-12 has been renamed SA-11
Strain improvement in the 1960s led to the replacement Trade name Subspecies and strain Target insect
of many of the early products with new Bt strains that
were up to 10-fold more potent than their predecessors, Biobit Bt kurstaki HD-1 Lepidoptera
Dipel Bt kurstaki HD-1 Lepidoptera
and the search for new and better strains continues to this
Florbac Bt aizawai Lepidoptera
day. Most Bt products are derived from kurstaki HD1 (e.g. Costar Bt kurstaki SA-12 Lepidoptera
Biobit, Dipel and Thuricide) although other strains are Delfin Bt kurstaki SA-11 Lepidoptera
used to tackle lepidopteran pests (kurstaki SA-11, kurstaki Thuricide Bt kurstaki HD-1 Lepidoptera

SA-12), diptera (israeliensis) and coleoptera (tenebrionis) Tekar Bt israeliensis Diptera

Javelin Bt kurstaki SA-11 Lepidoptera
(Kaur, 2000). A selection of Bt topical pesticides is listed in
Bactimos Bt israliensis Diptera
Table 1. Vectolex GC Bacillus sphaericus Diptera
Strain search and assessment programs initially involved Bactospeine Bt kurstaki HD-1 Lepidoptera
bioassays and biochemical testing, but this has been Acrobe Bt israliensis Diptera
Novodor Bt tenebrionis Coleoptera
replaced by PCR testing for specific toxin signatures. This
Trident Bt tenebrionis Coleoptera
can determine whether increased potency is achieved by
higher toxin expression levels or the presence of a novel
toxin, the latter offering the prospect of controlling differ- previously unknown combinations of existing toxins, a pro-
ent ranges of pests (Kuo and Chak, 1996; Porcar and cess that can be implemented by conjugation or direct
Juárez-Pérez, 2003). Tailoring the host range can be transformation (González et al., 1982; Kronstad et al.,
achieved not only through the discovery of new toxins, 1983; Carlton and Gonzalez, 1985) (Table 2). Examples of
but also by creating new bacterial strains carrying combination pesticides produced by conjugation include

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 283–300
288 Georgina Sanahuja et al.

Table 2 Bt topical products based on transconjugant and Table 3 Current pesticides based on Bt (modified from Kaur, 2000)
recombinant strains (Baum et al., 1999)
Trade names Bt subspecies and strain Host range
Product Strain ⁄ genes Insert order
Able, Bactospeina, Condor, kurstaki Lepidoptera
Transconjugant strains Costar, CRYMAX, Cutlass,
Agree aizawai Lepidoptera Futura, Lepinox, Thuricide,
Condor kurstaki Lepidoptera Steward
Cutlass kurstaki Lepidoptera Florbac, Agree, Design, aizawai Lepidoptera
Design aizawai Lepidoptera Xentari
Foil kurstaki Lepidoptera ⁄ Coleoptera Costar kurstaki SA-12 Lepidoptera
Recombinant strains Foil, Raven kurstaki Lepidoptera ⁄
Raven Cry1Ac (x2), Cry3A Lepidoptera Coleoptera
Cry3Bb (imported) Thuricide, Biobit, Dipel, Foray, kurstaki HD-1 Lepidoptera
CRYMAX Cry1Ac (x3), Cry2A Lepidoptera Javelin, Vault
Cry1C (imported) M-Trak Pseudomonas Coleoptera
Lepinox Cry1Aa, Cry1Ac (x2), Cry2A Lepidoptera Mattch, MVP Pseudomonas Lepidoptera
Cry1F-1Ac (imported) Novodor, Trident tenebrionis Coleoptera

Foil, a product based on strain EG2424, which produces ‘advantage’ needs to be cited with caution, because Bt is
Cry1Ac from Bt kurstaki (active against the European corn only effective when present on the plant organs on which
borer), and Cry3A from Bt tenebrionis (active against the insects feed. Usually Bt is applied when early instar larvae
Colorado potato beetle, Leptinotarsa decemlineata) (Carl- are present, because older larvae are more tolerant. Bt
ton and Gawron-Burke, 1993). Strain EG2348 (created by sprays persist for only a few days on the leaf surface
Ecogen) synthesizes a combination of Cry proteins particu- because UV light, weather, the chemical environment of
larly active against specific lepidopteran pests that infest the leaf surface and the presence of proteinases contrib-
soybean crops, and this is the active ingredient of Condor. ute to the degradation of Cry proteins. Many spores are
It is not always possible to introduce plasmids by conjuga- washed off the leaf surface into the soil. There is no evi-
tion, particularly if this results in the coexistence of two dence to suggest Bt is dangerous to humans and other
plasmids with similar origins of replication in the same cell, mammals, and indeed the studies performed thus far sug-
as they would be incompatible. In such cases, the relevant gest Bt is one of the safest microbial products known.
genes can be inserted onto a different plasmid in Escheri- Given its extensive use, it is remarkable that only a single
chia coli using standard cloning methods and that plasmid injury sustained from the use of Bt products in the field
can be introduced into an existing Bt strain by artificial has been reported, the identification of Bt spores in the
transformation (Arantes and Lereclus, 1991). Today, the Bt corneal ulcer of a farmer whose face was splashed with
biopesticide market is dominated by Abbott Laboratories Dipel (Burges, 2001). Laboratory mice can survive subcuta-
(Chicago, IL) (since the acquisition in 1995 of Novo- neous injections of 106 spores, or 107 spores administered
Nordisk’s biopesticide business) and Novartis (created intranasally (Siegel, 2001), and although limited mortality
through the merger in 1996 of Ciba and Sandoz), together is evident when 108 spores are delivered intranasally, this
accounting for >70% of global production. The other 30% is equivalent to an exposure level of 1012 in humans, or
is divided among approximately 30 companies with over one billion times higher than the maximum ever encoun-
100 different Bt product formulations, most containing a tered in the field during spraying. Even so, concerns about
single Bt toxin but some combining up to five. the environmental persistence of Bt spores in soil and
water have encouraged research into different formula-
tions, including pure protein crystals that are applied in
Advantages and disadvantages of topical Bt
the same way as the spores. As these are even more vul-
pesticides
nerable to degradation than the spores, Cry proteins have
Topical Bt sprays are advantageous in terms of their been encapsulated in the bacterium Pseudomonas fluores-
safety, specificity and potency compared to chemical cens (e.g. in the Mycogen products MVP which targets
sprays, and are also biodegradable, which provides for a lepidopteran pests and M-Trak which targets coleopteran
large and competitive market (Table 3). However, this last pests). This encapsulation strategy protects the Cry protein

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 283–300
 A century of Bacillus thuringiensis 289

from UV light and chemical degradation, and allows large

The development of Bt transgenic crops
amounts of each Cry protein to be produced using high-
yielding expression constructs, but the bacteria do not per-
Early development
sist in soil or water for as long as Bt spores. Crop Genetics
International developed a similar strategy using Clavibacter One of the major disadvantages of topical Bt pesticides is
xyli var. cynodontis, an endophytic bacterium that can their short window of effectiveness, and the fact that
penetrate the vascular system of plants, and this confers inclement weather can render Bt sprays useless within a
resistance to the European corn borer (Lampel et al., matter of hours. Topical Bt sprays must therefore be reap-
1994). The advantages and disadvantages of topical appli- plied several times in a growing season to reach the entire
cation are listed in Table 4 (Kaur, 2000). larval pest population, increasing the amount of product
that needs to be used and the fuel needed for spraying.
Furthermore, topical sprays have little impact on so-called
Bacillus thuringiensis subspecies israelensis in
cryptic pests, i.e. sap sucking insects and larvae that feed
mosquito control
near the roots.
Bt var israeliensis was isolated in Israel in 1976, and was A potential solution to this problem was developed in
shown to be effective against dipteran larvae including the mid-1980s when scientists introduced Bt cry genes
blackflies and mosquitoes (Goldberg and Margalit, 1977). into tobacco and tomato plants and expressed the pro-
Therefore, whereas most Bt strains have been developed teins directly in plant tissues (Table 5). The Belgian com-
as topical pesticides for use in agriculture, Bt subsp. pany Plant Genetic Systems pioneered commercial interest
israelensis has been applied to water courses and stag- in Bt transgenic technology, but no Cry protein could be
nant pools to prevent the spread of malaria (Margalith detected in the first generation of experimental transgenic
and Ben-Dov, 2000; Fillinger et al., 2003). The toxins are plants (Fischhoff et al., 1987; Vaeck et al., 1987; Perlak
encoded by a megaplasmid called pBtoxis which contains et al., 1991). Experiments to determine the cause of low
the genes for four Cry proteins (Cry4Aa, Cry4Ba, expression levels concentrated on differences between
Cry10Aa, Cry11Aa) and two Cyt proteins (Cyt1Aa and prokaryotic and eukaryotic systems because recombinant
Cyt2Ba) (Berry et al., 2002). The Cyt proteins interact Cry proteins were expressed at high levels in heterologous
directly with the lipid membrane in the larval midgut and bacteria.
act as receptors for the Cry toxins, so that the two act in A large difference in average GC content was noted
synergy. Product development has shown that formula- between Bt and plant DNA, plus differences in codon
tions containing smaller particles are the most effective preference that could account for low-efficiency protein
because they remain suspended for longer and are easily synthesis. The bacterial genes also contained frequent
ingested by the filter-feeding larvae. Heavier particles sink ATTTA sequences, which in plants act as signals to
and are covered in mud, quickly becoming ineffective, so increase the rate of mRNA turnover. Perlak et al. (1991)
specialist products that facilitate slow release have the modified the sequence of the cry1Ab and cry1Ac genes to
best performance, e.g. products with the bacteria sus- increase the GC content, replace sequences with four or
pended in ice chips, or formulations including Bacillus
sphaericus.
Table 5 Earliest Bt transgenic plants (data from Krattiger, 1996)

Crop Bt Target pest

Tomato Gene from Bt var. Lepidopteran pest: tomato

Table 4 Advantages and disadvantages of Bt sprays
kurstaki HD-1 fruitworm (Heliothis zea) and
Advantages Disadvantages tomato pinworm (Keiferia
lycopersicella)
Potent insecticidal activity Rapidly inactivated by UV light, Truncated forms of the Lepidopteran larvae
Specific host range heat and extreme pH gene from Bt var
Harmless to humans and Susceptible to proteases in leaf exudates kurstaki HD-1
other mammals Easily removed from plant surface Tobacco DNA encoding a toxin Manduca sexta (tobacco
Biodegradable by wind and rain from Bt var. kurstaki hornworm), and other
Therefore needs to be reapplied for full effect HD-1 Lepidopteran insects

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290 Georgina Sanahuja et al.

more consecutive adenine or thymine residues and shift and cotton crops. The first to reach the market was
codon preference towards that favoured by plants, Monsanto’s NewLeaf potato variety expressing Cry3A,
increasing protein levels by up to 100-fold and achieving swiftly followed by two transgenic corn hybrids express-
total yields equivalent to 0.02% total soluble protein ing Cry1Ab to protect against the European corn borer,
(TSP). This was still insufficient for adequate pest control, i.e. KnockOut by Syngenta (Basel, Switzerland) and Natu-
but expression levels could be increased still further using reGard by Mycogen (both containing event 176). Mons-
stronger promoters, more efficient polyadenylation and anto also released the cotton varieties Bollgard and
termination signals, and by including a heterologous intron Ingard (events 531, 757 and 1076) expressing a modified
in the expression construct. The development of synthetic Cry1Ac toxin. Two additional Bt corn varieties expressing
cry genes optimized for expression in plants meant that Cry1Ab were released shortly thereafter, namely Agrisure
Cry proteins were soon expressed at levels of 0.2–1% TSP CB by Northrup King (event Bt11) and the widely dis-
(Koziel et al., 1993) and this increased to over 5% when cussed YieldGard variety (event MON 810) by Monsanto.
cry genes were introduced into the chloroplast genome The early landscape of the biotech crop industry had thus
(McBride et al., 1995). been established (Box 1). NewLeaf potato and its succes-
sors (NewLeaf Y and NewLeaf Plus) were withdrawn
from the market in 2002 (Box 2), and corn varieties con-
Field trials and early commercial crops
taining event 176 were later withdrawn and replaced
After successful results in laboratory tests, the first field tri- with more profitable products.
als with Bt transgenic tobacco were conducted in the Uni-
ted States and France in 1986. The plants expressed a Growth and diversification of Bt crops
truncated gene encoding the N-terminal (toxic) portion of By 1998, the uptake of Bt crops had increased significantly
Cry1A from Bt var kurstaki HD-73 under the control of as data became available showing the positive impact of
the constitutive Cauliflower mosaic virus 35S promoter Bt transgenic technology on agriculture and the environ-
and protected the plants from leaf damage caused by ment (see below). In 1998, the EPA approved an insect-
Helicoverpa zea, a pest known variously as cotton boll- resistant tomato line (event 5345) expressing Cry1Ac, and
worm, corn earworm or tomato fruitworm, depending on in 2001 the Herculex corn variety jointly developed by Pio-
the crop it infests (Hoffmann et al., 1992). Whereas it was neer Hi-Bred (Johnston, IA) and Dow AgroSciences (India-
never likely that the Bt tobacco variety would be devel- napolis, IN) (event TC 1507) expressing Cry1F and
oped for commercial exploitation, transgenic potato plants protecting plants against black cutworm (Agrotis ipsilon),
expressing Cry3A from Bt var. tenebrionis were shown to fall armyworm (Spodoptera frugiperda) and the European
protect the crop against Colorado potato beetle in the corn borer. A new landmark was achieved in 2002, with
field much more efficiently than Cry3A topical sprays and the approval of Monsanto’s Bollgard II cotton (event
were earmarked for commercial development (Perlak 15985), which expressed two Bt toxins, Cry1Ac and
et al., 1993). Trials with cotton, maize and rice soon Cry2Ab, and later YieldGard Rootworm (event MON 863),
followed (Table 6). expressing a synthetic variant of the cry3Bb1 gene from Bt
In 1995, the US Environmental Protection Agency subsp. kumamotoensis, providing resistance against the
(EPA) approved the first registration of Bt potato, corn western corn rootworm (Diabrotica virgifera virgifera). The
first stacked variety developed by crossing two previously
released Bt varieties was also released in 2003. This was
Table 6 Early field trials with Bt transgenic plants (data from
Hilder and Boulter, 1999) Monsanto’s YieldGard Plus (event MON 810 + MON 863),
which expressed Cry1Ab1 and Cry3Bb1. During this per-
Crop Bt toxin Target pest
iod, there was significant turbulence in the market as both
Tomato Cry1A Pinworm large and small industry players manoeuvred to acquire
Tobacco Cry1A Helicoverpa zea strategic patents and technologies (Box 3). Tables S1–3
Potato Cry1A Tuber moth show, respectively, the current status of Bt patents, the
Cry3A Colorado potato beetle
companies currently engaged in the commercial develop-
Cotton Cry1A Pink bollworm
Maize Cry1A European corn borer ment of Bt and the commercial status of different Bt
Rice Cry1A Stemborers crops.

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 283–300
 A century of Bacillus thuringiensis 291

Box 1 Portrait of an industry

The early pioneers of commercial Bt technology were agrobiotechnology companies such as Monsanto, DuPont, Calgene and Agracetus, which had collab-
orated with academic research groups to carry out field trials and develop potential commercial products in the mid-to-late 1980s. As the 1990s
approached, these companies entered into agreements with major seed distributors such as Delta and Pine Land (DPL) to develop and test their enhanced
transgenic varieties. The predominant role of Monsanto in the early commercial landscape reflected their exclusive agreement with DPL to market trans-
genic cotton seeds internationally. Later, Monsanto forged agreements with several local companies in developing countries particularly China and India
(Huang et al., 2002a,b). Early in the 1990s, Monsanto purchased Dekalb Genetics, Asgrow and Holden’s acquiring elite germplasm through which to com-
mercialize its biotechnology products.

Figure in Box 1 Evolution of the commercial landscape for Bt crops. The five major companies currently selling Bt seeds have arisen through
a series of mergers, acquisitions and spin offs ⁄ demergers as larger companies segregate their agribusiness interests. Monsanto Co., in its cur-
rent incarnation, was an agribusiness spin-off from Pharmacia in 2002 following the merger of the original Monsanto Co. (established in 1901)
with Pharmacia and Upjohn in 2000. Pharmacia created the new Monsanto as an agribusiness subsidiary in late 2000, and then established it
as an independent company in 2002. Bayer CropScience is an agribusiness subsidiary of Bayer AG, formed following the acquisition of Aventis
CropScience in 2000. Syngenta formed from the merger of Novartis and AstraZeneca in 2000, both of which were agribusiness spin-offs gen-
erated in previous mergers. Dow AgroSciences is a wholly owned subsidiary of Dow Chemical Co., formed when Dow Chemical Co. purchased
Eli Lilly’s stake in Dow Elanco (an agribusiness spin-off from Dow Chemical Co. and Ely Lily & Co. formed in 1989). Finally, Pioneer Hi-Bred
International is now an agribusiness subsidiary of DuPont, which acquired 20% of the company in 1997 and the remaining 80% in 1999.
The Monsanto business model of assimilating companies with aligned strategic objectives was soon replicated by the other major players (see Figure
below). Although the 1990s was characterized by aggressive positioning, mergers and acquisitions, this has now given way to a more collaborative indus-
try. The recent approval of SmartStax corn, codeveloped by Monsanto and Dow AgroSciences is one example, and others include the Greenleaf Genetics
collaboration between Syngenta and Pioneer Hi-Bred International, and Monsanto licensing RoundupReady events to Syngenta so that the trait can be
incorporated into their hybrids (Marra et al., 2010).

amount of land in total used for Bt or Bt stacked crops.

International expansion
However, Bt crops are grown in 25 other countries and
The United States has enthusiastically embraced the devel- the number of countries adopting Bt crops and the
opment of Bt agriculture and has by far the largest amount of land set aside for their cultivation has shown a

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292 Georgina Sanahuja et al.

Box 2 The demise of NatureMark

NatureMark was a subsidiary of Monsanto created by the company in 1996 to market its new transgenic potato lines, beginning with NewLeaf (contain-
ing the gene for Cry3Aa) and subsequently NewLeaf Y (containing the Bt gene and an additional gene conferring resistance to Potato virus Y ) and New-
Leaf Plus (containing the Bt gene and an additional gene conferring resistance to Potato leaf roll virus). Despite rapid take up and hugely favourable
responses from growers, all the potato lines and the NatureMark brand itself were abandoned in 2002 after a highly successful potato growing season
in 2001. Why did this happen?

Figure in Box 2 NewLeaf Plus field (left) is protected from Potato leaf roll virus (PLRV) without sprays. No infection is evident. The conventional
potato field on the right is 100% PLRV infected, despite sprays. Figure used with permission from Peter Thomas.

The NatureMark story shows how misguided activism can have disastrous effects. Activists began a very public campaign of misinformation against
Monsanto’s potato products in 1999 and soon had the support of several major convenience food providers including McDonalds, which banned geneti-
cally engineered crops from its food to avoid negative publicity. This in turn put pressure on potato processers and growers, who had little choice but to
cancel contracts for transgenic potatoes they would not be able to sell. The transgenic varieties had all received appropriate clearance from the regulators
and were safe for human consumption, so it is clear that the entire chain of events was based on hysteria and not one shred of scientific evidence. Not-
withstanding the above, Monsanto was forced to abandon NatureMark and all potato-related research and development as there would be no market
for its products. The activists were successful, but it was a Pyrrhic victory. In their haste to save the environment by getting rid of ‘unnatural’ vegetables,
they ensured that tons of chemical pesticides would be required for future potato crops and tons of fuel would be required to spray them; they ensured
that farmers in North America would mourn the loss of a superior product, lose profit and make the agricultural sector as a whole less profitable; and
they ensured that many additional beneficial crops would never see the light of day, or would be developed instead in other countries.

Box 3 War and peace

Many aspects of Bt technology fall under the scope of intellectual property and can be protected with patents, although many of these aspects (process-
ing and formulation in the case of Bt sprays, gene transfer and expression in the case of Bt crops) are not specific to Bt and can have wider ramifications.
Many companies and academic departments working on Bt technology have sought patents, and in 1996 when the first Bt crops were commercialized
these were divided more or less equally between the ‘old guard’ companies that had developed Bt topical products and the ‘new wave’ of companies
expressing Bt genes in plants. Nearly 60% of the approximately 400 Bt patents were owned by Mycogen, Novartis, Abbot, Toa, AgrEvo, Ecogen, Mons-
anto and Zeneca. It is noteworthy that the five major companies involved in Bt crop development today have, since 1996, engaged in many cooperative
research and development agreements and this has continued to the present day. However, they have also litigated extensively to prevent patent
infringements. The figure below shows current and past collaborations and licensing deals whereas the arrows show litigation pursued either by the com-
pany as it exists today, or one of its predecessors. The current Bt patent landscape is summarized in Table S1.

Figure in Box 3 Early patent wars (arrows show litigation) and collaborations (dotted lines) in the commercialization of Bt.

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 283–300
 A century of Bacillus thuringiensis 293

continued upward trend for 15 years (Figure 3). Bt agricul- resistant mealmoths (Plodia interpunctella) were found in
ture is expanding on every continent except Europe, which grain stores that had been sprayed with Bt spores. The
persists with its absurdly byzantine approach to all geneti- selection pressure was recreated in the laboratory, showing
cally engineered crops. Remarkably, the small African the evolution of resistant strains after 15 generations of
nation of Burkino Faso grows more Bt crops than the sublethal selection (McGaughey, 1985). Resistance was also
whole of Europe. The total global area devoted to Bt crops observed in wild populations of the diamondback moth
in 2009 was >50 million hectares (36% of all biotech (Plutella xylostella) feeding on watercress in Hawaii that
crops), made up of 21.7 million hectares of Bt-only crops had been sprayed with Bt up to 400 times (Liu and Tabash-
and 28.7 million hectares of crops with Bt stacked with nik, 1997). Laboratory experiments were able to produce
herbicide tolerance (James, 2010). Bt-resistant varieties of several additional species that had
Although Argentina and Brazil currently hold second not evolved resistance in the wild, suggesting that the
and third place in the global rankings for Bt agriculture, intensive use of single Cry proteins was likely to result in
China and India have seen the most rapid adoption. This the evolution of resistant strains (Tabashnik et al., 2005).
is because both are major growers of cotton, and China in The Bt crop industry is aware of the danger of resistant
particular is a major grower of rice. Field trials of Bt rice pests, and many seed vendors insist on customer agree-
were first conducted in China in 1998. A series of trans- ments that mandate the use of preventative measures,
genic Bt rice lines transformed with modified cry1A, particularly the refugia strategy in which a proportion of
cry1Ab and cry1Ac genes were assessed in large-scale tri- any field containing Bt crops must be planted with the
als in 2007 (Huang et al., 2007) and were approved for nontransgenic variety to encourage the breeding of nonre-
commercial release in November 2009, although large- sistant pests. The widespread use of this strategy is proba-
scale cultivation is still pending. bly responsible for the remarkable lack of resistant
populations even in areas devoted to high-intensity Bt
agriculture for 15 years. Tabashnik and coworkers have
Ecological aspects of Bt crops
studied pest populations on Bt sites in the United States,
Australia, China and Europe and have found that among
Potential for the evolution of resistant insect
six major insect pests, field resistance occurred in only one
populations
species (H. zea) and only at a limited number of sites in
All insecticides create selection pressure on target popula- Arkansas and in Mississippi, not in, for example, North
tions, and the mode of action of Bt toxins (binding to a Carolina, where the refuge areas are typically larger (Ta-
specific receptor on midgut epithelial cells) presents a clear bashnik et al., 2008). The prolonged efficacy of the first
opportunity for pests to evolve resistance. The first generation of Bt crops for more than a decade against
evidence of this process was observed in 1985, when nearly all targeted pest populations has exceeded the

Figure 3 Sharing out the Bt pie. More than 50 million hectares of Bt crops were grown commercially in 2008, the vast majority (>33 million
hectares) in the USA. India, China, Argentina, Brazil, South Africa, Canada, Philippines, Australia and Uruguay were (in descending order by land
area) the other countries to grow >100 000 hectares. The minor growers (in descending order by land area) were Spain, Mexico, Colombia,
Honduras, Burkino Faso, Czech Republic, Romania, Portugal, Germany, Poland, Slovakia and Egypt. The data include Bt-only crops and Bt stacked
with other traits. Source: Brookes and Barfoot (2010).

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 283–300
294 Georgina Sanahuja et al.

expectations of many entomologists working on popula- in 2009 showed that the pest evolved resistance to Boll-
tion genetics (Bourguet, 2004). Although integrated pest gard I cotton (Cry1Ac) in four areas of Gujarat: Amreli,
management strategies have been carefully implemented Bhavnagar, Junagarh and Rajkot. However, no resistance
by growers, the absence of resistant populations in the was observed in fields growing the Bollgard II variety,
wild suggests that resistance may attract a fitness penalty which contains Cry1Ac and Cry2Ab (Monsanto, 2010).
in the absence of the toxin (Sanchis and Bourguet, 2008). Attempts have also been made to enhance toxin activity
Although current refugia strategies have worked better by coexpression or protein fusion. One example is the
than anticipated, a number of alternative or complemen- expression of Cry toxins along with fragments of their
tary approaches have been proposed to address the possi- receptors, which can potentiate their activity by allowing
bility of resistance evolving in target pests. High-dose and them to assemble into pore-forming complexes immedi-
low-dose approaches aim in one case to overwhelm insect ately (Chen et al., 2007). The fusion of two or more toxins
populations with immense toxin doses so that there is no has been used to generate artificial hybrids with a host
chance to evolve resistance, and in the other case merely range differing from that of the parent toxins. For exam-
to make them more susceptible to predators, but both ple, Naimov et al. (2003) created a fusion toxin comprising
make many assumptions and would be difficult to imple- a truncated version of Cry1Ba and domain II from Cry1Ia.
ment in the field (Gould, 1994). Similarly, targeted expres- Desiree potato plants expressing this recombinant toxin
sion (e.g. wound-inducible expression) or temporally were the first Bt plants resistant to both coleopteran and
restricted expression would provide the same advantages lepidopteran pests [Colorado potato beetle, potato tuber
of a refugia but would be more difficult to implement. moth (Phthorimaea operculella) and European corn borer]
A more robust approach is resistance pyramiding, i.e. the and the hybrid protein did not compete for binding sites
stacking of multiple genes in the same plant that target with either of the parent toxins, indicating it bound to a
the same pest via different mechanisms. In theory, resis- distinct receptor. More recently, Walters et al. (2010) cre-
tance against one Cry protein could arise through a single ated a hybrid Cry1Ab ⁄ Cry3A toxin (eCry3.1Ab) which was
point mutation in the gene encoding its receptor. Experi- toxic to the western corn rootworm, a pest unaffected by
ments have shown that mutations affecting the secondary, either of the parent toxins. In a related approach, Mehlo
high-affinity Cry toxin receptors do not induce resistance, et al. (2005) fused the Cry1Ac toxin to the galactose-bind-
but multiple resistance alleles can be identified in the ing domain of the nontoxic ricin B-chain, again expanding
cadherin genes encoding the primary receptors (Soberón its repertoire of potential receptors and therefore broaden-
et al., 2009; Zhao et al., 2010). However, the chances of ing its host range. Transgenic rice and maize plants
two mutations arising simultaneously in the receptors for expressing the fusion protein were significantly more toxic
two independently acting toxins would be much lower. in insect bioassays than those containing the Bt gene
This is probably why mosquito strains resistant to Bt var alone. They were also resistant to a wider range of insects,
israelensis have not evolved despite many years of including important pests that are not normally susceptible
deployment—the toxin crystals in this case comprise five to Bt toxins.
different toxins, one of which is Cyt1A which, as discussed
above, has a different action mechanism to the three-
Environmental impact
domain Cry toxins. Pyramiding resistance crops are there-
fore likely to require smaller refuges (Shelton et al., 2002). Although there is much debate both politically and publical-
The pyramiding strategy is supported by laboratory tests ly concerning the environmental impact of genetically engi-
with cotton bollworm and the recent analysis of pink boll- neered crops, it is clear that Bt crops have provided
worm (Pectinophora gossypiella) populations in Bt cotton immense environmental benefits. The deployment of Bt
fields in India. The laboratory studies showed that cotton crops has reduced the use of pesticides, also saving on fossil
bollworm can evolve resistance to MVP, a commercial Bt fuels required for spraying, reducing CO2 emissions by limit-
formulation based on Cry1Ac, but not to DiPel or XenTari, ing the need for ploughing, and conserving soil and mois-
which contain multiple Cry proteins (Akhurst et al., 2003). ture by encouraging no-tilling agriculture. The cumulative
The resistant strain was also resistant to Cry1Ab (which reduction in pesticide use for the period 1996–2008 was
binds to the same receptor as Cry1Ac in cotton bollworm) approximately 356 000 tonnes (8.4%), which is equivalent
but not to Cry2Aa or Cry2Ab which bind different recep- to a 16.1% reduction in the associated net environmental
tors (Liao et al., 2002). Field monitoring of pink bollworm impact as measured by the environmental impact quotient

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 283–300
 A century of Bacillus thuringiensis 295

(EIQ). The corresponding data for 2008 alone revealed a Cry3Bb1 and a stacked variety MON 89034 · MON
reduction of 34 600 tonnes of pesticides (9.6%) and a 88017, expressing Cry1A105, Cry2Ab2 and Cry3Bb1). The
reduction of 18.2% in EIQ (Brookes and Barfoot, 2010). In study showed that the predator and alternative prey popu-
countries such as India, China, Argentina and Brazil, which lations naturally adjusted to reflect the absence of the tar-
are the most enthusiastic adopters of Bt agriculture after geted pest (Faria et al., 2007). Bt maize increased the
the United States, the greatest impact of Bt has been the population of corn aphid (Rhopalosiphum maidis) which
reduction in the number of pesticide sprays (from 16 down resulted in more honey dew synthesis, which increased
to 2–3 per growing season) and a concomitant reduction in the number and longevity of the lepidopteran larval para-
the number of poisonings caused by chemical exposure. sitoid Cotesia marginiventris. Bt cotton appears to have no
These factors, together with average yield increases of up effect on the cotton aphid (Aphis gossypii) population,
to 10%, have raised net income by as much as 40% (Subra- and the Bt toxin was not detected in the honey dew,
manian and Qaim, 2010). which is an energy source for many arthropod species
Although the reduction in pesticide use has been bene- including predators and parasitoids. Bt cotton therefore
ficial to the environment and the economy, concern has has no negative impact on beneficial insects in the cotton
been expressed that the use of Bt transgenic plants could ecosystem (Lawo et al., 2009).
affect beneficial insects, upset the balance of natural eco-
systems and encourage the breeding of secondary pests. It Secondary pests
is unclear why those raising such concerns are not equally A secondary pest is a pest species whose numbers are
concerned about the impact of topical Bt sprays and usually kept in check by the presence of a primary pest,
chemical pesticides on these populations, because sprays such that no control measures are necessary. However,
are the only current alternative to transgenic crops that elimination of the major pest may elevate the secondary
will guarantee adequate food production. pest to primary status, perhaps even affecting surrounding
crops that are not usually troubled by either the primary
Beneficial insects or secondary pest species. Cotton bollworm is a primary
The potential impact of Bt crops on beneficial insects was pest of cotton, and it suppresses the population of mirid
brought into focus by the now discredited Monarch but- bugs, i.e. homopteran insects that feed on plant sap. Bt
terfly study, which suggested Monarch larvae feeding on cotton represents approximately 95% of all cotton in
leaves covered in pollen shed from Bt maize plants (event Northern China and is lethal to the cotton bollworm at
Bt176) did not grow as rapidly as those feeding on uncon- the larval stage, so a study was carried out to look at any
taminated leaves. This report was seized on by opponents impact on mirid bug populations (Lu et al., 2010). The
of genetic engineering technology and is still routinely study showed that mired bug populations have not
cited as an argument against the deployment of Bt crops increased in nontransgenic cotton because the species is
despite follow-up studies finding no evidence for a statisti- controlled by broad-spectrum pesticides that are also used
cally significant effect. What other evidence is there to kill bollworm larvae. In Bt cotton, the mirid bug popula-
regarding the impact of Bt crops on nontarget insects? tion has increased every year from 1997 to 2008 and has
Field studies on the NewLeaf potato (Cry3Aa) showed gained the status of a primary pest, a phenomenon that is
that the toxin specifically affects the Colorado potato bee- now impacting on unrelated crops such as dates, grape-
tle and has no deleterious effect on other insects in the vine, apple, peach and pear. Although this is an undesir-
potato field, including the beetle’s natural predators. In able outcome, it is somewhat balanced by the increased
contrast, chemical sprays killed both the beetle and its insect biodiversity observed in Bt cotton in China. Field
predators, leading to an explosion in the population of studies revealed 31 insect species in Bt plots (23 beneficial)
vectors carrying viral pathogens, thus increasing the risk of compared to 14 species in non-Bt plots, and only five of
potato virus diseases (Reed et al., 2001). Any impact on which were beneficial (Pray et al., 2002).
natural predators that normally keep pest populations in
check could have knock-on effects throughout the food Environmental diversity
web, so careful studies of these effects are required (Dut- In addition to insect populations, it is useful to study the
ton et al., 2002). One such study looked at nontarget impact of Bt on other parts of the ecosystem, particularly
arthropod predators in Bt maize fields (specifically events the soil as this is where Bt spores end up when washed
MON 810 expressing Cry1Ab, MON 88017 expressing from the plant surface, and is the destination of Bt toxins

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 283–300
296 Georgina Sanahuja et al.

Figure 4 Nine common pests of rice, cotton and maize that are controlled by Bt crops. Top row from left (rice pests): yellow stem borer
(Scirpophaga incertulas), rice leaffolder (Lerodea eufala), striped stem borer (Chilo suppressalis). Middle row from left (cotton pests): pink bollworm
(Pectinophora gossypiella), tobacco budworm (Heliothis virescens), American bollworm (Helicoverpa armigera). Bottom row from left (maize pests):
European corn borer (Ostrinia nubilalis), fall armyworm (Spodoptera frugiperda), northern corn rootworm (Diabrotica barberi). Image sources
from top left: (i) IRRI, (ii) IRRI, (iii) Bayer CropScience, (iv) USDA, (v) Cotton Corporation of India Ltd., (vi) USDA, (vii) Frank Peairs, Colorado State
University, Bugwood.org, (viii) Canadian Biodiversity Information Facility, (ix) Plant Management Network.

exuded from plant roots, released from pollen grains and mosquitocidal toxins, and Zhang et al. (2010) recently
released from decaying or residual plant biomass ploughed isolated strain LLP29 from the phylloplane of Magnolia
into the soil. Earthworms (oligochaetes) are good indica- denudate, identifying a novel toxin (Cyt1Aa6) which is
tors of general soil health and comparisons of earthworm lethal to mosquito larvae. Homologous genes have also
numbers in plots containing nontransgenic maize and Bt been identified in related bacteria, reflecting the fact that
maize expressing cry1Ab (events Bt11 and MON 810) and Cry proteins are members of a diverse superfamily.
cry3Bb1 (event MON 863) over 4 years showed no differ- Strain engineering efforts have continued to extend the
ences in development or biomass (Zeilinger et al., 2010). Bt host range. For example, Liu et al. (2010) recently
More earthworms were found within the rows of maize reported the construction of strain BIOT185 from the origi-
plants than between them in all plots, perhaps because nal strains HBF-1 and BTO 185 that express Cry8ca2 and
the soil is lighter and has more biological activity and Cry8Ea1, respectively. The new strain is toxic towards
therefore represents a better source of nutrients. scarab insects such as Atractaspis corpulenta. Similarly,
Wang et al. (2008) constructed a new strain by introducing
the cry3Aa7 gene into the UV17 strain, which produces
Looking to the future
Cry1Aa, Cry1Ac, Cry1Ca and Cry2Ab. The new strain was
The first generation of Bt crops has been extraordinarily toxic to both lepidopteran and coleopteran insects.
successful, with a few examples of pest populations evolv- The toxicity of Cry proteins can be enhanced by amino
ing resistance. These crops are already being supplanted acid substitutions, the introduction of cleavage sites in
with second-generation varieties with more resilient traits specific regions of the protein and the deletion of small
generated by stacking and pyramiding resistance genes. fragments from the N-terminal region (Pardo-López et al.,
Even so, this is not the time to be complacent and the 2009). For example, replacing residue N372 in Cry1Ab
search for more efficacious and potent strains must con- domain II with alanine or glycine increases its toxicity to
tinue (Christou et al., 2006; Crickmore, 2006). New strains the gypsy moth (Lymantria dispar) eightfold by inducing a
of Bt are reported on a regular basis, especially now prote- fourfold increase in binding affinity to its receptor (Raj-
omics methods can be used to screen for novel toxins on amohan et al., 2006). More recently, Muñóz-Garay et al.
a large scale. Sun and Park (2010) recently identified (2009) produced an engineered Cry1AMod toxin lacking
Cry60Ba from Bt serovar malayensis in a search for helix a-1, which did not need to bind the receptor cadher-

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 283–300
 A century of Bacillus thuringiensis 297

in and therefore killed even insects that were resistant to European Research Council Advanced Grant BIOFORCE;
the parent toxin, Cry1Ab. Center Consolider, MICINN, Spain; COST Action FA0804;
The commercial environment for stacked and pyramid- Associated Unit CAVA; and SmartCell, FP7 Integrated
ing traits in Bt crops was given a boost in 2010 with the Project.
approval of SmartStax corn, codeveloped by Monsanto
and Dow AgroSciences. Approvals were granted in the
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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 283–300
300 Georgina Sanahuja et al.

Zhao, J., Jin, L., Yang, Y. and Wu, Y. (2010) Diverse cadherin Table S2 Companies engaged in the commercial develop-
mutations conferring resistance to Bacillus thuringiensis toxin ment of Bt crops.
Cry1Ac in Helicoverpa armigera. Insect Biochem. Mol. Biol. 40,
Table S3 Commercial status of Bt crops. Data from Cen-
113–118.
ter for Environmental Risk Assessment (CERA) GM crop
database.
Supporting information
Please note: Wiley-Blackwell are not responsible for the
Additional Supporting information may be found in the content or functionality of any supporting materials sup-
online version of this article: plied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
Table S1 The distribution of Bt patents in 2010 (Updated
for the article.
from Krattiger (1996) with data from EU and US patent
databases).

ª 2011 The Authors

Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 283–300