Critical Reviews in Plant Sciences, 23(1):47–72 (2004)
Copyright
C
Taylor and Francis Inc.
ISSN: 0735-2689
DOI: 10.1080/07352680490273400
Genetic Transformation of Crops for Insect Resistance:
Potential and Limitations
Hari C. Sharma
∗
, Kiran K. Sharma, and Jonathan H. Crouch
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324,
Andhra Pradesh, India
Transgenic resistance to insects has been demonstrated in plants
expressing insecticidal genes such as δ-endotoxins from Bacillus
thuringiensis (Bt), protease inhibitors, enzymes, secondary plant
metabolites, and plant lectins. While transgenic plants with intro-
duced Bt genes have been deployed in several crops on a global scale,
the alternative genes have received considerably less attention. The
protease inhibitor and lectin genes largely affect insect growth
and development and, in most instances, do not result in insect
mortality. The effective concentrations of these proteins are much
greater than the Bt toxin proteins. Therefore, the potential of some
of the alternative genes can only be realized by deploying them in
combination with conventional host plant resistance and Bt genes.
Genes conferring resistance to insects can also be deployed as mul-
tilines or synthetic varieties. Initial indications from deployment
of transgenics with insect resistance in diverse cropping systems in
USA, Canada, Argentina, China, India, Australia, and South Africa
suggest that single transgene products in standard cultivar back-
grounds are not a recipe for sustainable pest management. Instead,
a much more complex approach may be needed, one which may in-
volve deployment of a combination of different transgenes in differ-
ent backgrounds. Under diverse climatic conditions and cropping
systems of tropics, the success in the utilization of transgenics for
pest management may involve decentralized national breeding pro-
grams and several small-scale seed companies. While several trans-
genic crops with insecticidal genes have been introduced in the tem-
perate regions, very little has been done to use this technology for
improving crop productivity in the harsh environments of the trop-
ics, where the need for increasing food production is most urgent.
There is a need to develop appropriate strategies for deployment of
transgenics for pest management, keeping in view the pest spectrum
involved, and the effects on nontarget organisms in the ecosystem.
Keywords biotechnology, genetic transformation, Bacillus thuring-
iensis, host plant resistance, insecticidal genes, pest man-
agement, nontarget effects, limitations of transgenics.
INTRODUCTION
There is a continuing need to increase food production, par-
ticularly in the developing countries of Asia, Africa, and Latin
∗
Corresponding author: E-mail: h.sharma@cgiar.org
America. Losses due to insect pests represent one of the sin-
gle largest constraints to crop productivity, estimated at 14%
of the total agricultural production (Oerke et al., 1994). In ad-
dition, insects also act as vectors of various plant pathogens.
The annual global cost of attempting to reduce pest damage
through insecticide application is currently valued at US$10 bil-
lion. Large application of insecticides for insect control results in
toxic residues in food and food products, in addition to adverse
effects on nontarget organisms and the environment in general.
Furthermore, the cost–benefit ratio of such practices can eas-
ily become negative in marginal cropping systems, particularly
when other factors such as diseases or drought also become rate
limiting in crop production.
The losses due to insect pests can be minimized effec-
tively through host plant resistance to insects (environmentally
safe seed-based technology through conventional plant breed-
ing and/or biotechnological approaches) compared to other pri-
mary constraints to crop production such as low soil fertil-
ity and drought. The ability to isolate and manipulate single
genes through recombinant DNA technology (Watson et al.,
1987), together with the ability to insert specific genes into a
chosen variety (Chilton, 1983) has opened a new era of tar-
geted plant breeding. Significant progress has been made over
the past two decades in introducing foreign genes into plants,
and this has provided opportunities to modify crops to increase
yields, impart resistance to biotic and abiotic stresses, and im-
prove nutritional quality (Sharma et al., 2002a). Genes encod-
ing δ-endotoxins from Bacillus thuringiensis (Bt) were cloned
in the early 1980s (Schnepf and Whiteley, 1981), and geneti-
cally modified plants with resultant resistance to insects were
developed by the mid-1990s (Hilder and Boulter, 1999; Sharma
et al., 2000). In this article, we focus on candidate genes con-
ferring resistance to insect pests and review the current progress
in developing transgenics with insect resistance and their lim-
itations in order to assess the future potential of this technol-
ogy, with particular reference to the genetic improvement of
crops for improving the livelihoods of poor people in developing
countries.
47
48 H. C. SHARMA ET AL.
GENETIC TRANSFORMATION OF CROP PLANTS
The efficiency of tissue culture and transformation proto-
cols is one of the most important components for successful
generation of transgenic crops (Sharma and Ortiz, 2000b). The
major components for the development of transgenic plants are:
(1) development of reliable tissue culture and regeneration sys-
tems, (2) preparation of gene constructs and transformation with
suitable vectors, (3) efficient techniques of transformation for
introduction of genes into the crop plants, (4) recovery and mul-
tiplication of transgenic plants, (5) molecular and genetic char-
acterization of transgenic plants for stable and efficient gene
expression, (6) transfer of genes into elite cultivars by con-
ventional breeding methods, and (7) evaluation of transgenic
plants for their effectiveness in alleviating biotic and abiotic
stresses without being an environmental hazard (Birch, 1997).
Although several approaches have been tried successfully for in-
tegrative transformation (Potrykus, 1991), only four approaches
are used widely and have enabled scientists to introduce genes
into a wide range of crop plants (Dale et al., 1993). These in-
clude (1) Agrobacterium-mediated gene transfer, (2) micropro-
jectile bombardment with DNA or biolistics, (3) microinjection
of DNA, and (4) direct DNA transfer into isolated protoplasts.
Of these techniques, the first two approaches have been used
quite successfully.
•
Agrobacterium tumefaciens has been used widely for
transforming the desired genes into crop plants. It
is a soil-inhabiting bacterium that has been impli-
cated in gall formation at the wound sites in many
dicotyledonous plants. This tumor-inducing capabil-
ity is due to the presence of a large Ti (tumor-
inducing) plasmid in virulent strains of Agrobacterium.
Likewise, Ri (root-inducing) megaplasmids are found
in virulent strains of Agrobacterium rhizogenes, the
causative agent of “hairy root” disease. The Ti and
Ri plasmids, and the molecular biology of crown gall
and hairy root induction, have been studied in great
detail (Zambryski et al., 1983; Zambryski, 1992).
Agrobacterium-mediated transformation is brought
about by incorporation of genes of interest from an in-
dependently replicating Ti plasmid within the A. tume-
faciens cell, which then infects the plant cell and trans-
fers the T-DNA containing the gene of interest into the
chromosomes of the actively dividing cells of the host
plant.
•
Genetically engineered DNA can also be directly in-
jected into nuclei of embryogenic single cells, which
can be induced to regenerate plants in cell culture
(Neuhaus et al., 1987). This requires micromanipu-
lation of single cells or small colonies of cells under
the microscope, and precise injection of small amounts
of DNA solution with a thin glass micro pippette. In-
jected cells or clumps of cells are subsequently raised
in in vitro culture systems and regenerated into plants.
•
In the particle bombardment (biolistics) method, tung-
sten or gold particle microprojectiles are coated
with the DNA to be inserted, and bombarded into
cells/tissues capable of subsequent plant regeneration.
Acceleration of heavy microprojectiles (0.5 to 5.0 µm
diameter tungsten or gold particles) coated with DNA
carries genes into virtually every type of cell and tissue
(Klein et al., 1987; Sanford, 1990). The DNA-coated
particles enter the plant cells, the DNA is incorporated
in a small proportion of the treated cells, and the trans-
formed cells are selected for plant regeneration.
•
In the protoplast transformation, the cell wall of the
target cells is removed by enzymatic treatment, and the
cells are bounded by a plasma membrane (Zhang and
Wu, 1988). The DNA can be added into cell suspension,
which can be introduced by affecting the plasma mem-
brane by polyethylene glycol or by passing an electric
current through the protoplast suspension. The DNA
gets incorporated into the genome of a few cells. A
suitable marker may be inserted to select the trans-
formed protoplasts and the cell colonies that develop
from them (Shimamoto et al., 1989). The Cry2Aa2
operon expressed in tobacco chloroplasts resulted in
Bt protein content of up to 45.3% of the total protein in
mature leaves, which resulted in 100% mortality of cot-
ton bollworm and beet armyworm (Cosa et al., 2002).
GENE EXPRESSION
Efficient genetic engineering relies on being able to gener-
ate a specific gene product at the desired level of expression,
in the appropriate tissues, at the right time. This can be ac-
complished by creating gene constructs that include promoters
and/or transcription regulation elements that control the level,
location, and timing of gene expression. A major constraint in
the development of effective transgenic products has been the
lack of promoters that can offer a high level of gene expres-
sion at this degree of specificity in the crop species of interest.
Traditionally transgene expression has been driven by strong
constitutive promoters such as cauliflower mosaic virus 35S pro-
moter (CaMV35S) (Benfey and Chua, 1989, 1990) and Actin 1
(McElory et al., 1990). Although CaMV35S has been widely
used in a number of dicotyledonous plant transformation sys-
tems, it has low activity in monocotyledonous systems (Wilmink
et al., 1995). Moreover, the pattern of CaMV35S promoter activ-
ity in different tissues of transgenic plants is difficult to predict
(Benfey and Chua, 1990). In general, it has been found that
monocot promoters are more active in monocot tissues than in
dicot tissues (Wilmink et al., 1995).
More recently, tissue-specific promoters have been success-
fully employed for driving transgene expression solely in pith tis-
sue. Phosphoenolpyruvate carboxylase (PEPC) from maize can
be used for gene expression in green tissue (Hudspeth and Grula,
1989). From a crop-yield–potential perspective, insect-resistant
GENETIC TRANSFORMATION OF CROPS FOR INSECT RESISTANCE
49
transgenes should be expressed only in those organs likely to
be attacked by the insects. Otherwise, plants may be highly
resistant, yet the metabolic cost may substantially reduce the
crop yield. This approach also reduces the probability of unex-
pected negative effects on nontarget organisms. Often, it may
not be possible to extrapolate results on gene expression lev-
els from one species to another, and each crop should be tested
with a set of promoters. Although the constitutive promoters
such as CaMV35S are effective in providing high levels of gene
expression, such expressions in some cases are not only un-
necessary, but could also have unanticipated negative conse-
quences towards nontarget organisms. On the contrary, a more
targeted expression of insecticidal genes by using tissue- and
organ-specific promoters can form an important component for
developing transgenic plants with resistance to insects (Wong
et al., 1992; Svab and Maliga, 1993; McBride et al., 1995).
Transposon-mediated repositioning of transgenes is an attrac-
tive strategy to generate plants that are free of selectable markers
and T-DNA inserts (Cotsaftis et al., 2002). By using a minimal
number of transformation events, a large number of transgene
insertions in the genome can be obtained so as to benefit from
position effects in the genome that can contribute to higher levels
of expression. Cry1B gene expressed under the control of maize
ubiquitin promoter between minimal terminal inverted repeats
of the maize Ac-Ds transposon system was cloned in the 5
un-
translated sequence of a gfp gene used as an excision marker.
The results indicated that transposon-mediated relocation of the
gene of interest is a powerful method for generating T-DNA
integration site-free transgenic plants and exploiting favorable
position effects in the plant genome.
BIO-EFFICACY OF TOXIN GENES EXPRESSED IN
TRANSGENIC PLANTS AGAINST INSECT PESTS
Genes from bacteria such as Bacillus thuringiensis (Bt) and
Bacillus sphaericus, protease inhibitors, plant lectins, ribosome-
inactivating proteins, secondary plant metabolites, and small
RNA viruses have been used alone or in combination with con-
ventional host plant resistance to develop crop cultivars that
suffer less damage from insect pests (Hilder and Boulter, 1999).
Genes conferring resistance to insects have been inserted into
crop plants such as maize (Zea mays), rice (Oryza sativa), wheat
(Triticum aestivum), sorghum (Sorghum bicolor), sugarcane
(Saccharam officinarum), cotton (Gossypium hirsutum), potato
(Solanum tuberosum), tobacco (Nicotiana tabacum), broccoli
(Brassica oleracea var italica), cabbage (Brassica oleracea
var capitata), chickpea (Cicer arietinum), pigeonpea (Cajanus
cajan), cowpea (Vigna unguiculata), groundnut (Arachis hy-
pogea), tomato (Lycopersicon esculentum), brinjal (Solanum
melongena), and soybean (Glycine max) (Sharma et al., 2000).
Genetically transformed crops with Bt genes have been deployed
for cultivation in USA, Argentina, Canada, China, South Africa,
Australia, Romania, Mexico, Bulgaria, Spain, Germany, France,
Uruguay, Indonesia, Ukraine, Portugal, and India.
While several transgenic crops with insecticidal genes have
been introduced in the temperate regions, very little has been
done to use this technology for improving crop productivity in
the harsh environments of the tropics, where the need for in-
creasing food production is most urgent. Transgenic Bt cotton
and maize have largely been grown on a commercial scale un-
der high input temperate or subtropical cropping systems. The
most urgent need to use this technology is in the tropical regions,
where soil fertility, water availability, insect pests, and diseases
severely constrain crop production. For transgenic plants to be
successful in integrated insect pest management they have to
substitute, completely or partially, for the use of insecticides
in crop production, and then result in increased crop produc-
tion and environment conservation. The bioefficacy of different
toxin genes expressed in transgenic plants has been discussed
below.
δ-Endotoxins from Bacillus thuringiensis
Bacillus thuringiensis was isolated from diseased larvae of
Mediterranean meal moth (Ephetia kuhniella) (Berliner, 1915).
It is a gram-positive bacterium that produces proteinaceuos crys-
talline (Cry) inclusion bodies during sporulation. It also pro-
duces cytotoxins that synergize the activity of Cry toxins. There
are several subspecies of this bacterium that are effective against
lepidopteran, dipteran, and coleopteran insects. The identifica-
tion of the kurstaki strain, which is highly effective against the
lepidopteran insects, provided a boost for commercialization of
Bt. Since then, a vast array of Bt strains have been isolated, of
which HD 1 strain is the most important product in the mar-
ket (Dulmage, 1981). The Bt toxins were earlier classified into
four types, based on insect specificity and sequence homology
(Hofte and Whiteley, 1989). Cry-I–type genes encode proteins
of 130 kDa and are usually specific to lepidopteran larvae, type II
genes encode for 70 kDa proteins that are specific to lepidopteran
and dipteran larvae, and type III genes encode for 70 kDa pro-
teins specific to coleopteran larvae. Cry-IV–type genes are spe-
cific to the dipteran larvae. The system was further extended
to include Cry-V–type genes that encode for proteins effec-
tive against lepidopteran and coleopteran larvae (Tailor et al.,
1992). The Bt δ-endotoxins are now known to constitute a fam-
ily of related proteins for which 140 genes have been described
(Crickmore et al., 1998), with specificities for Lepidoptera,
Coleoptera, and Diptera. Expression of Bt genes in tobacco and
tomato provided the first example of genetically modified plants
with resistance to insects (Barton et al., 1987; Fischhoff et al.,
1987; Vaeck et al., 1987). Progress made in developing trans-
genic plants with Bt genes has been discussed below.
Cotton
Considerable progress has been made in developing cotton
cultivars with Bt genes for resistance to bollworms, and there
is a clear advantage of growing transgenic cotton in reduc-
ing bollworm damage and increasing cottonseed yield (Hilder
and Boulter, 1999). Cotton plants with Bt genes are effective
50 H. C. SHARMA ET AL.
against pink bollworm (Pectinophora gossypiella) (Wilson et al.,
1992). Cotton cultivar Coker 312, transformed with the Cry1A(c)
gene (having 0.1% toxin protein), has shown high levels of
resistance to cabbage looper (Trichoplusia ni), tobacco cater-
pillar (Spodoptera exigua), and cotton bollworms (Helicoverpa
zea/Heliothis virescens). In transgenic cotton, cotton bollworm
damage was reduced to 2.3% in flowers and 1.1% in bolls com-
pared to 23% damage in flowers and 12% damage in bolls in
the commercial cultivar, Coker 312 (Benedict et al., 1996). The
cottonseed yield was 1050 kg ha
−1
in Coker 312 compared to
1460 kg in Bt cotton. In China, cotton cultivars Shiyuan 321
and Zhongmiansuo 19, 3517, and 541 (transformed with Bt
genes) have resulted in up to 96% mortality of cotton bollworm
(Helicoverpa armigera) (Guo et al., 1999). Transgenic cotton
lines S 545, S 591, S 636, and S 1001 from Simian 3 and 1109
from Zhongmiansuo 12 with Bt genes have shown adverse af-
fects on survival and development of H. armigera (Ni et al.,
1996). Cotton bollworm (H. zea and Heliothis virescens) sur-
vival has been found to be greater on squares and flower anthers
than on other floral structures in Deltapine 5415 conventional
cotton and transgenic NuCOTN 33B (Cotsaftis et al., 2002).
ELIZA tests indicated that Cry1A(c) expression varied in dif-
ferent plant parts, but bollworm survival did not correlate with
the protein expression (Gore et al., 2001). Trends in Bollgard
II were similar to Bollgard I and nontransgenic cotton. These
data support the observations under field conditions that white
flowers and small bolls of cotton suffer greater damage than the
older bolls.
First- to fourth-instar larvae of H. armigera died on trans-
genic Bt cotton, while in fifth- and sixth-instar larvae the pu-
pation decreased by 48.2 and 87.5%, and adult emergence by
66.7 and 100%, respectively. Egg laying decreased by 50.1 to
69.7%, and egg hatching by 80.6 to 87.8% (Cui and Xia, 1999).
Feeding dust of transgenic cotton to the adults decreased the
number of eggs and egg hatching by 59.8 and 72.1%, respec-
tively. Cry1A(c) levels can be quantified by cotton budworm
(H. virescens) growth inhibition bioassay through concurrently
run concentration-response curves using purified Cry1A(c) pro-
tein (Greenplate, 1999). The assay is amenable to large number
of samples, uses small amounts of plant tissue, and avoids some
of the concerns associated with immuno-based quantitative as-
says. The bioassay sensitivity ranged from 0.1 to 10.0 ng per
ml. Accurate measurements of Bt toxins through immunoassay
requires the production of quality antibodies, as well as opti-
mization and validation of protein extraction from the specific
tissue. Bioassays over the crop-growing season give a better in-
dication of active toxins in the plant than the immunological
recognition of the toxins, which may be influenced by other
chemical constituents of the plant.
Maize
Bacillus thuringiensis toxins expressed in maize plants are
highly effective against the European corn borer (ECB) (Ostrinia
nubilalis) (Koziel et al., 1993; Armstrong et al., 1995; Archer
et al., 2000). Transgenic maize expressing Cry9C (from Bacil-
lus thuringiensis subsp. tolworthi) is highly effective against
ECB (Jansens et al., 1997). Maize plants transformed with Bt
genes have also been found to be effective against the spotted
stem borer (Chilo partellus) and the maize stalk borer (Busseola
fusca) in Southern Africa (Rensburg van, 1999). Spotted stem
borer is more susceptible than the maize stalk borer to trans-
genic maize with Bt genes. Maize plants with Cry1A(b) gene are
also resistant to the sugarcane borers (Diatraea grandiosella
and Diatraea saccharalis) (Bergvinson et al., 1997). The Bt-
transformed plants exhibit greater resistance to D. grandiosella
than those derived from conventional host plant resistance breed-
ing. Williams et al. (1997) developed transgenic corn hybrids,
which sustained significantly less leaf feeding damage by fall
armyworm (Spodoptera frugiperda) and Southwestern corn
borer (Diabrotica undecimpuncta howardi) than the resistant
cultivars derived through conventional breeding. Resistance to
fall armyworm and near immunity to Southwestern maize borer
observed in these transgenic maize hybrids is the highest level of
resistance documented for these insect pests. Transgenic trop-
ical maize inbred lines with Cry1A(b) or Cry1A(c) genes with
resistance to corn earworm, fall armyworm, Southwestern corn
borer, and sugarcane borer have also been developed (Bohorova
et al., 1999). A binary insecticidal crystal protein (bICP) from
B. thuringiensis strain PS149B1, composed of a 14-kDa protein
(Cry34Ab1) and a 44-kDa protein (Cry35Ab1), have been coex-
pressed in transgenic maize plants and provide effective control
of Western maize rootworm, (Diabrotica virgifera virgifera) un-
der field conditions (Herman et al., 2002). The 14-kDa protein is
also active alone against the southern maize rootworm (Diabrot-
ica undecimpunctata howardi), and was synergized by a 44-kDa
protein.
Bt-maize is quite effective in preventing ECB damage, and
generally produces higher grain yields than the nontransgenic
crop (Clark et al., 2000). First generation ECB damage is re-
duced or eliminated with the use of the Bt hybrids. In the ab-
sence of ECB pressure, the performance of transgenic hybrids is
similar to their nontransgenic counterparts. Yield of isoline hy-
brids is 10% lower than the standard and Bt hybrids regardless
of ECB infestation (Lauer and Wedberg, 1999), but Bt hybrids
generally yield 4 to 8% greater than the standard hybrids un-
der severe ECB pressure. Transgenic crops have also been ob-
served to have beneficial effects on nontarget pests; for example,
maize hybrids with CryIA(b) also suffer less Fusarium ear rot
than their nontransgenic counterparts (Munkvold et al., 1999).
Novartis Sweetcorn and GH 0937 hybrids containing the Bt gene
are highly resistant to H. zea and S. frugiperda (Wiseman et al.,
1999; Lynch et al., 1999b).
Rice
Rice plants having 0.05% toxin of the total soluble leaf pro-
tein have shown high levels of resistance to the striped stem borer
(Chilo suppressalis) and rice leaf folder (Cnaphalocrosis medi-
nalis) (Fujimoto et al., 1993). Scented varieties of rice (Basmati
370 and M 7) have been transformed with Cry II(a) and are resis-
tant to yellow rice stem borer (Scirpophaga incertulas) and the
GENETIC TRANSFORMATION OF CROPS FOR INSECT RESISTANCE
51
rice leaf folder (Mqbool et al., 1998). Truncated Cry1A(b) gene
has been introduced into several indica and japonica rice cul-
tivars by microprojectile bombardment and protoplast systems
(Datta et al., 1998). Rice lines transformed with the synthetic
Cry1A(c) gene are highly resistant to yellow stem borer (Nayak
et al., 1997), and those with the Cry1A(b) gene are resistant to
the striped stem borer and the yellow stem borer (Ghareyazie
et al., 1997). Cry1A(b) gene has also been inserted into the
maintainer line, R 68899B, with enhanced resistance to yel-
low stem borer (Alam et al., 1999). Khanna and Raina (2002)
developed Bt-transgenics of elite indica rice breeding lines (IR
64, Pusa Basmati 1 and Karnal Local) with synthetic Cry1A(c)
gene. Selected Bt-lines of IR 64 and Pusa Basmati 1, having
Bt-titres of 0.1% (of total soluble protein), showed 100% mor-
tality of yellow stem borer larvae within 4 days of infestation
in cut-stems as well as at the vegetative stage in whole plant
assays. Husnain et al. (2002) expressed Cry1A(b) in Basmati
rice under the control of three promoters (PEPC, ubiquitin, and
pollen-specific promotor derived from Bp10 gene of Brassica
napus in pGEM 4Z). Toxin protein expression was 0.05% of
the total protein in stems under the control of PEPC promo-
tor alone or in combination with the pollen-specific promotor,
but was nearly 0.15% of the total protein under the control of
ubiquitin promotor, suggesting that a specific promotor can be
used to limit the expression on Cry1A(b) gene in desired plant
parts. The GUS histochemical assay, larval mortality, leaf area
consumed, and leaf disc and whole-plant bioassays have been
found to give similar results (Ye et al., 2000). Detached leaf
assay for evaluating the resistance of transgenic rice to striped
stem borer overcomes the difficulty of maintaining fresh stems
for a long time and frequent escape of striped stem borer larvae
(Yao et al., 2002).
Sorghum
Toxins from B. thuringiensis var morrisoni have shown bi-
ological activity against the sorghum shoot fly(Atherigona
soccata). Cry1A(c), Cry1C, Cry1E, and Cry2A are moder-
ately effective against spotted stem borer (C. partellus), while
Cry1A(c) is effective against H. armigera (Sharma et al., 1999).
Sorghum plants having the Cry1A(c) gene have been developed
at ICRISAT and are presently being tested for their resistance to
the spotted stem borer (Harshavardhan et al., 2002).
Sugarcane
The truncated Cry1A(b) gene in sugarcane has shown signifi-
cant activity against the sugarcane borer (D. saccharalis) despite
low expression of the Bt protein (Arencibia et al., 1997).
Oilseed Crops
A codon-modified Cry1A(c) gene has been introduced into
groundnut by using microprojectile bombardment (Singsit et al.,
1997). The immunoassay of plants selected with hygromycin has
shown the expression of Cry1A(c) protein up to 0.16% of the
total soluble protein. Complete mortality or up to 66% reduction
in larval weight has been recorded in the lesser corn stalk borer
(Elasmopalpus lignosellus). There is a negative correlation be-
tween larval survival and larval weight of the lesser corn stalk
borer with the amount of Bt protein.
Grain Legumes
A tissue culture and regeneration protocol has been devel-
oped for chickpea, which has been found to be useful for genetic
transformation of this crop (Jayanand et al., 2003). Chickpea
cultivars ICCV 1 and ICCV 6, transformed with Cry1A(c) gene,
have been found to inhibit the development of and feeding by
H. armigera (Kar et al., 1997). Pigeonpea plants with Cry1A(b)
and soybean trypsin inhibitor (SBTI) genes developed at
ICRISAT are being tested against H. armigera.
Tobacco
Expression of Bt genes in tobacco provided the first example
of genetically modified plants with resistance to insects (Barton
et al., 1987). Synthetic CryIII genes in tobacco are effective
for the control of Colorado potato beetle (Leptinotarsa decem-
lineata) (Perlak et al., 1993). Tobacco plants containing the
CryIIa5 gene are highly resistant to H. armigera (Selvapandian
et al., 1998), and the effectiveness of this toxin is comparable to
Cry1A(b) or Cry1A(c).
Potato
Synthetic CryIII gene has been expressed in potato plants
with resistance to Colorado potato beetle (L. decemlineata)
(Jansens et al., 1995). Transgenic potato plants containing the
Cry1A(b) gene (Bt 884), and a truncated gene Cry1A(b)6 re-
sulted in less damage to the leaves by the potato tuber moth
(Pthorimaea opercullela). However, the size of the leaf tunnels
increased over time in plants containing only the Bt 884 gene,
while there was no increase in tunnel length in those contain-
ing Cry1A(b)6 (Arpaia et al., 2000). The latter also resulted in
100% mortality of the insects in tubers stored up to six months.
Transgenic LT 8 and Sangema tubers remained uninfested by
P. operculella for 6 months. However, no significant effects were
observed on the nontarget species such as Liriomyza huidobren-
sis, Russelliana solanicola and Myzus persicae. Damage to the
4th terminal leaf by Epitrix cucumeris was 20 to 31% lower than
in nontransgenic plants (Stoger et al., 1999). Davidson et al.
(2002) developed transgenic lines of Ilam Hardy and Iwa with
Cry1Ac9 gene. A transgenic line from each cultivar inhibited lar-
val growth of P. opercullela by over 40%, and the line derived
from Ilam Hardy prevented pupation of all larvae. A modified
gene of B. thuringiensis var tolworthi (CryIIIB) has shown insec-
ticidal activity toward neonate larvae of Colorado potato beetle
(Arpaia et al., 1997). Picentia and the wild species, Solanum
integrifolium, have also been transformed with a wild-type (wt)
and four mutagenized versions of Bt 43 belonging to the CryIII
class (Innacone et al., 1997). Adult males feeding on high-level
Bt-expressing transgenic potatoes were able to mate and produce
mobile sperm, but the females were impaired in their reproduc-
tive ability since their ovaries were not fully developed (Stewart
et al., 1999). New Leaf Bt-transgenic potatoes provide substan-
tial ecological and economic benefits to potato growers (Hoy,