ARTICLE IN PRESS

Crop Protection 22 (2003) 1071–1086

Review
Advances in genetically engineered (transgenic) plants in pest
management—an over view
R. Mohan Babua,b,*, A. Sajeenab, K. Seetharamanb, M.S. Reddyc
a
Department of Biology, Erindale College, University of Toronto at Mississauga, Canada L5L 1J7
b
Department of Plant Pathology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai 625 104, Tamil Nadu, India
c
Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA 36849
Received 9 August 2002; received in revised form 11 May 2003; accepted 20 May 2003

Abstract

Transgenic plants are produced via Agrobacterium mediated transformation and other direct DNA transfer methods. A number
of transgenes conferring resistance to insects, diseases and herbicide tolerance have been transferred into crop plants from a wide
range of plant and bacterial systems. In the majority of the cases, the genes showing expression in transgenic plants are stably
inherited into the progeny without detrimental effects on the recipient plant. More interestingly, transgenic plants under field
conditions have also maintained increased levels of insect resistance. Now, transgenic crops occupy 44.2 million hectares on global
basis. During the last 15 years, transformations have been produced in more than 100 plant species; notable examples include maize,
wheat, soybean, tomato, potato, cotton, rice, etc. Amongst these herbicide tolerant and insect tolerant cotton, maize and soybean
carrying Bacillus thuringiensis (Bt) genes are grown on a commercial scale. Genetic transformation and gene transfer are routine in
many laboratories. However, isolation of useful genes and their expression to the desired level to control insect pests still involves
considerable experimentation and resources. Developing pest resistant varieties by insertion of a few or single specific gene(s) is
becoming an important component of breeding. Use of endotoxin genes such as Bt and plant derived genes (proteinase inhibitors) to
the desired levels offers new opportunities to control insects and strategies involving combination of genes. Transgenic technology
should be integrated in a total system approach for ecologically friendly and sustainable pest management. Issues related to
Intellectual property rights, regulatory concerns, and public perceptions for release of transgenics need to be considered. Providing
wealth of information on gene expression in higher plants by switching the gene on and off as and when required, makes gene
manipulation a more direct process for genetic improvement of crops.
r 2003 Elsevier Ltd. All rights reserved.

Keywords: Bt genes; Gene transfer methods; Pest management; Recombinant DNA technology; Transgenic crops

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1072
2. Methods for producing transgenic plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073
2.1. Agrobacterium-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073
2.2. Direct gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073
2.3. Polyethylene glycol (PEG) mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . 1073
2.4. Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1074
2.5. Microinjection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1074
2.6. Microprojectile bombardment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1074
2.7. Approaches for developing genetically engineered plants resistant to insects . . . . . . . . . . . . 1074

*Corresponding author. Present address: Agricultural College and Research Institute, Department of Plant Pathology, Tamil Nadu Agricultural
University, Madurai 625 104, India. Tel.: +91-452-422956; fax: +91-452-422785.
E-mail address: mohanbabu r@yahoo.com (R. Mohan Babu).

0261-2194/03/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0261-2194(03)00142-X
 ARTICLE IN PRESS
1072 R. Mohan Babu et al. / Crop Protection 22 (2003) 1071–1086

2.8. Bacillus thruingiensis (Bt) genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1074

2.9. Genetic manipulation of Bt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076
2.10. Plasmid curing and conjugal transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076
2.11. Recombinant DNA technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076
2.12. Field trial testing of Bt crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077
2.13. Plant derived genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077
2.14. Proteinase inhibitor genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077
2.15. a-Amylase inhibitor genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078
2.16. Lectin genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078
2.17. Other novel genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079
2.18. Gene pyramiding (combination of multiple gene effect) . . . . . . . . . . . . . . . . . . . . . . 1080
2.19. Commercialized transgenic crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1080
3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081

1. Introduction A number of methods are now available to produce

transgenic plants, which include a modified Ti plasmid
The global population exceeded 6 billion in 2000 and is system in Agrobacterium tumefaciens and direct gene
expected to reach approximately 8.5 billion by 2025. Thus transfer including PEG-induced DNA uptake, micro-
to meet the increasing needs of the growing human injection of DNA into cultured cells, electroporation and
population, it will be necessary to produce 50% more microprojectile bombardment. Efficient methods of gene
food by 2025. Crop varieties with higher yield and cloning, transformation, plant regeneration, availability
stability are required. During the last few decades, major of new gene constructs, improved vector systems based
progress has been made in increasing crop productivity on Ti and Ri plasmids of Agrobacterium, appropriate
worldwide. As an example the world rice production has organ-specific promoters for gene expression, series of
more than doubled from 25 million tonnes in 1966 to 92 selectable marker genes and a large number of cloned
million tonnes in 1999 (Khush, 1999, 2001). Despite DNA constructs are among the important breakthroughs
extensive plant breeding efforts, over $10 billion is spent which have contributed to the large scale production of
worldwide each year on the management and chemical transgenic plants (Sanchis et al., 1997; Ding et al., 1998;
control of insect damage (Khush, 1999). Heavy reliance Peloquin et al., 2000; Walker et al., 2000; Zhao et al.,
on chemical pesticides, may not be viable as they provide 2001a–c; Brotman et al., 2002; Puterka et al., 2002; De
ephemeral benefits, often with adverse side effects, and in Leo and Gallerani, 2002). Transgenic plants have been
some instances actually worsen farmer’s overall pest produced in several major crop plants such as maize,
problems (Way and Heong, 1994; Manichon, 1996; wheat, barley, rice, cotton, potato, tomato and soybean.
Estruch et al., 1997; Kogen, 1998; Matteson, 2000; Cook, A series of genes governing agronomically important
2000; Sharma and Ortiz, 2002). Thus, the major challenge traits have been transferred through various transforma-
is how to increase and sustain crop productivity with less tion techniques (Table 1) (Brar and Khush, 1993; Brar
use of chemicals. Until recently, pest resistant varieties et al., 1995; Khush and Brar, 1998). Notable examples
have been developed mainly through the application of include Bt and proteinase inhibitor genes for insect
principles of classical Mendelian genetics and conven- resistance, coat protein (CP) genes, antisense-CP and
tional plant breeding methods. Recent advances in satellite RNA for virus resistance; chitinase and ribosome
cellular and molecular biology have opened new avenues inactivating protein genes (RIP) for fungal resistance;
for production of genetically engineered (transgenic) bar, ALS genes for herbicide resistance (Brown, 2000;
plants with new genetic properties. Such crops often Christeller et al., 2002; Tabashnik et al., 2002; Datta et al.,
referred to as genetically modified (GM) crops, represent 2002; Hudak et al., 2002; Butterfield et al., 2002; Tranel
a promising opportunity to make an important contribu- et al., 2002; Burd et al., 2003; Volpicella et al., 2003; Qu
tion to integrated pest management (IPM) programs. et al., 2003; Lin et al., 2003; Zhang and Simon, 2003).
Since the first transgenic tobacco plants expressing These developments in genetic engineering have also
foreign proteins obtained in 1984 (Horsch et al., 1985), provided a wealth of information on gene silencing in
transgenic plants have been produced in more than 100 transgenic plants, which helps to understand the genome
species (Brar et al., 1995; Khush and Brar, 1998). The organization and regulation of gene expression. The
area under transgenic crops has increased from 1.7 development of transgenic pest resistant varieties having
million hectares in 1996 to 44.2 million hectares in 2000 strong insecticidal activities has raised concerns on the
(James, 2000). development of resistance to pests including effect on
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Table 1
Insertion and expression of chimeric genes in genetically engineered plants

Classification Chimeric genes

Abiotic stress tolerance gpat, sod, MtID, sod

Salinity tolerance betA, p5cs, hval, codA, afp, imt 1
Seed storage protein Phaseolin, phytohemagglutinin, conglycinin, patatin, zein glutenin
Nutritional quality psy, crtl, Icy
(pro vitamin A)
Male sterility fertility Ribonuclease gene (barnase), Ribonuclease inhibitor (barstar)
restoration
Photosynthesis Chlorophyll a/b-binding protein, ribulose-1, 5-bisphosphate carboxylase small subunit
Nodulation Lectin, leghemoglobin
Transposon Ac, Ds, Tam, En-l
Fungal resistance Chitinase, ribosome inactivating protein (RIP)
Virus protection Coat protein genes, antisense-coat protein, satellite RNA
Insect resistance Bt toxin; crylA(a), crylA(b), crylA(c), crylC, crylllA, proteinase inhibitor (CpTi), corn cystatin (CC), oryza cystatin
1 (OCI) a! ai, gna, chicken avidin gene
Herbicide tolerance aroA and EPSP (glyphosate), bar (phosphinothricin), bxn (bromoxynil), ALS (sulfonylurea), tfdA (2,4-D)
Visible coloration b-glucuronidase, luciferase, b-galactosidase
Opine synthesis Octopine synthase, nopaline synthase
Drug resistance Neomycin phosphotransferase, chloramphenicol acetyltransferase, dehydrofolate reductase, hygromycin
phosphotransferase, streptomycin phosphotransferase
Pigmentation Dehydroquercetin reductase, chalcone synthase
Others Polygalacturonase, heat shock protein, chitinase, tubulin, ATP synthase, isopentenyl transferase, metallothionein,
pathogenesis-related protein, phytochrome

environment (Fan et al., 1999; Carriere et al., 2001; formation method’ (Horsch et al., 1985) and selectable
Stewart et al., 2001). The expressions of multiple genes in marker genes encoding for kanamycin resistance
plants for long-term durable resistance to insects are also (Cheng et al., 1998; Li et al., 1999; Huang et al., 2001)
emphasized (Porter et al., 2002; Odintsova et al., 2002; have become available and are being used extensively in
Datta et al., 2002). gene transfer into higher plants. The method has been
used successfully for transformation of numerous dicot
species (Atkinson, 2002; Wimmer, 2003). More
2. Methods for producing transgenic plants recently, Agrobacterium-mediated gene transfer method
has also been used in transformation of agronomically
The refinement in plant regeneration from cultured important monocots like maize, wheat and rice (Hiei
cells, efficient vector constructs and availability of et al., 1994).
defined selectable marker genes and biolistic method
of transformation have resulted in the production of
2.2. Direct gene transfer
transgenic plants in more than 100 species (Wimmer,
2003). Various methods for plant transformation have
Although A. tumefaciens has been found to be a very
become available (Wimmer, 2003).
effective DNA transfer system for genetic transforma-
tion, direct DNA transfer methods have been developed
2.1. Agrobacterium-mediated gene transfer
for transformation of plant species (Christou, 1997a, b;
Kohli et al., 1998; Ghareyazie et al., 1997; Tu et al.,
Agrobacterium tumefaciens is a soil bacterium which
2000). Some methods are discussed below.
infects a wide range of dicot plant species and is capable
of transferring a piece of DNA (T-DNA) into the genome
of host plants. The genes inserted into the T-DNA region 2.3. Polyethylene glycol (PEG) mediated gene transfer
through Ti plasmids are co-transferred and integrated
into the host genome. It is now well known that for DNA PEG has been employed as a fusion agent in somatic
transfer only the T-DNA borders and some flanking cell hybridization of various plant and animal species
sequences are needed. Thus by deleting T-DNA encoded (Barreto et al., 1997; Zhao et al., 2001a–c; Szarka
genes and adding selectable markers, ‘disarmed plasmid et al., 2002). Using the PEG method, foreign genes in
vectors’ can be used to transfer foreign genes without the form of plasmid DNA were inserted into plant
disturbing the endogenous hormone balance. Further- protoplasts, resulting in the recovery of fertile regener-
more, simple co-cultivation techniques; ‘leaf disc trans- ated tobacco (Paszkowski et al., 1984). The procedure
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involves several tissue culture steps to produce transgenic sugarbeet and wheat. Several reports have demonstrated
plants. the successful transformation of rice for pest resistance
using this biolistic method of transformation (Wuhn
2.4. Electroporation et al., 1996; Nayak et al., 1997; Ghareyazie et al., 1997;
Tu et al., 2000).
Electroporation involves the application of high-
voltage electrical pulses to a solution containing a 2.7. Approaches for developing genetically engineered
mixture of protoplasts and foreign DNA. Such electrical plants resistant to insects
treatment is known to reversibly permeate membranes of
mammalian, plant and microbial cells (Sorokin et al., Both A. tumefaciens-mediated gene transfer and direct
2000; Rowan et al., 2000; Koscianska and Wypijewski, DNA transfer methods have been used to produce
2001; Delaitre et al., 2001; Gehl, 2003). The method transgenic plants with new genetic properties (Christou,
offers a unique opportunity to introduce DNA which 1997a; Ghareyazie et al., 1997; Cheng et al., 1998; Kohli
enters the cells through reversible pores created in the et al., 1998; Li et al., 1999; Tu et al., 2000; Huang et al.,
membrane by the action of short electrical pulses. 2001; Atkinson, 2002; Wimmer, 2003) Several genes for
Electroporation has been used to produce transgenics in insect resistance have been transferred through genetic
species such as wheat, rice, sugarcane, tobacco and others engineering techniques (Table 1). Use of delta-endotoxin
(Sorokin et al., 2000; Koscianska and Wypijewski, 2001; coding sequences originating from Bacillus thuringiensis
Brumbley et al., 2002). (Bt) and use of plant derived genes, such as those
encoding enzyme inhibitors or lectins are the two main
2.5. Microinjection approaches to produce such genetically engineered
plants (Hua et al., 1998; Sehnke and Ferl, 1999; Zhu
Microinjection of DNA into intact plant tissue such as et al., 2000).
meristems, including cultured cells has been used to
produce transgenic plants (Russell and Fromm, 1997; 2.8. Bacillus thruingiensis (Bt) genes
Dresselhaus et al., 1999; Newell, 2000; Holm et al., 2000).
The method is simple and can be used on whole plants Bacillus thuringiensis, commonly known as Bt, is a
without any tissue culture steps (Newell, 2000). However, bacterium that occurs naturally in the soil. It has been
few reports are available to support production of stable used for more than 50 years as a biological insecticide
transformants using microinjection (Newell, 2000; Holm (Musser and Shelton, 2003; Carriere et al., 2003; Qaim
et al., 2000; Kimura, 2001). and Zilberman, 2003). The bacterium was first discovered
in Japan in 1902. It produces a number of insect toxins,
2.6. Microprojectile bombardment the most distinctive of which are protein crystals formed
during sporulation. The crystal proteins are specifically
The biolistic method of particle bombardment has been toxic to lepidopteran insects (Olsen and Daly, 2000;
a major development in direct gene transfer of DNA Stewart et al., 2001; Adamczyk et al., 2001a, b; Gore et al.,
(Nayak et al., 1997; Ghareyazie et al., 1997; Tu et al., 2001, 2002) and act by disrupting the midgut cells of the
2000; Taylor and Fauquet, 2002). It has enabled insect. The crystal proteins are dissolved by the alkaline
transformation of many plant species which were not gut juices in the midgut lumen, and are converted by gut
amenable to Agrobacterium or protoplast based gene proteases into toxic core fragments. Thereafter, the
transfer techniques. The method is considered to be midgut epithelia cells swell and eventually burst. Micro-
independent of genotype and the tissue (Cheng et al., bial preparations of Bt spores and crystals have been used
1998; Tu et al., 2000; Taylor and Fauquet, 2002). It as commercial insecticides for over 20 years. Individually,
consists of delivering DNA into cells of intact plant the Bt proteins deployed in transgenic crops show specific
organs or cultured tissues via a projectile bombardment activity against narrow groups of pest species and usually
process. Small, high density particles (microprojectiles) have little or no direct effect on non-target species,
are accelerated to high velocity by a particle gun including beneficial insects (Perlak et al., 1990; Carriere
apparatus (Ghareyazie et al., 1997; Tu et al., 2000; et al., 2003; Qaim and Zilberman, 2003). This specificity
Taylor and Fauquet, 2002; Wimmer, 2003). These dense offers many advantages over unconventional pesticides as
particles thus acquire sufficient kinetic energy to pene- tools for integrated pest management.
trate plant cells and membranes and carry foreign DNA At least 90 genes encoding protoxins (Digestive
into the interior of bombarded cells. Christou (1997a) enzymes cutting the inactive protein (protoxin) into
reviewed several reports on transformation through fragments, some of which may bind to ‘‘receptor’’
microprojectile bombardment of major crop plants proteins already present in the insect’s midgut) from B.
including banana, barley, bean, canola, cassava, maize, thuringiensis isolates have been isolated and sequenced
cotton, papaya, peanut, soybean, squash, sunflower, (Mazier et al., 1997; Zhao et al., 2001a, b). Initially, Bt
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toxins were classified into 14 distinct groups and 4 content of their encoding genes (Koziel et al., 1993;
classes based on their host range. These include cry1 Perlak et al., 1991). The first results on transfer of Bt
(active against Lepidoptera), cry II (Lepidoptera and genes in tobacco and tomato were published in 1987
Diptera), cry III (Coleoptera) and cry IV (Diptera). (Fischhoff et al., 1987; Barton et al., 1987). Since then Bt
Transgenic plants carrying Bt genes have been produced genes have been transferred to many crops including
in tobacco, potato, tomato, cotton, maize and rice with cotton, maize, rice and potato. Fischhoff et al. (1987)
different crystal protein genes (Table 2). A critical factor constructed a chimeric gene (A gene or DNA sequence
following transformation is the desired expression of the not found in nature but has been constructed in a
insecticidal gene. Bt genes are Adenine–Thymine rich laboratory. This includes genes which have only been
while plant genes tend to have a higher Guanine– slightly altered from their original form) containing the
Cytosine content. The expression of insecticidal proteins CaMV 35S promoter and the B. thuringiensis protein-
have been enhanced by increasing Guanine–Cytosine coding sequences. The transgenic tomato plants

Table 2
Examples to show Bt genes insertion and expression in transgenic plants for insect resistance

Crop target Inserted Origin of Useful trait Reference(s)

gene transgene

Oryza sativa L. cryIA(b) B. thuringiensis Resistance to striped stem borer (Chilo suppressalis Walker), Fujimoto et al. (1993)
and leaf folder (Cnaphalocrocis medinalis Guenee)
cryIA(b) B. thuringiensis Resistance to yellow stem borer (Scirpophaga incertulas Wuhn et al. (1996)
Walker) and striped stem borer (C. suppressalis Walker)
cryIA(c) B. thuringiensis Resistance to yellow stem borer (S.incertulas Walker) Nayak et al. (1997)
cryIA(b) B. thuringiensis Resistance to yellow stem borer (S. incertulas Walker) and Ghareyazie et al. (1997)
striped stem borer (C. suppressalis Walker)
cryIA(b) B. thuringiensis Resistance to yellow stem borer (S. incertulas Walker) Data et al. (1998)
cryIA(b) B. thuringiensis Resistance to yellow stem borer (S. incertulas Walker) Alam et al. (1999)
cryIA(b) B. thuringiensis Resistance to leaf folder (C. medinalis Guenee) and yellow Tu et al. (2000)
cryIA(c) stem borer (S. incertulas Walker)
Zea mays L. cryIA(b) B. thuringiensis Resistance to European corn borer (Ostrinia nubilalis Koziel et al. (1993)
.
Hubner),
Glycine max L. cryIA(c) B. thuringiensis Resistance to bollworm (Helicoverpa zea Boddie) and Stewart (1996)
bud worm (H. virescens F.)
Meidcago sativa L. cryIC Synthetic gene Resistance to leaf worm (Spodoptera littoralis Boisd.) Strizhov et al. (1996)
Lycopersicon cryIA(c) B. thuringiensis Resistance to tobacco hornworm (Manduca sexta L.) Fischhoff et al. (1987)
esculentum L.
Bt(k) B. thuringiensis Resistance to tobacco hornworm (M.sexta L.), tomato Delannay et al. (1989)
pinworm (Keiferia lycopersicella Walshingham) and tomato
fruit worm H. zea Boddie)
Solanum cryIA(b) B. thuringiensis Resistance to tuber moth (Phthorimaea operculella Zeller) Peferoen et al. (1990)
tuberosum L.
cryIIIA B. thuringiensis Tolerance to Colorado beetle (Leptinotarsa decemlineata Perlak et al. (1993)
Say)
cryV Bt B. thuringiensis Potato tuber moth (P. operculella Zeller) Douches et al. (1998)
cryIA (b)
Gossypium cryIA(b) B. thuringiensis Resistance to cotton boll worm (H. Armigera Hubn) Perlak et al. (1990)
hirsutum L. cryIA(c)
Brassica napus L. cryIA(c) B. thuringiensis Resistance to (H. zea Boddie) and (S. exigua Hubner) Stewart (1996)
Nicotiana cryIA(a) Bacilus Resistance to tobacco hornworm (M.sexta L) Barton et al. (1987)
tobaccum L. thuringiensis
cryIA(b) B. thuringiensis Resistance to tobacco hornworm (M.sexta L) and budworm Vaeck et al. (1987)
(H. virescens Fabricius)
cryIA(c) B. thuringiensis Resistance to tobacco hornworm (M.sexta L) Perlak et al. (1991)
cryIA(c) B. thuringiensis Resistance to tobacco budworm (H. virescens Fabricius) McBride et al. (1995)
cryIA(c) B. thuringiensis Resistance to tobacco budworm (H. virescens Fabricius) Strizhov et al. (1996)
cry2aa2 B. thuringiensis Resistance to cotton bollworm (H. zea Boddie) De Cosa et al. (2001)
Saccharum cryIA(b) B. thuringiensis Resistance to stem borer (Diatraea saccharalis F.) Arencibia et al. (1997)
officinarum L.
Solanum cry(III)b B. thuringiensis Resistance to Colorado potato beetle (L. decemlineata Say) Arencibia et al. (1997)
melongena L.
Populus cryIA(a) B. thuringiensis Resistance to forest tent caterpillar (Malacosoma disstria Mc Cown et al. (1991)
tremuloides Michx. and gypsy moth Lasiocampidae)
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expressed the Bt toxin gene. Transgenic plants showed was introduced into several varieties of rice (indica and
little feeding damage by the larvae of Heliothis virescens japonica) by microprojectile bombardment and proto-
Fabricius (tobacco budworm). Similarly, leaves of plast systems. Out of 1800 putative Bt transgenics, 100
transgenic potato engineered with cry IA (b) gene proved plants were confirmed for integration of cry IA (b).
highly resistant to feeding and tunneling damage by Transgenics showed high level of resistance to S.
Phthorimaea operculella Zeller (tuber moth) larvae incertulas Walker (yellow stem borer). McBride et al.
(Peferoen et al., 1990). (1995) integrated a native Bt gene into tobacco chlor-
Perlak et al. (1990) produced insect resistant cotton. Bt oplast genome by homologous recombination (exchange
coding sequence was modified to increase the levels of of genetic material between two DNA fragments pre-
both crylA (b) and crylA (c) insect control protein senting sequence homologies). The integration of the
expression to 0.05–0.1% of the total soluble proteins. toxin Bt gene (3–5% of leaf soluble protein) showed
These truncated forms of the insect control protein genes tolerance to insects.
Bt provided effective control. Plants assayed under high
insect pressure with cotton bollworm showed effective 2.9. Genetic manipulation of Bt
square and boll protection. The plants with the modified
cry IA (b) gene had 10–100 fold higher level of insect The genetic manipulation of Cry genes in Bt offers a
control protein compared with the wild type gene. Similar promising means of improving the efficacy and cost-
results were obtained with the cry IA (c) gene (Perlak effectiveness of Bt based bioinsecticide products. Certain
et al., 1991). combinations of Cry proteins have been reported to
Fujimoto et al. (1993) transformed rice variety exhibit synergistic toxicity towards lepidopteran and
Nipponbare with cry IA (b) gene under the control of dipteran pests (Becker and Rettich, 1994; Wilcox et al.,
35S promoter. Two independent lines carried a single 1994; Lee and Dean, 1996; Wirth et al., 1997; Roush,
stable inherited and functional cry IA (b) gene. Trans- 1997). In addition, the presence of spores can also
genic plants had increased resistance to striped stem borer synergize the activity of cry proteins against certain
(Chilo suppressalis Walker) and leaf folder (Cnaphalocro- lepidopteran pests, and may forestall the development of
cis medinalis Guenee). Many laboratories have produced insect resistance to Cry proteins (Li et al., 1987; Benoit
transgenic rice carrying Bt genes for tolerance to stem et al., 1995; Liu et al., 1998). Other factors may
borer (Wuhn et al., 1996; Nayak et al., 1997; Ghareyazie contribute to the entomopathogenic character of Bt,
et al., 1997; Tu et al., 2000). Wuhn et al. (1996) including the vegetative insecticidal proteins (VIP), a-
introduced cry IA (b) gene into rice variety IR 58 (IR endotoxin and a variety of secondary metabolites,
stands for IRRI, Phillipines) through particle bombard- including Zwittermycin may be amenable to genetic
ment. The transgenic plants showed significant insectici- manipulation (Schnepf et al., 1998; Federici, 1999; Glare
dal effect on several lepidopterous insect pests. Feeding and Callaghan, 2000; National Academy of Sciences,
studies showed up to 100% mortality for Scirpophaga 2000; Aronson and Shai, 2001; James, 2001; National
incertulas Walker (yellow stem borer) and C. suppressalis Agricultural Biotechnology Council, 2001; Shelton et al.,
Walker (striped stem borer). Nayak et al. (1997) 2002).
transformed IR64 through particle bombardment using
cry IA (c) gene placed under the control of the maize 2.10. Plasmid curing and conjugal transfer
ubiquitin 1 promoter. The transferred synthetic cry IA (c)
gene was stably expressed in the T2 of these lines and the Using plasmid curing and conjugal transfer techniques,
transgenic rice plants were highly toxic to S. incertulas a Bt strain EG-2424, the active ingredient in the soil of
Walker (yellow stem borer) larvae which caused little bioinsecticide was derived by Ecogen Inc. for application
feeding damage. Ghareyazie et al. (1997) transformed on potatoes, eggplant and other Solanaceous crops
aromatic rice variety ‘‘Tarom Molali’’ carrying a (Carlton and Gawron-Burke, 1993). This strain produces
synthetic truncated cry IA (b) toxin gene. The transgenic both cry IIlA and lA (c) proteins, which are responsible
lines showed high levels of tolerance to S. incertulas for the lepidopteran–coleopteran activity (Carlton and
Walker (yellow stem borer). Alam et al. (1999) intro- Gawron-Burke, 1993).
duced cry IA (b) gene driven by 35S promoter through
biolistic method. The integration of the cry IA (b) gene 2.11. Recombinant DNA technology
in the transgenic rice (IR68899B) showed resistant to
S. incertulas Walker (yellow stem borer). Certain combinations of naturally occurring insectici-
Datta et al. (1998) introduced the truncated chimeric dal crystal protein (ICP) genes cannot be achieved
Bt gene, cry IA (b) of B. thuringiensis, driven by two through conjugal transfer since not every ICP-encoding
constitutive promoters, 35S from CaMV and Actin-1 plasmid can be transferred via conjugation (Dankocsik
from rice and two tissue-specific promoters, pith tissue et al., 1990). In addition, genes coding for ICP’s with
and pepcarboxylase (PEPC) for green tissues from maize, superior insecticidal activity against a specific target pest
 ARTICLE IN PRESS
R. Mohan Babu et al. / Crop Protection 22 (2003) 1071–1086 1077

may be present on the same plasmid as other ICP genes protein detected was 20 ng/mg soluble protein. Field
coding for proteins with much less desirable insecticidal testing of the transgenic rice lines showed high protection
activities (Peferoen, 1992; Merlin et al., 1993; Estruch to leaf folder (Cnaphalocrocis medinalis Guenee) and
et al., 1996; Sims et al., 1996; Vaughan and Donald, Scirpophaga incertulas Walker (yellow stem borer). The
1999). This necessitates the use of in vitro molecular percentage of plants with whiteheads (stem borer injury)
biological techniques to accomplish the optimal ICP was significantly lower on the Bt Shanyou63 (11%) as
gene combinations (Estruch et al., 1996; Vaughan and compared to control Shanyou63 (44%) plants. Similarly
Donald, 1999; Sugita et al. 2000; De Cosa et al., 2001). transgenic plants showed no leaf folder attack (0.0%) as
Recombinant DNA techniques can also be used to compared with non-transgenic (Shanyou63) where, 60%
improve the yield of an ICP by altering certain of the plants were affected by leaf folder. Bt proteins have
controlling regions (promoters) of the ICP gene been commercialized in cotton (expressing the cry IA(c),
(Dankocsik et al., 1990). This can be particularly maize (cry IAb), and potato (cry3A). The cry IAc protein
important only, in which highly active ICP’s are in Bt cotton provides insecticidal activity against many
produced in limited amounts by a naturally occurring lepidopteran species. However, there were no effective
strain. controls for O. nubilalis Hubner (European corn borers).
Potatoes have been engineered for control of Leptinotarsa
2.12. Field trial testing of Bt crops decemlineata Say (Colorado potato beetle), a significant
pest in many production areas (Duncan et al., 2001).
The first field trial with genetically engineered plants More Bt crops are under development, including rice,
expressing Bt toxin was conducted in 1986 with tobacco sorghum, lupins, peas and other legumes, and several tree
(Johnson et al., 1989; James, 1997, 2000). Since then, crops (Duan et al., 1996; James, 1997, 2000). Bt cotton
transgenic corn, tomato and cotton have been field tested varieties have been developed and commercialized as
in USA, Argentina and Australia. In 1996, Bt crops BOLLGARD (Bryant et al., 1997, 1999; Edge et al.,
occupied 1.2 million hectares (James, 1997, 2000). 2001) in the USA, China, South Africa, and Argentina,
Delannay et al. (1989) evaluated transgenic tomatoes and as INGARD (Pyke and Fitt, 1998; Wilson et al.,
expressing Bt insect control protein under field conditions 1998; Finnegan et al., 1998) in Australia.
in 1987 and 1988. The transgenic plants showed very
limited feeding damage after infestation with the tobacco 2.13. Plant derived genes
hornworm (Manduca sexta) whereas control plants
showed heavy feeding damage and were almost comple- The second category of transgenes having insecticidal
tely defoliated within two weeks. Significant control of activity is plant derived genes such as proteinase
tomato fruit worm and tomato pinworm was also inhibitors. Proteinase inhibitors from plants are of
observed. Bioassay showed that transgenic plants pro- particular interest because these inhibitors are part of
duce 1 ng of Bt protein per mg of soluble protein. Koziel the natural plant defense system against insect attack
et al. (1993) produced transgenic maize plants and (Casaretto and Corcuera, 1995; Heath et al., 1997;
obtained high levels of resistance to Ostrinia nubilalis Jongsma and Bolter, 1997; Larry and Richard, 2002).
Hubner (European corn borer). A synthetic gene encod- Many insect species possess serine-type proteinase, such
ing a truncated version of the cry IA (b) protein derived as trypsin and chymotrypsin-like enzymes in their
from B. thuringiensis was introduced. Modification of the digestive systems for digestion of food protein. Proteinase
native cry IA (b) coding region and increasing Guanine– inhibitors can be detrimental to the growth and develop-
Cytosine content from 38% to 65% greatly enhanced its ment of many insects (Heath et al., 1997; Jongsma and
expression in maize. The first field trials with Bt Bolter, 1997; Larry and Richard, 2002). These proteinase
transgenic cotton were conducted in USA in 1988 inhibitors are common components of plant derived food
(Jenknis et al., 1991). The cry IA proteins expressed in for humans and animals, and they are easily inactivated
Bt cotton and Bt corn have been extensively tested for by cooking, thus introduction of the proteinase inhibitor
toxicological analysis in the laboratory and field. These genes into crop plants can be regarded as safe (Duan
studies (Huang et al., 1999; Thomas et al., 1995a; et al., 1996; Colfalonieri et al., 1998; Mochizuki et al.,
McGaughey et al., 1998) strong1y support the specific 1999; Larry and Richard, 2002).
activity spectrums of Bt proteins, which are largely
mediated by the gut conditions required for activation 2.14. Proteinase inhibitor genes
of the proteins, and by the need for specific binding to
receptors in the mid-gut before toxicity is demonstrated. Another strategy for achieving insect resistance is to
Tu et al. (2000) produced transgenic indica rice CMS engineer proteinase inhibitor genes and transfer such
restorer line Minghui 63 (T5I-I) expressing a Bt fusion genes into commercial cultivars (Duan et al., 1996;
gene derived from cry IA (b) and cry I (c) under the Colfalonieri et al., 1998; Mochizuki et al., 1999; Larry
control of the rice actin 1 promoter. The level of Bt fusion and Richard, 2002). The presence of antimetabolic
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proteins, which interfere with the processes of digestion in tion of the interaction between protease inhibitor and its
insects, is a strategy for defence that plants have used target protease will lead to increased protection for
extensively (Thomas et al., 1995a, b). Proteins can occur transgenic plants expressing that inhibitor. The strategy
in tissues that are particularly vulnerable to attack, such to use more than one foreign inhibitor in transgenics to
as seeds, or can be induced by mechanical wounding in affect different digestive proteases in the pest seems
tissues attacked by chewing insect pests, such as leaves appropriate. Combination of transgenes could lead to
(Datta et al., 1998; Mochizuki et al., 1999; Larry and higher levels of insect control (Zhao et al., 1998; Cornu
Richard, 2002). Plants can now be transformed with et al., 1996; Sharma et al., 2000).
proteinase inhibitor gene with strong promoters to
express the inhibitor proteins in relatively high levels at 2.15. a-Amylase inhibitor genes
specific times (Duan et al., 1996; Colfalonieri et al., 1998;
Mochizuki et al., 1999). The presence of serine proteinase a-Amylase inhibitors might be toxic to insects through
inhibitors in plants can reduce insect attack. They are of interference in the digestion of dietary carbohydrates. It
interest because of their potent inhibitory activities is widely assumed that the proteinase inhibitors and a-
against proteolytic enzymes of insects. Analysis of the amylase inhibitors that accumulate in plant tissues are
effects of dietary proteinase inhibitors has shown that produced as a normal defense against insects (Ishimoto
these are detrimental to the growth and development of et al., 1996; Shade et al., 1999). Other genes that might
insects, from a variety of genera including Helicoverpa, confer enhanced insect resistance are genes encoding a-
Spodoptera and Diabrotica (Thomas et al., 1994; amylase inhibitors, lectins and chitinases. Many lectins
Edmonds et al., 1996; Wu et al., 1997). are toxic to insects, presumably through some deleterious
The first gene of plant origin to be successfully interaction with intestinal glycoproteins. Chitinases
transferred to another plant species resulting in enhanced might be toxic to insects if they are able to degrade the
insect resistance was isolated from cowpea encoding a chitin layer of the peritrophic membrane, which protects
trypsin inhibitor (CpTi) (Hilder et al., 1987). Since then, the insect’s gut epithelium (Shade et al., 1999; Schroeder
many plant derived genes for insect resistance have been et al., 1995; Ishimoto et al., 1996).
transferred to produce transgenic plants (Table 3).
Cowpea trypsin inhibitor (CpTi) gene has provided 2.16. Lectin genes
protection for lepidopteran pests including H. zea,
Spodoptera littoralis and Manduca sexta (Gatehouse Lectins are carbohydrate binding proteins and are
et al., 1992). Resistance to bruchid beetle, Callosobruchus abundant in seeds and storage tissues of some plant
maculatus is due to an elevated level of trypsin inhibitor. species. Lectins such as those purified from snowdrop or
Duan et al. (1996) used serine proteinase inhibitor garlic are toxic to insects but not to mammals (Powell
genes from cowpea and potato and produced transgenic et al., 1993, 1995; Gatehouse et al., 1995; Rao et al.,
rice through biolistic method of transformation. Over 1998; Sharma et al., 2000). The most effective protein
70% transformants were fertile. The transgenic plants tested is the lectin from snowdrop (Galanthus nivalis
showed enhanced resistance to striped stem borer. agglutinin; GNA), which gave approximately 80%
Similarly, Xu et al. (1996) introduced CpTi gene driven mortality at a concentration of 1 g l 1 in the diet, when
by the active promoter of the actin I gene into rice. The used in assays with first and third instar nymphs. GNA
transgenic rice carrying CpTi gene showed increased also had antimetabolic effect on brown plant hopper
resistance to striped stem borer and pink stem borer. (BPH), and green leafhopper pests of rice (Powell et al.,
Marchetti et al. (2000) transferred three soybean genes 1993).
(Kti3, C-II and PI-IV) coding for serine proteinase Rao et al. (1998) introduced snowdrop lectin gene (gna)
inhibitors in potato and tobacco through Agrobacterium through biolistic method of transformation in rice.
mediated transformation. Biochemical assays confirmed To evaluate the potential of the gna gene to confer
that transgenic plants synthesized serine proteinase resistance towards BPH, transgenic rice (Oryza sativa L.)
inhibitors. plants were produced, containing the gna gene in
The protease inhibitor genes have certain limitations constructs where its expression was driven by a phloem-
(Sharma and Ortiz, 2000; Miflin, 2000) i.e. with a given specific promoter (from the rice sucrose synthase RSs1
insect species, some inhibitors are found to be effective gene) and by a constitutive promoter (from the maize
antimetabolites, whereas others are not (Edmonds et al., ubiquitin ubi1 gene). PCR and Southern analyses on
1996; Wu et al., 1997; Sharma and Ortiz, 2000). Inhibitor DNA from these plants confirmed their transgenic
can show varying degrees of effectiveness as an anti- status, and that the transgenes were transmitted to
metabolite against different insects, and the protease progeny after self-fertilization. Western blot analysis
inhibitor genes are not as effective as Bt genes for insect revealed expression of GNA at levels up to 2% of total
control (Edmonds et al., 1996; Wu et al., 1997; Larry and protein in some of the transgenic plants. The GNA
Richard, 2002). On the basis of these results, optimiza- expressed transgenic plants showed decreased survival
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Table 3
Examples of plant derived genes insertion and expression in transgenic plants for insect resistance

Crop target Gene inserted Origin of transgene Useful trait Reference(s)

Oryza sativa L. Pin 2 Potato and pink stem Resistance to striped stem borer (Chilo Duan et al. (1996)
borer suppressalis Walker)
Corn cystatin Corn Sitophilus Resistance to brown plant hopper Nilaparvata Irie et al. (1996)
(CC) zeamais lugens (Stal)
GNA Snowdrop Resistance to brown plant hopper N. lugens (Stal) (Rao et al. (1998)
Zea mays L. avidin Chicken avidin Resistance to storage insect pest Kramer et al. (2000)
Pisum sativum L. a-AI Bean a-amylase I Resistance to Bruchid beetle Bruchus pisorum (L.) Shade et al. (1994)
Vigna angularis L. a-AI Bean a-amylase I Resistance to pea weevil B. pisorum (L.) Schroeder et al. (1995)
a-AI Bean a-amylase I Resistance to Bruchid beetle B. pisorum (L.) Ishimoto et al. (1996)
Populus OCI Rice cystein Resistance to Chrysomela tremulae F. Leple et al. (1995)
tomentosa C.K.
Schneid.
Solanum CpTi Cowpea Tolerance to tomato moth Lacanobia oleracea L. Gatehouse et al. (1997)
tuberosum L.
aai Phaseolus vulgaris Insecticidal activity on Tenebrio moliator L. Altabella and
Chrispeels (1990)
Kti3, C-II, PI-IV Soybean Resistance to Spodoptera littoralis Boisd. Marchetti et al. (2000)
cpTi Cowpea and pink Resistance to striped stem borer (Chilo Xu et al. (1996)
stem borer suppressalis Walker)
GNA Snowdrop Resistance to L. oleracea L. Bell et al. (2001)
— Bean chitinase Tolerance to aphid (Macrosiphum euphorbiae Gatehouse et al. (1996)
Thomas)
GNA Snowdrop lectin Tolerance to tomato moth L. oleracea L. Gatehouse et al. (1996)
— Wheat b-amylase Tolerance to potato aphid (M. euphorbiae Gatehouse et al. (1996)
Thomas)
Nicotiana CpTi Cowpea Resistance to tobacco budworm (H. virescens Hilder et al. (1987)
tobaccum L. Fabricius) and tobacco hornworm (Manduca
sexta L.)
Proteinase Potato Resistance to tobacco hornworm (M. sexta L.) Johnson et al. (1989)
inhibitor II
Chitinase Insect chitinase gene Resistance to tobacco budworm (H. virescens Ding et al. (1998)
Fabricius)

and fecundity of insects, retarded insect development levels in transgenic tobacco with the CaMV35S promo-
and had a deterrent effect on BPH feeding. The gna is ter. Larval biomass of H. virescens including leaf
the first transgene to exhibit insecticidal activity towards damage was reduced on transgenic plants (Boulter
sap-sucking insects in an important cereal crop plant. et al., 1990).
Bell et al. (2001) reported reduced damage on leaves
of transgenic potato carrying gna gene. Expression of 2.17. Other novel genes
GNA 1.0% total soluble protein by transgenic signifi-
cantly reduced the level of pest damage. Furthermore, Ding et al. (1998) introduced insect derived chitinase
the presence of the parasitoid (Eidopus pennicornis) genes into tobacco. The cDNA encoding a tobacco
significantly reduced the levels of damage incurred hornworm (Manduca sexta L) chitinase was transferred
either by the transgenic or control plants when compared through Agrobacterium mediated transformation. A
to those plants grown in the absence of the parasitoid. truncated but enzymatically active chitinase was present
For the GNA expressing plants the presence of the in plants expressing the gene. Segregating progeny of
parasitoid resulted in further reductions (ca 21%) in the high-expressing plants were compared for their ability to
level of damage caused by the insect pest Lacanobta support growth of tobacco budworm (Heliothis vires-
oleracea. cens) larvae and for feeding damage. Both parameters
The lectin (arcelin-l) found in beans has toxic effects were significantly reduced when budworms fed on
on an insect pest of bean, Zabrotes subfasciatus transgenic tobacco plants expressing high levels of the
(Christine et al., 1998). Introduction of lectin gene aai chitinase gene. In contrast, hornworm larvae showed no
into other plants could protect seed from the larvae of significant growth reduction when fed on the chitinase-
Coleoptera (Altabella and Chrispeels, 1990). Genes expressing transgenics. However, both budworm and
encoding the pea lectin (P-lec) have expressed at high hornworm larvae, when fed on chitinase-expressing
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1080 R. Mohan Babu et al. / Crop Protection 22 (2003) 1071–1086

transgenic plants coated with sublethal concentrations copies of the first transgenes (nptII and uidA genes). The
of a Bt toxin, were significantly stunted relative to transgene-stacked marker-free transgenic tobacco plants
larvae fed on toxin-treated non-transgenic controls. were generated from ca. 20% of excision-positive ipt-
Foliar damage was also reduced. Thus, the chitinase shooty explants within 5 months of Agrobacterium
expressing transgenic tobacco showed reduced infection. The presence of uidA, nptII, GFP genes and
damage when fed to budworm larvae. Kramer et al. the absence of the ipt gene were verified by PCR analyses.
(2000) produced transgenic maize containing avidin Furthermore, Southern blot analysis showed that no
gene. Avidin, is a glycoprotein found in chicken egg chromosomal rearrangements were introduced between
white, that sequesters the vitamin biotin. The avidin the first and second transformations. De Cosa et al.
X100 ppm is toxic to and prevents development of insects (2001) introduced several genes into the chloroplast
that damage grains during storage. Avidin expression in genome. Stable integration of the cry2Aa2 operon by
food or feed grain crops can be used as a biopesticide PCR and Southern blot analyses in T0 and Tl transgenic
against a spectrum of storage insect pests (Kramer et al., plants was confirmed. Thus pyramiding of single genes or
2000). multiple genes through new transformation procedures
could be important for long-term durable resistance to
2.18. Gene pyramiding (combination of multiple gene insects.
effect)
2.19. Commercialized transgenic crops
Many of the candidate genes, that have been used in
genetic transformation of crops, are either too specific or The available data strongly support that insertion of
are only mildly effective against the target insect pests. one or a few foreign genes does not adversely affect the
Some insect species are also insensitive to some of these agronomic characteristics of the host cultivars except for
genes. Therefore, to convert transgenics into an effective the expression of the introduced gene (Lee and Dean,
weapon in pest control, e.g. by delaying the evolution of 1996; Sanchis et al., 1997; Wirth et al., 1997; Roush,
insect populations resistant to the target genes, it is 1997; Ding et al., 1998; Li et al., 1999; Sharma et al.,
important to deploy genes with different modes of action 2000; Peloquin et al., 2000; Walker et al., 2000; Zhao
in the same plant. Pyramiding of different genes would et al., 2001a, b; Brotman et al., 2002; Puterka et al.,
reduce the likelihood of resistance development, since 2002; De Leo and Gallerani, 2002; Wimmer, 2003).
multiple mutations would be needed concurrently Thus, breeding of insect resistant varieties by insertion
in individual insects (Zhao et al., 1998; Cornu et al., of specific single or few genes would be a good
1996; Sharma et al., 2000). To increase the protective supplement to conventional crop improvement pro-
efficacy, spectrum of activity and durability of resis- grams. During 1986–1997, 25,000 transgenic field trials
tance, it is suggested to combine or pyramid different have been conducted on more than 60 crops, represent-
transgenes. With Bt alone, there are over 50 genes whose ing 10 engineered traits, in 45 countries (James, 1997).
insecticidal activities are known (Lee and Dean, 1996; Most of the trials were conducted on corn, tomato,
Wirth et al., 1997; Roush, 1997; Li et al., 1999; Sharma soybean, canola, potato and cotton and some of the
et al., 2000). Several of these could be deployed introduced traits were herbicide resistance, insect resis-
simultaneously to increase the level of the protection tance and virus resistance including product quality
from insect damage and possibly reduce the risk of (James, 2000). The People’s Republic of China was the
insect developing resistance. Each component of pyra- first country to commercialize transgenics in the early
mids should act on different targets within the insect, 1990s with the introduction of a virus resistant tobacco,
thus mimicking the multiple mechanisms of resistance, followed by virus resistant tomato (James, 2000). The
which occur in nature (Cornu et al., 1996; Sharma et al., United States Food and Drug Administration (USDA)
2000). approved the commercial sale of gene-spliced ‘Flavr Savr’
Integration of single transgenes to plant genomes has for the delayed ripening tomatoes produced by the
been valuable, however, multiple integration is essential multinational Calgene Inc. in May 1994. In 1997, the
to allow manipulation of complex metabolic pathways global area of transgenics increased 4.5 fold from 2.8
and to combine various agronomic characteristics in million hectares in 1996–12.8 million hectares with 7
the next generation of plant molecular breeding. Sugita crops grown in 6 countries. Herbicide tolerance ac-
et al. (2000) reported that MAT-vector system used counted for 63%, insect resistance 30% and virus
for generating marker free transgenic plants is also an resistance 7% (James, 2000).
efficient and reliable transformation system for the An initial assessment of the benefits from transgenic
repeated introduction of multiple transgenes independent crops has been reviewed by James (1997). Insect
of sexual crossing. The GST-MAT vector can be used resistant Bt cotton in the USA in 1996 resulted in
successfully to introduce a second transgene (GFP) into a insecticide savings, with 70% of Bt cotton planted in 1996
marker-free transgenic tobacco line containing single requiring no insecticides to control the targeted insect
 ARTICLE IN PRESS
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