Introduction

Cotton is the most important commercial crop of our country contributing upto 75% of total
raw material needs of textile industry and provides employment to about 60 million people.
India has the largest area under cotton cultivation with relatively low productivity primarily
due to the large area under rainfed cultivation with inadequate supply of inputs. Area wise,
India ranks first in world, whereas, it ranks second in production next to China. Only in
India, all the four spinnable fibre yielding species of Gossypium viz., Gossypium hirsutum,
G. barbadense, G. arboreum and G. herbaceum are cultivated commercially. Hybrid cotton
cultivation in about 45% of total cotton area contributing 55% of production is a significant
milestone achievement in Indian Cotton scenario.

Qualitative and quantitative transformation has taken place in cotton production in India,
since independence. The production increased from a meager 2.79 million bales of 170 kg
each in 1947-48 to a record of 24.0 million bales in 2005-06 .At the time of independence,
mostly short and medium staple cottons were produced. Today, India produces widest range
of cottons from 6 to 120s counts, from non-spinnable coarse to medium, long, extra long and
superfine cotton. The cotton productivity reached a plateau of around 300-330 kg lint per
hectare during the past one decade, whereas it has been improved to more than 460 kg lint per
hectare during the past two years.

Cotton is attacked by several insect pests reducing the crop yield to a greater extent. The
insect pests that attack cotton crop may be classified into sap sucking insects (Aphids, Jassids
and White fly) or chewing insects (Bollworms, leaf eating caterpillars etc.). Of the total
pesticides used in Indian Agriculture, about 45 per cent is sprayed on cotton crop alone. To
reduce pesticide usage in cotton, several strategies like use of Genetic Resistance to insect
pests, Integrated Pest Management (IPM), Insecticide Resistance Management (IRM) etc. are
advocated. In recent times, Bt cotton technology is found to be one of the best strategies to
manage bollworms, the most important pest of cotton.

1
The genetic resistance, one of the important pest management strategies, is available in cotton
gene pool against the sap sucking pests such as jassids, whitefly etc and using this several
resistant / tolerant varieties and hybrids have been developed and released in India. However,
such kind of known resistance is not available against the bollworms. Hence, an alternate
strategy is explored to circumvent this problem by cloning and transferring the genes
encoding the toxic crystal δ - endo toxin protein from the soil bacterium Bacillus
thuringiensis. The Bt transgenic cotton (Bollgard of Monsanto) has thus been developed
successfully in USA, which has the ability to control the bollworms at the early stages of crop
growth (upto 90 days) effectively.

The first commercial Bt cotton variety was released in USA by M/S. Monsanto (Bollgard),
which contains Cry 1Ac gene of Bacillus thuringiensis. Bt cotton is commercially grown in
several countries like China, Australia, Mexico, South Africa, Argentina, India, Indonesia
etc. Worldwide the area under Bt cotton keep increasing year by year. As per data from the
Cotton Corporation of India and Cotton Association of India, there has been a rise of 37% in
the area under cotton cultivation and 40% rise in the cotton harvests when data from 2005-06
is compared with that of the year 2015-16. Also, in 2005-06, of the total cotton harvest, the
proportion of Bt cotton was just 11.70% as compared to 92.17% in 2015-16. However,
despite this, in a decade the yield has risen just by 2.5%.

2
Review of literature

Genetically modified crops (GM crops) are plants used in agriculture, the DNA of which
has been modified using genetic engineering methods. Plant genomes can be engineered by
physical methods or by use of Agrobacterium for the delivery of sequences hosted in T-DNA
binary vectors. In most cases, the aim is to introduce a new trait to the plant which does not
occur naturally in the species. Examples in food crops include resistance to certain pests,
diseases, environmental conditions, reduction of spoilage, resistance to chemical treatments
(e.g. resistance to an herbicide), or improving the nutrient profile of the crop.

Farmers have widely adopted GM technology. Acreage increased from 1.7 million hectares in
1996 to 185.1 million hectares in 2016, some 12% of global cropland. As of 2016, major crop
(soybean, maize, canola and cotton) traits consist of herbicide tolerance (95.9 million
hectares) insect resistance (25.2 million hectares), or both (58.5 million hectares). A 2014
meta-analysis concluded that GM technology adoption had reduced chemical pesticide use by
37%, increased crop yields by 22%, and increased farmer profits by 68%. Currently available
food derived from GM crops poses no greater risk to human health than conventional
food but that each GM food needs to be tested on a case-by-case basis before introduction.
Nonetheless, members of the public are much less likely than scientists to perceive GM foods
as safe. The legal and regulatory status of GM foods varies by country, with some nations
banning or restricting them, and others permitting them with widely differing degrees of
regulation.Tobacco, corn, rice,cotton and some other crops have been engineered to express
genes encoding for insecticidal proteins from Bacillus thuringiensis . The introduction of Bt
crops during the period between 1996 and 2005 has been estimated to have reduced the total
volume of insecticide active ingredient use in the United States by over 100 thousand tons.
This represents a 19.4% reduction in insecticide use.

History of Bacillus thuringiensis

Bacillus thuringiensis was first discovered in 1901 by the Japanese biologist Shigetane
Ishiwatarias a cause of sotto disease that was killing silkworms and named it Bacillus sotto .
In 1911, Ernst Berliner isolated this bacterium from dead Mediterranean flour moth in
Thuringia, Germany, and named it Bt. In 1915, Berliner reported the existence of a parasporal
body, or crystalline inclusion (called crystal) close to the endospore within Bt spore , but the

3
activity of the crystal was not then discovered . In 1956, it was found that the main
insecticidal activity against lepidopteran insects was due to the parasporal crystal. Zakharyan
(1979) reported the presence of a plasmid in a strain of Bt and suggested that the plasmids
involved in formation of endospore and crystal.

Bacillus thuringiensis is a Gram-positive, soil-dwelling bacterium(fig-1), the most

commonly used biological pesticide worldwide. B. thuringiensis also occurs naturally in the
gut of caterpillars of various types of moths and butterflies, as well on leaf surfaces, aquatic
environments, animal feces, and insect-rich environments. During sporulation, many Bt
strains produce the proteinaceous inclusions crystal proteins, called delta endotoxins, that
have insecticidal action. This has led to their use as insecticides, and more recently
to genetically modified crops using Bt genes,

The crystal
During the sporulation phase Bt produce insecticidal proteins as parasporal crystalline
inclusions that have diverse sizes, shapes and crystal proteins. These crystals are
predominantly comprised of one or more proteins (Cry and Cyt toxins). The most well-
studied B. thuringiensis insecticidal proteins are called δ-endotoxins. These crystalline
proteins are mainly encoded by extra-chromosomal genes located on the plasmids. The
parasporal crystalline proteins produced during the stationary (senescence) phase of its
growth cycle account for 20% - 30% of the dry weight of the cells of this phase. Cry proteins
are parasporal inclusion (Crystal) proteins from Bacillus thuringiensis that are selectively
toxic against a wide variety of insects and pests. Similarly, Cyt proteins are parasporal
inclusion proteins from Bacillus thuringiensis that exhibits hemolytic (Cytolitic) activity.
Among these crystal toxin, Cry gene is one of the most widely used insecticidal genes
throughout the world (Chen et al., 2011). . Therefore, Bt is a viable alternative for the control
of insect pests in agriculture. Despite the limited use of Bt products as sprayable insecticides,
Cry toxins have been introduced into transgenic crops and recent strategy uses plants that
produce two or more toxins that kill the same pest, providing a more targeted and effective
way to control insect pests in agriculture.

The B. thuringiensis nomenclature system currently contains several hundred individual

sequences, divided between 74 classes of Crystal (Cry) toxin, 3 classes of Cytolitic (Cyt)
toxin, 4 classes of Vegetative insecticidal protein (Vip) toxin and one Secreted insecticidal

4
protein (SIP) toxin.Bt Cry and Cyt toxins belong to a class of bacterial toxins known as pore-
forming toxins (PFT) that are secreted as water-soluble proteins undergoing conformational
changes in insect. There are two main groups of PFT: 1) The α-helical toxins, in which α-
helix regions form the trans-membrane pore. They are colicins, exotoxin A, diphtheria toxin
and also the Cry three-domain toxins.2) The β-barrel toxins that insert into the membrane by
forming a β-barrel composed of β-sheet hairpins from each monomer. This class include
toxins such as aerolysin, α-hemolysin, anthrax protective antigen, cholesterol-dependent
toxins as the perfringolysin O and the Cyt toxins.

Cry proteins can be sub-divided into groups according to their homology and molecular
structure. Cry toxins are classified by their primary amino acid sequence and more than 500
different Cry gene sequences have been classified. To date, the Bt Toxin Nomenclature
Committee has classified 73 holotype sequences and 133 crystal proteins, into 73 groups
(Cry1 to Cry73). The Cry proteins comprise 75 primary subgroups of Cry genes and their
length vary from 369 (Cry34) to 1344 amino acids (Cry43). Based on experimentally derived
structures and molecular modelling, 89% of Cry toxins possess three-domain fold .

The mode of action of the three domain Cry toxin family involves sequential interaction of
these toxins with midgut proteins facilitating the formation of a pre-pore oligomer structure
and subsequent that leads to the killing of insect by osmotic shock. The insecticidal crystal
proteins (Cry proteins) are solubilized in the larval midguts and are acted upon by midgut
proteases and converted into activated Cry toxins. These toxins bind to specific epithelial
receptors in the midgut and cause alteration of ion channels and disruption of the membrane,
finally resulting in larval death. Several types of Cry proteins have been isolated that are toxic
to different orders of the insect families like lepidopteran, dipteran and coleopteran. These
crystal proteins, however, are found to be highly unstable in the environment and expression
of the highly AT-rich Cry genes is very poor in unrelated plant.

5
Fig.1 Bacillus thuringiensis under microscope Fig.2 Internal structure ofBacillus
thuringiensis

INSECT-RESISTANT BIOTECH CROPS

Currently commercialized
Insect-resistant transgenic crops were first commercialized in the mid-1990s with the
introduction of GM corn (maize), potato and cotton plants expressing genes encoding
the entomocidal δ-endotoxin from Bacillus thuringiensis . In 2010, 148 million ha of
biotech crops were grown in 29 countries, representing 10% of all 1.5 billion hectares
of cropland in the world. The global value of this seed alone was valued at US $11.2
billion in 2010, with commercial biotech maize, soybean grain and cotton valued at
approximately US $150 billion per year. The concept of using genes encoding Cry
proteins was not novel as Bt formulations have been used commercially for
approximately four decades to control insect pests, and in particular Lepidoptera.
Early commercial varieties of insect-resistant transgenic crops expressed single Cry
proteins with specific activity against lepidopteran pests, as illustrated by Bollgard
cotton expressing Cry1Ac developed by Monsanto and attribute maize expressing
Cry1Ab developed by Syngenta. Subsequently, other lepidopteran-active Bt toxins,
such as Cry1F and Cry2Ab2, were introduced and often presented as pyramided genes
in a single variety (Widestrike cotton expressing both Cry1F + Cry1Ac developed by
Dow Agrosciences and Bollgard II cotton expressing Cry1Ac + Cry2Ab2 developed

6
by Monsanto). Cry3 toxins with activity against coleopteran pests are also being used
in commercial transgenic crops, particularly maize, to protect against chrysomelid
rootworms (e.g. Monsanto's Yieldgard Rootworm maize expressing Cry3Bb1, Dow
Agrosciences' Herculex RW maize expressing Cry34Ab1 and Cry35Ab1, stacked with
a HT gene, and Syngenta's Agrisure RW maize expressing a modified version of
Cry3A). More recently released transgenic maize varieties express genes encoding
Cry proteins active against Lepidoptera and Coleoptera which, in some cases, are
stacked with HT genes. Furthermore, Syngenta has recently launched Agrisure
Viptera trait-stacked maize, the first commercially available variety to exploit a non-
Cry Bt protein (Vip3) for the provision of multiple pest resistance. In China, Bt cotton
cultivars expressing Cry1Ac together with a modified cowpea trypsin inhibitor (CpTI)
were commercially released in 2000 and in 2005 accounted for approximately 15 per
cent of the cotton crop.

Insect and pest resistance popular GMO crops

Several studies showed that Bt crops, which provide resistance to insect species like
Lepidopteran (Cry type I), Lepidoptera and Diptera (Cry type II), Coleopteran (Cry type III)
and Diptera (Cry type IV), help to reduce the use of chemical pesticide and increase total
yield.

Bt BRINJAL

The genetically modified brinjal is a suite of transgenic brinjals (also known as an eggplant
or aubergine) created by inserting a crystal protein gene (Cry1Ac) from the
soil bacterium Bacillus thuringiensis into the genome of various brinjal cultivars. The
insertion of the gene, along with other genetic elements such as promoters, terminators and
an antibiotic resistance marker gene into the brinjal plant is accomplished
using Agrobacterium-mediated genetic transformation. When fruit and shoot borer larvae
feed on Bt brinjal plants, they ingest the Bt protein Cry1Ac along with plant tissue. In the
insect gut, which is alkaline with a pH >9.5, the protein is solubilized and activated by gut
proteases. The Bt protein binds to specific receptor proteins present in the insect membrane,
resulting in pore formation in the membranes. This leads to disruption of digestive processes,

7
paralysis, and subsequent death of the fruit and shoot borer larvae.The Bt brinjal has been
developed to give resistance against lepidopteron insects, in particular the Brinjal Fruit and
Shoot Borer (Leucinodes orbonalis).

Mahyco, an Indian seed company has developed the Bt brinjal.Some of the cultivars of
brinjal include: Malpur local, Manjari gota, Kudachi local, Udupi local, 112 GO, and Pabkavi
local.It was approved for commercialization in India in 2009, but - after an apparent public
outcry and rounds of debates in which representatives from Mahyco, the scientific
community, and NGO's spoke on the topic - the then Indian Environment Minister, Jairam
Ramesh, facilitated a moratorium on its release until further, unspecified, tests were
conducted.

Bt CORN

Bt corn is a variant of maize that has been genetically altered to express one or
more proteins from the bacterium Bacillus thuringiensis including Delta endotoxins. The

8
protein is poisonous to certain insect pests. Approved Bt genes for corn are Cry1A.105
(MON89034), CryIAb (MON810), CryIF (1507), Cry2Ab
(MON89034), Cry3Bb1 (MON863 and MON88017), Cry34Ab1 (59122), Cry35Ab1
(59122), mCry3A (MIR604), and Vip3A (MIR162). The Bt protein is expressed throughout
the plant. When a vulnerable insect eats the Bt-containing plant, the protein is activated in
its gut, which is alkaline. In the alkaline environment, the protein partially unfolds and is cut
by other proteins, forming a toxin that paralyzes the insect's digestive system and forms holes
in the gut wall. The insect stops eating within a few hours and eventually starves.

Cultivation of Bt corn started in the USA, Canada, and Europe (Spain) in 1997, and by 2009,
it was commercially planted in 11 countries. It was then representing 85% of the total area of
corn in USA, 84% in Canada, 83% in Argentina, 57% in South Africa, 36% in Brazil, 20% in
Spain, and 19% in Philippines. In 2016, GM corn in the world (in 16 countries) reached 60.6
million ha, out of which 6 million (10%) were Bt corn, 7 million (11.7%) were herbicide-
tolerant corn, and 47.7 million (78.7%) were combined Bt and herbicide-tolerant corn. The
crop was produced to resist the infestation by the European corn borer, Ostrinia nubilalis, but
later in the 2000s, it has been produced against the corn earworm, H. zea, and the corn
rootworm, Diabrotica virgifera in addition to O. nubilalis (James 2016).

GM maize has also caused controversy with respect to possible health effects, impact on
other insects and impact on other plants via gene flow. One strain, called Starlink, was
approved only for animal feed in the US but was found in food, leading to a series of
controversy.

9
Bt Potato

In 1995, Bt-potato became commercialized in USA against Colorado potato beetles on

Cry3A protein. Insecticide use was reported to be reduced by up to 40% for Btplants in 1997.

Transgenic of Indian potato cultivar Kufri Badshah expressing synthetic, modified cry1Ab
gene were developed against potato tuber moth (Phthorimaea opercullela Z.) a destructive
pest. The cry1Ab gene was in spatial and temporal expression under the control of tuber-
specific GBSSi promoter. The trans-formation vector pBinCG1 was developed harbouring
transgene expression cassette comprising cry1Ab gene under the control of potato GBSSi
promoter, castor bean catalase intron (5UTR) and OCS termi-nation signals.

Bt soybean

Bt varieties expressing Cry1Ac+ Cry1Ab were approved for commercial use in Latin
America to control lepidopteran insects in soybean.Soybean crops, containing Bt toxins, were
planted in almost 100 million ha in USA.

Bt Tobacco

Among the insect species causing infestations and serious damages to stored commodities,
the cigarette beetle, Lasioderma serricorne (F.) and the tobacco moth, Ephestia
elutella (Hübner) are the major pests of both raw and manufactured tobacco. Post-harvest
tobacco control is achieved through sanitation, insect monitoring, and fumigation with
phosphine. However, insect resistance to phosphine and control failures have been reported,
and increasing regulatory pressure is being exerted on fumigants. Biological control agents
such as Bacillus thuringiensis appear to be environmentally sound and potentially viable
alternatives to chemical control. Bt is a bacterium that produces insecticidal crystal proteins
during the sporulation phase and has been, for more than 40 years, the microorganism of
choice for the biocontrol of phytophagous insect pests. It produces insecticidal crystal
proteins that display specific activity against certain orders of insects and become active upon
ingestion by the insect.

10
Bt Cotton
For cotton growers, there was a lot of pressure from pests before the introduction
of Bt cotton. Due to synthetic insecticide resistance, farmers were losing much of their cotton
because of H. virescens and pink bollworm, Pectinophora gossypiella. According to USDA,
94% of the cotton cultured in USA is genetically modified.

A study in University of California revealed that the average cost reduction in pesticides
applied in Bt cotton fields from 1996 to 1998 was between 25 and 65 dollars per acre; the
yield estimated, in the same period, was 5% more, on average, than the traditional cotton. In
addition, Bt cotton significantly decreased the number of foliar sprays, against other cotton
pests and consequently the cost of insecticides.

First generation Bt-cotton

In 1996, Bollgard cotton (a trademark of Monsanto Company) was the first Bt cotton to be
marketed in the USA.

The most prevalent Bt-gene on a global basis Cry I A(c) was incorporated into Coker
312 cotton designated MON 531 by Monsanto and later named Bollgard cotton (first
generation Bt-cotton). The high transformation efficiency was achieved in Coker 312
with Agrobacteium tumefaciens. The transformed Coker was then back crossed with lines
from Delta and Pine land and other companies that had necessary agronomic qualities for
commercial acceptance.

The advantages of the Cry IA (c) in bollgard over the Bt-cotton spray are as follows:

1. Active protein expressed in all plant parts.

2. Active protein expressed throughout the season, hence timing of insecticide
applications in relation to an infestation is not an issue.
3. Less farmer exposure to insecticide.
4. Labour saving technology due to elimination or reduction of insecticide sprays.
5. Contribution to and provides the foundation for an IPM strategy.

11
Second generation Bt-cotton (Bollgard II cotton)

The Insect Resistance Management (IRM) strategy for Bt-cotton that Monsanto in
conjunction with USDA developed second generation of improved Bt-cotton with two Bt-
genes, now designated Bollgard II. The new product bollgard II, Event 15985 was developed
using particle acceleration plant transformation procedures to add the Cry II A(b) gene to the
cotton line DP 50B that already had the cry 1 A(c) gene. Hence the bollgard II cotton
contains Cry 1 A(c) and Cry II A(b). The dual gene cultivars are expected to provide growers
with a broader control over a wide variety of insects.
In 2002, Dow Agrosciences announced the development of new Bt-cotton with traits that
confer broad spectrum resistance to lepidopteran pest of cotton. The new Bt cotton product
contains the dual genes Cry IA(c) and Cry IF, transformed with Agrobacterium
tumefaciens and incorporated through back crossing into several high quality commercial
varieties of cotton.

Third generation Bt-cotton

The most recent 3rd generation of Bt cotton contained three genes: Bollgard 3 (Cry1Ac +
Cry2Ab + Vip3A), Twin Link Plus (Cry1Ab + Cry2Ac + Vip3Aa19), and Wide Strike 3
(Cry1Ac + Cry1F + Vip3A).

12
 Crystal shape Protein size (KD) Insect actively
Gene
Cry I A(a), A(b), A(c), B, Bipyramidal 130 – 138 Lepidopteran larva
C, D, E, F, G
Cry II (Sub group) Cuboidal 69 - 71 Lepidoptera, diptera
A, B, C
Cry III (Sub group) Flat irregular 73 – 74 Coleoptara
A, B, C
Cry IV (Sub group) Biopyrimidal 73 – 134 Diptera
A, B, C, D
Cry V – IX Various 35 – 129 Various
The bacteria Bacillus thuringiensis produce two types of toxins namely 1. Cry (Crystal) toxin
encoded by different cry genes and 2. Kytolytic toxin. Over 80 genes have been noted to
encode for cry toxin and they are sequenced for various studies. The various genes and their
properties are tabulated here.

Chronology of Bt Cotton in India

March 10, 1995: Department of Biotechnology (DBT) of the Government of India permits
import of 100 gm of transgenic Cocker-312 variety of cottonseed cultivated in the United
States by Mahyco. This variety contained the Cry 1 Ac gene from the bacterium Bacillus
thuringiensis.

April 1998: Monsanto-Mahyco tie up. Monsanto given permission for small trials of Bt
cotton 100 g per trial by Department of Biotechnology (DBT).

January 8, 1999: RCGM expresses satisfaction over the trial results at 40 locations and on
April 12th directs MAHYCO to submit applications for trials at 10 locations before MEC.

2000-2002: ICAR trials were conducted at different AICCIP centres of Central and South
Zone locations.

February 20, 2002: The Indian Council of Agricultural Research (ICAR) submits a positive
report to the Ministry of Environment on the field trials of Bt cotton. It is now expected that
the Genetic Engineering and Approval Committee (GEAC) of the environment ministry will
approve commerical use of Bt cotton within a month.

March 25, 2002: Approval given for commercial cultivation to three Bt Cotton hybrids of
M/s. MAHYCO.

Bt cotton hybrid has increased from a mere 29,307 ha during 2002 to 12, 50833 ha in 2005.

13
 During 2006, the area under cultivation of Bt cotton has increased up to 3000,000 hectares.

In 2011, India grew as the largest producer of GM cotton crop at 10.6 million hectares.

By 201595% of cotton grown in India was GM

So far, 52 different Bt cotton hybrids were released for cultivation in India

Company’s Name Gene utilised

Mahyco cry1Ac
Monsanto cry1Ac+2Ab
Nath seeds cry1Ab+Ac fusion (China)
JK seeds cry1Ac modified (IIT Khargpur)
Syngenta vip3A +cry1 Ab
Dow Agri. Science cry1 Ac + cry 1F,
Metahelix cry1 C
ICAR cry1 Aa3
cry1F
cry1 Ia5
cry1 Ab

Genes utilized for the development of transgenic cotton hybrids in India

14
Methodology for developing of a transgenic crop

Five important steps for developing a transgenic crop are

(a) Identification of effective gene or genes

(b) Gene transfer technology

(c) Regeneration ability from protoplasts, callus or tissues

(d) Gene expression of the product at desired level

(e) Proper integration of genes so that are carried for generations by usual means of
reproduction.

Once identification of bollworm inhibiting genes has been achieved, molecular biologists
have step by step solved the problems to achieve perfect transgenics. In case of cotton,
Agrobacterium-mediated gene transfer technique has been essentially used. Although now for
direct gene transfer to protoplast, biolistic gene transfer techniques are available. The
regeneration of cotton plants from callus and somatic embryogenesis has so far been
restricted to few ‘Coker’ genotypes. All cotton genotypes are not amenable to regeneration
and that is one big hurdle in gene transfer. There are reports of induction of somatic
embryogenesis has also been reported from china and Australia but in India, attempts to
repeat it with Indian genotypes have been unsuccessful .To circumvent the problem of
genotype-limited regeneration of callus or leaf tissues, transformation and regeneration from
meristematic tissues was attempted which was found useful. Using Cry 1 Ab and Cry 1 Ac
genes, transgenic cottons with perfect integration, expression and reproduction was achieved
first in USA in 1987. Subsequently, there are reports from china and Australia.

There are four important methods of foreign gene (DNA) transfer in crop plants viz. plasmid
method, particle bombardment, direct DNA uptake and micro-injection. These methods are
also known as systems of DNA delivery for genetic transformation. The soil borne bacterium
Agrobacterium tumifaciens (termed as Nature’s Genetic Engineering) is used for
development of transgenic plants. This method has three main limitations viz. host
specificity, somaclonal variation and slow generation. There are two main advantages of
Agrobacterium mediated DNA transfer method. Firstly, this method has some control over
the copy number and site of integration of transgene which is not possible in particle

15
bombardment method. Secondly, this is a cheaper method of genetic transformation than
particle bombardment method. Perlak et.al. (1991) transferred successfully the Cry 1 Ac gene
to cotton via Agrobacterium with CaMV promoter and the Cry protein produces by
transgenic cotton was found highly toxic to bollworms. This method was later used
extensively by others. The particle bombardment method in which the foreign DNA is
delivered into plant cells through high velocity metal particles, has some advantages over the
Agrobacterium mediated method of DNA transfer, This method does not exhibit host
specificity. Hence, it can be effectively used for the development of transgenic plants in
various plant species. Moreover, this method is technically simple than Agrobacterium
mediated DNA transfer method. In this method, there is no need of isolating protoplast. The
other two method viz. direct DNA transfer and microinjection technique are rarely used for
developing transgenics in cotton. Currently, two DNA delivery system, viz.(1)
Agrobacterium mediated gene transfer, and (2) bombardment of cells with plasmid DNA
coated particles, are widely used for development of transgenic (genetically engineered)
plants in cotton. The first two workers used Agrobacterium method while the last workers
used biolistic method of gene transfer in cotton for developing transgenic plants. More than
37 transgenic plants have been developed in cotton so far by these two methods.

16
Creation of Bt cotton
Biotechnologists created Bt cotton by inserting selected exotic DNA, from a Bt bacterium,
into the cotton plant’s own DNA. DNA is the genetic material that controls expression of a
plant’s or an animal’s traits. Following the insertion of modified Bt DNA into the cotton
plant’s DNA, seed companies moved the Cry-protein trait into highperformance cotton
varieties by traditional plant breeding methods. Agronomic qualities for yield, harvest ability,
fiber quality, and other important characteristics were preserved at the same time the Cry-
protein gene was added to commercial varieties.

The three primary components of the genetic package inserted into cotton DNA include:

Protein gene

The Bt gene, modified for improved expression in cotton, enables the cotton plant to produce
Cry-protein. The first varieties of Bt cotton produced in the United States contained one Cry-
protein gene—Cry1Ac. Other varieties contain a “stacked” gene complex, for example— one
gene for insect control (Cry1Ac) and one gene to protect the cotton from application of the
herbicide glyphosate. Future cotton varieties may include these genes, other genes that allow
the plant to produce different Cry-proteins, or insecticidal proteins from sources other than
Bt. There are many possible combinations for crop improvement traits.

Promoter

A promoter is a DNA segment that controls the amount of Cry-protein produced and the plant
parts where it is produced. Some promoters limit protein production to specific parts of the
plant, such as leaves, green tissue, or pollen. Others, including those used in Bt cotton and
certain Bt corn varieties, cause the plant to produce Cry-protein throughout the plant.
Promoters can also be used to turn on and turn off protein production. Current varieties of Bt
cotton produce some Bt protein throughout the growing season.

Genetic marker

A genetic marker allows researchers to identify successful insertion of a gene into the plant’s
DNA. It also assists plant breeders in identifying and developing new cotton lines with the Bt
gene. A common marker is an herbicide tolerance gene linked to the Bt gene. Following a
transformation attempt to place the Bt and marker gene into the plant’s DNA, plants are
treated with herbicide. Plants that were successfully transformed have the Bt gene and the
herbicide resistance gene and will survive herbicide treatment; plants without the marker
gene, and hence without the linked Bt gene, will be killed by the herbicide. This genetic
package—a Bt gene plus a promoter and marker—can be inserted into cotton plant DNA
through a variety of plant transformation techniques. Transformed plants may be affected by

17
the genetic package, as well as the location of the new genes in the plant DNA. The insertion
site may affect Bt protein production and other plant functions as well. So biotechnology
companies carefully scrutinize each transformation to ensure adequate production of Bt
protein and to limit possible negative effects on agronomic traits. Following a successful
transformation, plants are entered into a traditional backcross breeding program with the
variety chosen to receive the foreign Bt gene package. The final product, a Bt cotton variety,
is developed after four or five backcross generations. Even though the new transgenic Bt
cotton variety may be named after the parent variety, agronomic qualities can be considerably
different.

Bioinsecticides like Bt that are sprayed on crops may perform as well as synthetic
insecticides in very limited situations, but the performance of Bt insecticides has been
inconsistent in many instances. The erratic performance in cotton is attributed to four reasons

1.The toxin is rapidly degraded by ultraviolet light, heat, high leaf pH, or desiccation.

2.Caterpillars must eat enough treated plant tissue to get a lethal dose of the toxin, since the
toxin has no contact effect.

3.The sites where tobacco budworms and bollworms feed are difficult to cover with the
foliar-applied sprays.

4.Bt Cry-proteins are less toxic to older larvae.

18
Mode of action of Bt

1. Ingestion

2. Solubilization & proteolytic activation

3. Binding to target site

4. Formation of toxic lesions

A cotton plant modified to produce Cry-protein within the plant tissues that caterpillars eat
overcomes most of the aforementioned limitations. The plant-produced Bt proteins are
protected from rapid environmental degradation since they are not directly exposed to the
environment. Incomplete coverage is not usually a problem because the plants produce the
proteins in all tissues where larvae feed, thus ensuring that the larvae will eat the Cry-protein.
The protein is always present whenever newly hatched larvae feed, eliminating the timing
problem associated with foliar application. The result is that Bt cotton has a built-in system
that efficiently and consistently delivers Cry-toxins to the target pests from the time a newly
hatched larva takes its first bite (fig. 1). Bt cotton offers a vastly improved method for
delivering Cry-insecticides to target insects, compared to traditional Bt sprays. Bt cotton may
also be considered a form of host plant resistance, in that the Cry-protein trait is carried in the
plant’s genes, as is traditional plant resistance to insects.

Benefits of Bt Cotton
The introduction of Bt cotton has provided growers with a new tool for managing bollworms
in cotton.Numerous benefits of this technology accrue to the grower, the global cotton
industry, and society on many levels-economic, environmental and social. These benefits
include direct benefits, such as reduced pesticide use, improved crop management

19
effectiveness, reduced production costs, improved crop management effectiveness, reduced
production costs, improved yield and profitability, reduction in farming risk and
improvement opportunity to grow cotton in areas of severe pest infestation. Indirect
significant benefits of the technology include improved populations of beneficial insects and
wildlife in cotton field, reduced pesticides runoff, air pollution and waste from the use
insecticides, improved farm worker and neighbor safety, reduction in labour costs and time,
reduction in fossil fuel use and improved soil quality.

Advantages of Bt cotton
1. The Bt cotton has inbuilt genetic resistance to bollworms and is very effective in
controlling the yield losses caused by bollworms to a considerable extent). The resistance is
governed by a single dominant gene.

2. Use of Bt cotton reuces use of pesticides resulting in reducing the cost of cultivation.

3. It results in improvement of yield levels and also improves margin of profit to the farmers.

4. It provides opportunities to grow cotton in areas of severe bollworm incidence.

5. It promotes ecofriendly cultivation of cotton and allows multiplication of beneficial

insects i.e. parasites and predators of bollworms.

6. It also reduces environmental pollution and risk of health hazards associated with use of
insecticides because in Bt cotton the insecticides are rarely used. An average reduction of 3.6
sprays per crop season has been reported in Bt varieties as compared to non-Bt.

The Risks and Potential Impacts of Bt cotton on Human Health

Effect of Bt cotton on the health of animals, poultry, human and environment are summarized below:

1. The feeding of Bt cotton seed to animal may have any adverse effect.

2. Seed of Bt cotton can have any adverse effect on digestion of animals. It may cause allergic or
toxic effect on animals.

3. The oil extracted from the seed of Bt cotton can have any adverse effect on human health.

4. It may have adverse effect of Bt cotton on non target beneficial insects \.

5. There is possibilities of cross pollination of Bt cotton to other species of Gossypium.

6. It may outcross with tetraploid wild species such as G.tomentosum which are found either
in cultivated areas or extremely isolated species gardens maintained at different research
institutes.

20
7. The upland cotton in which Bt gene has been inserted can have cross compatibility with
outer genera of the family of Malvaceae.

Growth of Bt cotton in India

In India, the basic research on Bt transgenic cotton is being carried out at the following research
institutes / centres:

1. National Botanical Research Institute (NBRI), Lucknow

2. National Research Centre on Plant Biotechnology (NRCPB), New Delhi

3. International Centre for Genetic Engineering & Biotechnology (ICGEB, New Delhi)

4. Central Institute for Cotton Research, Nagpur.

5. National Chemical Laboratory (NCL),Pune

6. Bhabha Atomic Research Centre (BARC), Mumbai

7. University of Agricultural Sciences, Dharwad

The work on Bt cotton in India was first initiated in 1994 at CICR, Nagpur with World Bank aided
Biotechnology Project. The Programme was undertaken with an objective to standardize the
regeneration protocol in Indian cultivars. Large number of cultivated varieties from different agro-
climatic zones were investigated for regeneration. Hypocotyl tissues from invitro germinated
seedlings of cvs. LRA 5166, LRK 516, Bikaneri Narma, CNH 36, PKV 081, MCU 10, MCU 5,
Suman, Khandwa 2, Khandwa 3, Coker 100 st, Coker UTT 68, Soneville 213 and some hybrids such
as H4, H6, PKV Hy2 and NHH 44 were utilized for callus induction and regeneration. Callus
induction was standardized in most of the cultivars and differentiation of roots from callus culture was
also obtained. However, development of explants was hampered by browning. Strategy to reduce
browning was developed by modifying carbohydrate source. Still complete regeneration of plantlets
was not possible. Two cultivars, viz., PKV 081 and Khandwa 2 are identified to be embryogenic in
nature but the frequency and recovery of plants from somatic embryo was very low. New approaches
of callus induction and differentiation from anther / pollen were also successfully examined. There
appeared certain factors for non-differentiation of callus into somatic embryos. Therefore,
simultaneous regeneration of shoot tip and cotyledonary node were attempted. Several studies were
carried out at CICR and a standard protocol best suited for Indian cultivars was developed for
micropropagation and transformation using Agrobacterium mediated and particle gun gene transfer.

0% Rise In Cotton Harvests, 37% Rise In Area Under Cultivation, 2.5% Rise In Yield In

21
Decade

Year Area (In million Production (In Yield (In kg per Proportion Of Bt

hectares) million bales) hectare) Cotton (In %)

2002- 7.67 13.6 302 0.38%

03

2003- 7.63 17.9 399 1.22%

04

2004- 8.79 24.3 470 5.67%

05

2005- 8.68 24.1 472 11.70%

06

2006- 9.14 28 521 41.56%

07

2007- 9.41 30.7 554 67.28%

08

2008- 9.41 29 524 80.80%

09

2009- 10.31 30.5 503 82.44%

10

22
2010- 11.14 33.9 517 90.83%

11

2011- 12.18 36.7 512 91.89%

12

2012- 11.98 37 525 93%

13

2013- 11.96 39.8 566 92.98%

14

2014- 12.85 38.6 511 93%

15

2015- 11.88 33.8 484 92.17%

16

2016- 10.5 35.1 540.8 83.33%

17

Source: Cotton Association of India Weekly Publication (December

2016), Cotton Corporation of India,

Future Thrusts
The genetic resistance is the cheapest and the most efficient method of protecting crop plants

from pests. The Bt transgenic cotton with inbuilt genetic resistance to bollworms will help in

23
protection of natural enemies of insect pests i.e. predators and parasites. It will also help in

reduction the cost of cultivation by reducing the use of pesticides. Moreover, it will reduce

environmental pollution and health hazards caused by pesticidal use. Transgenic cottons with

Bt endotoxin protein does reduce expenditure on insecticides and create eco-friendly

environment without reduction in yield. The future research work on Bt transgenic cottons

needs to be directed towards following thrust areas:

1. Through widespread cultivation of Bt transgenic cotton, the main risk is development of

insect resistance against Bt toxin. Hence, multiple sources of resistance should be identified

and used in developing bollworm and herbicide resistant Bt transgenic cottons to avoid the

risk of developing insect resistance and herbicide resistant weeds.

2. Recently, some transgenic Bt cotton hybrids have been released for commercial

cultivation in India. The seed of these transgenic hybrids is very costly. The price of seed is

Rs.1600/- for a packet of 450 g. which cannot be afforded by small and marginal farmers.

Hence, there is need to provide Bt transgenic seeds at cheaper rate, which can be afforded by

small and marginal farmers.

3. Cotton crop also suffers from abiotic stresses such as drought and salinity. There is need

to develop Bt transgenic cottons with resistance to drought and salinity conditions.

4. In case of hybrids, the farmer has to purchase fresh seed every year at a very high cost.

Hence, efforts should be made to develop Bt transgenic straight cotton varieties, the seed of

which can be used by the farmers for 3-4 years.

5. Cotton is a fibre, oil and protein yielding crop. There is need to improve the quality of

proteins and oil through genetic engineering besides fibre quality improvement.

6. Besides, Bt gene, several other genes can be used in future for developing resistant

genotypes of cotton to various insects. For example, cholesterol oxidaze gene from

Streptomycetes fungus can be used for developing boll weevil resistant genotypes.

24
7. The spider and scorpion venom genes can also be used for developing insect resistant

genotypes of cotton.

8. The Helicoverpa armigera stunt virus contains three genes which attack midgut of

Heliothis and ceases its feeding.

9. Protease inhibitor gene from cowpea, soybean and rhizomes of African Taro are being

used for development of transgenic cotton.

10. Diploid cottons cover about 25% of cotton area in India. Hence there is need to develop

transgenic Bt varieties and hybrids of diploid cotton.

Conclusion
Cotton fiber is a more challengeable fiber than synthetic fiber; it is inevitable that the per unit

yield of cotton will increase to fulfill the basic human need for clothing. Bt technology is one

of the best approaches in developed countries. The benefits from Bt cotton in India included

an increase in yield from 31 per cent to 67 per cent owing to effective control of bollworms,

25
decrease in chemical insecticide use from 25 per cent to 55 per cent, net profit to farmers

from ₹7,800 to ₹30,000/ha and, moreover, India turned from an importer to an exporter of

cotton. In spite of such extensive cultivation, Bt cotton has not caused any scientifically

validated ill-effects on humans, animals, other non-target organisms or the environment

anywhere in the world. Yet, the opponents continue to make the same old allegations that Bt

cotton is not safe, undaunted by its impeccable safety records. Their pre-decided agenda is to

oppose the technology.

26