TITLE of ASSIGNMENT:

Genetically Modified Crops:

Bt Corn the Insect Resistance Crop

PROGRAM and GROUP:

AS2572C

CODE:
BMS531

Student’s Name: Nurulanis Farhah Binti Jamil Razif

ID: 2020996953

Phone Number: 011-12080762

Email Address: farhah466@gmail.com

INTRODUCTION

Corn, also known as maize with a scientific name Zea mays L. becomes one of the important
crops for food, animal feed and source of energy worldwide (Liu et al.,2018). It contains high
nutrition, especially carbohydrate, and there are many products produced by corn. For instance,
the famous cereals which are the CocoCrunch, popcorn, dresser, cooking oil and many more.
The production of corn becomes a high demand in the world, but the presence of insects causes
significant yield loss. The major lepidopteran pests on maize, such as Ostrinia furnacalis,
Helicoverpa armigera, and Mythimna separate, dipteran pest which is flies and mosquitoes and
coleopteran pest which is beetles cause the yield loss (Liu et al., 2016). To overcome this
problem, the scientist came with a solution by producing transgenic corn that can increase
resistance to insect and herbicide called Bt corn. Bt corn is a type of genetically modified
organism that termed as GMO. A GMO has resulted from a genetically engineered organism
when a gene from one organism is transferred to improve the desired characteristic in another
organism, and this method done in the laboratory (Abbas, 2018). In 1994, FlavrSavr was the
first tomato crop that was genetically modified to ripen without softening for the market. Then,
in 1995, Bt corn was developed. The examples of GMO maize are DK 404SR, StarLink corn,
MON 810 and many more (Priyadarshan et al.,2019). Commonly, Bt corn derived from
Bacillus thuringiensis, a gram-positive soil bacterium that is spore-forming that synthesize
parasporal crystalline inclusion contain Cry protein or Bt delta-endotoxin release when the
protein breakdown that can be used as a bioinsecticide (Palma et al., 2014). Different type of
Bt Cry genes will produce different crystalline protein or toxin and toxic to different insect.
When Bt genes successfully inserted into the DNA of a crop, the process is called
transformation. The result is called an event.

OBJECTIVES

1. To show how the process of Bt corn was made using recombinant DNA technology.
2. To determine the material and element that need to be used in the process.
3. To show the expected result from the process.
4. To determine the advantages of the Bt corn to human and environment.
5. To show the mode of action of the toxin that can protect the corn from an insect.

METHODOLOGY
 The first step that is crucial in this method is the selection of the gene of interest and
plasmid. The gene of interest is the Crystal (Cry) protein gene extracted from the DNA of
Bacillus thuringiensis in order to generate Bt corn (Yadava et al., 2017). Crystal proteins are
formed as parasporal crystalline inclusions during the stationary phase of growth (Palma et al.,
2014). This is extracted by using a restriction enzyme from the plasmid or chromosomal DNA
of the bacteria.

Figure 1: The spore of Bacillus thuringiensis Figure 2: The images of crystal of electron microscope
by Abbas, 2018, retrieved from under 100x magnificent by Lone et al., 2017, retrieved
https://doi.org/10.1186/s41938-018-0051-2 from https://doi.org/10.1002/mbo3.484

Vector is important in recombinant DNA technology as it is used as a vehicle to carry

foreign genetic material. A cloning vector with Ti plasmid is used, and it is a piece of DNA of
Agrobacterium tumefaciens bacteria. Ti plasmid contains T-DNA that will be incorporated into
the genome of the plant, and the gene interest will be cloned into the T-DNA. This vector is
environmentally friendly (Priyadarshan et al., 2019). Some of the scientists used expression
vector as the expression of a gene can synthesize large quantities of Cry protein that benefit in
insect insectile. It must be included with an additional component such as a promoter,
selectable marker, promoter, terminator and selective marker (Que et al., 2014). The promoter
used to control Cry protein and can see the level of protein produced. Promoter effect depends
on the selectable marker that was used. For example, the maize pZmUbi-1, P35S and CaMV
35S are the promoters that widely used in monocotyledonous plants (Liu et al.,2016).

Figure 3: Diagram of Ti plasmid used in Bt corn by Gordon & Christie, 2014,

retrieved from https://doi.org/10.1128/microbiolspec.PLAS-0010-2013
 Vector and Bt gene that had obtained will be cut using the same restriction enzyme that
produces sticky ends such as EcoR1 and BamH1 so that the gene can hybridize to the vector
by complementary base pairing. BamH1 search for sequence GGATCC in double-stranded
DNA, and it will digest the phosphodiester backbone between the pair of G nucleotide on each
strand. It will leave 5'end overhang becomes G GATCC (Priyadarshan et al.,2019). After that,
the plasmid and gene of interest, which is Cry protein gene will be ligated together using DNA
ligase. This process will form a recombinant plasmid. Then, the recombinant plasmid will be
inserted into a host which is the corn cell and this process called transformation process.

There are many transformation techniques that can be used to insert the new gene into
its genome that be done by the vector. For instance, DNA transfer to protoplast by
electroporation, particle bombardment, whisker-mediated plant transformation, and
Agrobacterium tumefaciens-mediated method. Agrobacterium tumefaciens-mediated
transformation is the most common technique that been used as it had successfully produced
many commercial events of Bt corn from the first of Bt corn produced until now. This
transformation method used Agrobacterium tumefaciens which is a soil pathogen to transform
plant. It is an indirect method where the bacterium has the ability to infect plant cell with a
piece of its DNA. It will take over the plant's cellular machinery and use it for the proliferation
of the bacteria when bacterial DNA integrates into plant chromosome (Abbas, 2018). The
scientist transforms A. tumefaciens with the plasmid of interest and lets the bacterium do what
it does naturally to get the Ti plasmid-Bt gene into the corn DNA. The particle bombardment
is the other method which it is a direct method using gene gun which is a device where the
gene of interest transfer through cell wall penetration using tungsten that called
microprojectiles coated with plasmid DNA so it can integrate into genomes. MON 810 corn
that is really famous corn which is one of the product by Monsanto form using this method
(Mookkan, 2018).
 Figure 4: Steps on the production of Bt corn using the Agrobacterium tumefaciens-mediated
gene transfer system by Cusson, 2008, retrieved from https://doi.org/10.1641/B580806

After the cell multiple with the interested gene, selection of the cell will be made to
identify which cell has taken up the transgenic gene and ensure successful transgenic event. It
will only allow transform cell to proliferate while the non-transformant growth will be
suppressed or killed. There is only 3 type of selection system which are antibiotic resistance,
herbicide resistance and sugar metabolism. The inclusion of a selectable marker gene can let
us know whether the Bt gene incorporated into corn DNA. In antibiotic resistance, neomycin
phosphotransferase (npt II) derived from Escherichia coli were used with kanamycin as
selection agent was used in the from the early of corn transformation. In contrast, herbicide
resistance used a selectable marker, and this technique is common and widely used in maize
transformation system as it successfully commercialized 83 events. Bar and Pat selectable
marker are one of the first herbicide selectable marker genes where Bar gene derived from
Streptomyces hygroscopicus while Pat gene from Streptomyces viridochromogenes. Both
genes encode phosphinothricin acetyltransferase (PAT) that eliminate herbicidal activity by
acetylation. This selectable marker was found more efficient than kanamycin (Yadava et al.,
2017). Selectable marker gene expression can be improved by optimizing their protein-coding
sequence to enhance transformation efficiency in plants (Que et al.,2014). Lastly, the sugar
metabolism selection is where the corn transformant selected on media that contain mannose
(Yadava et al., 2017). It is called Phosphomannose isomerase (PMI) system where it is based
on sugar or amino acid metabolism. PMI selectable marker is where expression E.coli manA
gene encoding mannose-6-phosphate isomerase. It enables efficient transgenic corn event on
media to contain mannose. The advantage of using this marker is high transformation
frequency, less time to produce callus and can site-directed integration (Que et al.,2014).
Figure 5 : The list of selection system for generating transgenic event by category
by Que et el., 2014, retrieved from https://doi.org/10.3389/fpls.2014.00379
Flow chart

Figure 6 : The summary of the

whole step of Bt corn by
Cabrera, 2019, retrieved from
https://doi.org/10.1016/B978-
0-12-811971-6.00003-6

Selection gene of interest which is Cry The gene of interest and the plasmid
protein gene and plasmid which is Ti will be cut using the same restriction
plasmid. enzyme.

The recombinant plasmid will be

introduced in the corn cell using
The gene of interest and the plasmid
transformation technique which is
ligated together by DNA ligase to
Agrobacterium tumefaciens-mediated
form recombinant plasmid.
method.

The selection method which is Cells that carry Bt gene and

antibiotic resistance method will be antibiotic resistance will survive.
done to ensure the take up of
transgenic gene.

The cell will grow as Bt corn crops.

RESULTS AND DISCUSSION

A) B)

Figure 7: This is the result of recombinant DNA technology to produce Bt corn. A) Image of Bt
corn with inserted of Cry genes. B) Image of non Bt corn without the inserted of Cry genes by
Google, on 25/5/2020, retrieved from https://images.app.goo.gl/d2Jb685ZYFThzw3Z8

When the gene inserted in the host plant, it will grow with the gene. To cause mortality towards
the pest, Bt spore needs to be ingested first by the pest. And now it is ready to attack the insect
that eats the plant. In alkaline gut juice which at pH 8–10, the Cry toxin becomes active by
proteolytic enzymes. The protein breaks down to release a toxin, known as delta-endotoxin.
Most cry toxins are pro-toxins of about 130 to 140 kDa, and after activation, they become 60–
70 kDa which is low but enough to cause the insect to die. The activated toxin will pass through
the peritrophic membrane of the pest and binds to specific receptors on the intestinal lining
membrane of epithelial cells of the midgut. Then it will make pores where the toxin can
penetrate the cells and become swollen. The swelling continues until the cells lyse and separate
from the membrane of the midgut epithelium. The alkaline gut juices will leak into the
hemocoel of the insect body cavity, causing the hemolymph pH rises that leads to paralysis and
death of the insect. Some scientist said that the cause of the death is because the naturally
occurring bacteria in the gut which are E.coli and Enterobacter enter the disrupted epithelium
caused by Bt toxins and penetrate to the hemocoel. Then, it will multiply, causing sepsis of the
hemolymph and death of the insect. When the insect eats the part of the plant that contain Bt
protein, it attaches to wall gut of the insect. Bt will not kill the insect or any living organism
wherein their gut epithelial cells does not have a receptor (Abbas, 2018). The highest Bt protein
level in maize normally was detected in leaves followed by stems and roots, and the least in
seeds and the high content of Cry protein detected in husks, kernels, tassels, and silks of Bt
corn (Liu et al., 2016).
Figure 8: Mechanism of mode of action of insect when ingest Bt toxin by Sheikh et al., 2017, retrieved from
https://www.researchgate.net/publication/314151170_An_overview_on_resistance_of_insect_pests_against_Bt_Crops

An inbred line is the ideal maize transformation line for trait research with excellent
agronomic characteristics (Que et al., 2014). This can ensure the successful event can occur
and minimize the failure per cent. Although it harmful to the insect but it not affected human
and animal as it is environmentally friendly. Based on laboratory research, Cry toxins are
supposed to be active against selected insect only. The pollen has high doses of Cry toxins.
However, it is not toxic to lady beetles, green lacewings, or honeybees. Within a few days, Cry
proteins will deposit into the soil and degraded, but there will be no effect on soil bacteria,
actinomyces, fungi, protozoa, algae, nematodes, or earthworm. Thus, the remains of pollen of
Bt corn had an effect on the non-target plants in the fields of Bt crops. The insecticidal active
toxin presents in Bt maize have been proved that there were no toxicity or allergic response
towards a human. There was research about this when twenty-eight people showed allergic
reactions related to eating corn products that may have contained the StarLink toxin. StarLink
is one of the products of Bt corn produced by Aventis Company in the USA. However, the US
Centers for Disease Control studied the blood of these people and concluded that there was no
evidence that the allergic reaction was related to the StarLink toxin (Abbas,2018). Other than
that, the use of conventional insecticides will be decreased as the farmer do not have to waste
their time and money on pesticides (Liu et al.,2018).
CONCLUSION
In conclusion, the DNA of the corn can be genetically modified to produce Bt corn that
can kill pest by using recombinant DNA technology. The result of the process which produces
Bt corn is better than ordinary corn as it can resistance to an insect. The production of Bt corn
can make the farmer less work, but high production can be obtained compared to the
conventional method. It also will not put a risk on our environment and human health as it is
environmental-friendly. For the sake of future, the technique needs to be further improved as
it has remained the same for the last 20 years and nanotechnology can be applied in this method
The method will become simpler, cheaper, robust, high productivity, and can improve the
quality of the product.

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