Table of Contents

1 BT Crops ............................................................................................................................................... 2
1.1 Why Is It Used In Crops? .............................................................................................................. 2
1.2 BT Crops Available In The Market .............................................................................................. 2
1.3 How Does The Cry Protein Work? ............................................................................................... 2
2 RNA Interference (RNAi) ..................................................................................................................... 3
2.1 Discovery of RNAi ....................................................................................................................... 3
2.2 How RNAi Works? ....................................................................................................................... 4
2.3 Engineering Plant Metabolic Pathways through RNAi ................................................................ 5
3 Gene Gun .............................................................................................................................................. 5
3.1 Protocol For Gene Gun ................................................................................................................. 6
3.2 Applications of Gene Gun in Plants .............................................................................................. 6
4 Golden Rice .......................................................................................................................................... 7
4.1 Metabolic Engineering .................................................................................................................. 7
5 FLAVR SAVR Tomato ........................................................................................................................ 8
5.1 Development of the Modified Plant .............................................................................................. 8
6 Green Revolution .................................................................................................................................. 9
6.1 Factors Responsible For Green Revolution .................................................................................. 9
6.2 Impact Of Green Revolution ......................................................................................................... 9
7 Nobel Prize.......................................................................................................................................... 10
7.1 Nobel Prizes for Research in Plant Science ................................................................................ 10
8 References ........................................................................................................................................... 12

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1 BT Crops
BT stands for Bacillus thuringiensis. BT Crops are defined as crops which are genetically transformed to
prompt one or more proteins from the bacterium. In this process, BT genes are inserted which helps the
plant to produce proteins to fight against the insects or pest which results in destroying the yield. The
proteins produced by the plants are known as cry protein (Bates et al., 2005).

1.1 Why Is It Used In Crops?

Bacillus thuringiensis is a gram-positive, spore-forming bacterium which is mainly found in the soil and
hence it is also known as soil dwelling bacterium. This bacterium produces a protein which acts as a toxin
for those insects destroying the yield. This bacterium is mainly used in the sprays for commercial agriculture
and for organic farming. The use of this spray on crops are safe for the environment and causes no harm to
the consumers (Hammond et al., 2004)

The practice of using BT started in the year 1996 and began with using small quantities of genes from BT.
With the help of this genetic transformation, plants used to create the necessary proteins to protect the crop
from pests. All over the globe, in a land spanning 29 million acres, BT corn, BT potato, and BT cotton were
grown in the year 1999. Relying on this technology alone, approximately 92 million dollars was saved by
the United States. Here is the list of few pests which destroy the yield like:

1. European and southwestern corn borer

2. Tobacco and cotton budworm.Pink
3. bollworm and the Colorado potato beetle.

1.2 BT Crops Available In The Market

The first Bt crop deregulated in the U.S. were seven lines of Colorado Potato Beetle Resistant Bt Potato by
Monsanto. Since then, many more Bt crops have been deregulated, engineered to produce a variety of
different Bt proteins from various subspecies of Bt. These crops include corn, potato, cotton, cottonwood,
brinjal, rice, soy. These crops are called as genetically modified crops as they are transformed and modified
through genetic engineering (Hammond et al., 2004).

1.3 How Does The Cry Protein Work?

When an insect feeds on the plants, the cry protein present in the plants crystallizes the digestive system of
insects and it starves to death since the cry protein is toxic to organism’s digestive tract.Remember that it
affects insect’s digestive system and has no effect on human’s digestive system (Bates et al., 2005).

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Table 1.1: BT Cry proteins in GM crops authorized for cultivation in one or more countries.

Protein Insect type controlled Crop species approved

Cry1Ab Lepidoptera Maize

Cry1Ac Lepidoptera Cotton

Cry1A.105 + Cry2Ab2 Lepidoptera Maize

Cry1Ac + Cry2Ab2 Lepidoptera Cotton

Cry1Ac + Cry1F Lepidoptera Cotton

Soy

Cry1Fa2 Lepidoptera Maize

Cry1Ab + Cry2Ae Lepidoptera Cotton

mCry3A Coleoptera Maize

2 RNA Interference (RNAi)

RNA interference (RNAi) is a method of blocking gene function by inserting short sequences of ribonucleic
acid (RNA) that match part of the target gene’s sequence, thus no proteins are produced. Since Science
named it as “Breakthrough of the Year” and Fortune magazine hailed it as “Biotech’s Billion Dollar
Breakthrough” in 2003, RNAi has significantly gained prominence as the method of choice for researchers
sleuthing the structure and function of important genes (Tang G. et al., 2007).

RNAi has the potential to become a powerful therapeutic approach toward targeted and personalized
medicine. Even more exciting is the potential of RNAi in agriculture. RNAi has provided a way to control
pests and diseases, introduce novel plant traits and increase crop yield. Using RNAi, scientists have
developed novel crops such as nicotine-free tobacco, non-allergenic peanuts, decaffeinated coffee, and
nutrient fortified maize among many others (Saurabh S. et al., 2014)

2.1 Discovery of RNAi

Complete plant genome sequences has brought new dimensions in genetic modification to improve plant
characteristics. Scientists believed that they could produce any gene products by just introducing foreign
genes in plants, which was not always the case (Tang G. et al., 2007). Plant biologists found out that
introducing multiple copies of a gene that codes for purple petunia flowers led, not as expected to a deeper

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purple hue, but rather to plants with white or variegated flowers. Through an unknown mechanism, the
introduced transgenes were silenced as well as the plant’s ‘purple-flower’ gene (Novina and Sharp, 2004).
In another research, gene silencing was also observed when plants were infected with RNA viruses
engineered to contain fragments of the plant gene.

The mechanism causing these effects was not known until American scientists Andrew Fire and Craig
Mello discovered that injecting double stranded ribonucleic acids (dsRNA) into the worm Caenorhabditis
elegans triggered the silencing of genes with sequences identical to that of the dsRNA4. They called the
phenomenon RNA interference. Re-examining the co-suppression pathway in petunia and virus-induced
gene silencing revealed that all these processes led to the accumulation of dsRNAs, hence the RNAi
pathway. Fire and Mello were awarded the 2006 Nobel price for Physiology or Medicine for their discovery.

2.2 How RNAi Works?

1. The entry of long double stranded RNA, such as an introduced transgene, a rogue genetic element
or a viral intruder, triggers the RNAi pathway of cells. This results in the recruitment of the enzyme
Dicer.
2. Dicer cleaves the dsRNA into short, 20-25 base pairs long, fragments, called small interfering RNA
(siRNA).
3. An RNA-induced silencing complex (RISC) then distinguishes between the two siRNA strands as
either sense or antisense. The sense strands (with exactly the same sequence as the target gene) are
degraded.
4. The antisense strands on the other hand are incorporated to the RISC. These are used as guide to
target messenger RNAs (mRNA) in a sequence-specific manner.
5. Messenger RNAs (mRNA), which codes for amino acids, are cleaved by RISC. The activated RISC
can repeatedly participate in mRNA degradation, inhibiting protein synthesis.

Fig 1.1 : Mechanism of RNAi

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2.3 Engineering Plant Metabolic Pathways through RNAi
RNAi has been used to modify plant metabolic pathways to enhance nutrient content and reduced toxin
production .The technique takes advantage of the heritable

Table 1.2: Examples of novel plant traits engineered through RNAi.

Trait Target Gene Host Application

Enhanced Lyc Tomato Increased concentration of lycopene

nutrient content

DET1 Tomato Higher flavonoid and b-carotene

contents

FAD2 Canola, Peanut, Cotton Increased oleic acid content

SAD1 Cotton Increased stearic acid content

Reduced alkaloid CaMXMT1 Coffee Decaffeinated coffee

production
COR Opium poppy Production of non-narcotic alkaloid,
instead of morphine
Heavy metal ACR2 Arabidopsis Arsenic hyperaccumulation for
accumulation phytoremediation
Reduced Arah2 Peanut Allergen-free peanuts
allergenicity
Lolp1, Lolp2 Ryegrass Hypo-allergenic ryegrass

Reduced production of lachrymatory Onion "Tearless" onion

lachrymatory factor factor synthase
synthase gene

3 Gene Gun
Some cells, tissues and intracellular organelles are impermeable to foreign DNA, especially plant cells.
biolistics, including particle bombardment, is a commonly used method for genetic transformation of plants
and other organisms. To resolve this problem in gene transfer, the gene gun was made by Klein at Cornell
University in 1987 (Klein, 1987; Kikkert, 2005). On the gene gun technique, Klein and Sanford et al
published papers, obtained patents and formed a company called Biolistics.

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The gene gun is part of the gene transfer method called the biolistic (also known as biobalistic or particle
bombardment) method. In this method, DNA or RNA adhere to biological inert particles (such as gold or
tungsten). By this method, DNA-particle complex is put on the top location of target tissue in a vacuum
condition and accelerated by powerful shot to the tissue, then DNA will be effectively introduce into the
target cells (Klein, 1987).

Uncoated metal particles could also be shot through a solution containing DNA surrounding the cell thus
picking up the genetic material and proceeding into the living cells. The efficiency of the gene gun transfer
could be depended on the following factors: cell type, cell growth condition, culture medium, gene gun
ammunition type, gene gun settings and the experimental experiences, etc. Briefly for gene gun practice,
the target cells or tissues on the polycarbonate membranes could be positioned in a Biolistic PDS-1000/HE
Particle

3.1 Protocol For Gene Gun

The detail protocol for the gene gun transfection is described as follows:

(1) Prepare gold or tungsten particles: 60 mg gold or tungsten in 1 ml 70% ethanol, centrifuge at 10,000
rpm for 10 seconds and collect particles, and wash with H2O three times by centrifugation.
(2) Prepare DNA-coated particles: Mix 50 l (about 3 mg) metal, 2.5 l plasmid DNA (about 2.5 g),
CaCl2 50 l (2.5 M), spermidine 20 l (0.1M). Vortex and stand for 5 min. Centrifuge, remove
supernatant, and add 140 l 70% ethanol over the pelleted particles, and repeat the ethanol and
centrifugation three times, then add 50 l ethanol.
(3) Place a macrocarrier in the metal holder of gene gun and wash twice with ethanol.
(4) Vortex and spread 0.5 mg pellet slurry on the macrocarrier.
(5) Load the macrocarrier into the gene gun, and shoot it. Repeat the shoot until all the areas are shot.
(6) For transient expression, examine cells 48 hours after the shooting, by immunology or other
methods.
(7) For stable transfection, continue culture the transfected cells or tissues.

3.2 Applications of Gene Gun in Plants

Gene gun technology may be used to genetically modify plants for various reasons.

1. Many foods in the United States, including corn, soybeans, and canola, are genetically modified
using various techniques including gene gun technology. The incorporation of iron and vitamins
into rice, for example, may reduce the prevalence of malnutrition globally. Crops may also be
genetically modified to withstand harsher temperatures than non-modified foods. As a result, these
crops may survive in areas of the world where crops are difficult to grow.

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 2. Not all genetically modified plants are grown as food crops. Using gene gun technology, plants,
including trees, have been genetically modified to help reduce groundwater pollution. These plants
are designed to reduce the amount of heavy metal in contaminated soil, thereby reducing pollution.
3. In addition, crops such as rice, corn, soybeans, sweet potatoes, apples, tomatoes, cantaloupes, and
other fruits and vegetables, have been genetically modified using gene gun technology to improve
taste, color, size, and overall quality; reduce maturation time; increase nutritional value; increase
tolerance to extreme temperatures; and improve resistance to diseases, pests, and herbicides.

4 Golden Rice
Rice is the major staple food for hundreds of millions of people. It is generally consumed in its milled form
with outer layers (pericarp, tegmen and aleurone layers) removed. The main reason for milling is to remove
the oil-rich aleurone layer, which turns rancid upon storage, especially in tropical and subtropical areas. As
a result, the edible part of rice grains consists of the endosperm, filled with starch granules and protein
bodies, but it lacks several essential nutrients for the maintenance of health, such as carotenoids exhibiting
provitamin A-activity. Thus, reliance on rice as a primary food staple contributes to vitamin A deficiency,
a serious public health problem in at least 26 countries including highly populated areas of Asia, Africa and
Latin America (Sommer, 1988).

4.1 Metabolic Engineering

Golden Rice technology is based on the simple principle that rice plants possess the whole machinery to
synthesise β-carotene, and while this machinery is fully active in leaves, parts of it are turned off in the
grain. By adding only two genes, a plant phytoene synthase (psy) and a bacterial phytoene desaturase (crt
I), the pathway is turned back on and β-carotene consequently accumulates in the grain (Beyer, P. et al.,
2002)

Carotenoids and their derivatives include a vast number of molecules and accordingly a great number of
enzymes and cofactors. Only a small number of carotenoids namely those with at least one unsubstituted
β-ionone ring, such as β-carotene have provitamin A activity. Compounds derived from this important
pathway include plant hormones, like abscisic acid, the strigolactones and gibberellins. Tocopherols
(vitamin E), chlorophylls and quinones employ the pathway intermediate GGPP as a building block for
their synthesis (Beyer, P. et al., 2002).

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 Fig 1.2: Pathway overview.

5 FLAVR SAVR Tomato

The FLAVR SAVR tomato (Lycopersicon esculentum) was developed through a specific genetic
modification to exhibit decreased polygalacturonase (PG) activity. The novel variety was developed by
insertion of an additional copy of the PG encoding gene in the "antisense" orientation, resulting in reduced
translation of the endogenous PG messenger RNA (mRNA). The PG enzyme is the chief mechanism of
pectin degradation in tomato fruit leading to fruit softening. The transgenic variety ripens normally but
experiences less pectin breakdown and, therefore, has increased thickness and consistency that benefits all
stages of harvesting and processing (Bruening, and Lyons, 2000)

5.1 Development of the Modified Plant

The FLAVR SAVR tomato was created by Agrobacterium-mediated transformation in which the transfer-
DNA (T-DNA) contained a copy of the tomato PG encoding gene in the antisense orientation. In addition,
the T-DNA contained sequences encoding the enzyme neomycin phosphotransferase II (NPTII).

Fig 1.3: Flavr Savr tomato mechanism.

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The expression of NPTII activity was used as a selectable trait to screen transformed plants for the presence
of the antisense-PG gene. Transcription of the antisense-PG gene did not result in the expression of any
novel protein. There was no incorporation of translatable plasmid DNA sequences outside of the T-DNA
region (Bruening, and Lyons, 2000).

6 Green Revolution
The term ‘Green Revolution’ refers to the adoption in the mid 1960s of the new high yielding varieties
(HYV) of food grains. The Green Revolution technology enabled a three-fold increase in the output of food
grains between 1967 to 1992, thereby accelerating economic growth during the period (Shahid, 1999). The
most important feature of the new technology was that it required the timely application of a combination
of HYV seeds, chemical fertilizers and irrigation water3. This meant that rich farmers who had the financial
capability to ensure the right quantities of the input package and its timely application could achieve greater
cropping intensity and higher yields per acre compared to poorer farmers. In the context of Pakistan’s
agrarian structure, this was to have profound economic, social and ecological consequences.

6.1 Factors Responsible For Green Revolution

1. Miracle Seeds
The primary factor which brought agricultural revolution is the introduction of high yielding
variety, (HYV) seeds. The use of new variety of seeds has very much increased the agricultural
yields per hectare. For example, in Pakistan, the yield of wheat was 1189 Kg per hectare in 197172
and has increased to 2769 kg per hectare in 2006-07. Similarly, the yield of price which was 1554
kg per hectare in 1971-72 which has gone up to 2107 Kg per hectare in 2006-07
2. Agricultural Research
The agricultural research on higher yielding plant varieties, better methods of controlling insects
and deceases have resulted in higher production output.
3. Fertilizer
The increased use of chemical fertilizer is now playing a key role in raising the agricultural
production
4. Multiple Cropping
Due to new seeds maturing early, it has now become possible to get three or even four crops instead
of one or two from the same piece of land in a year.

6.2 Impact Of Green Revolution

The main effect of green revolution on the economy of a country are as under (Bruening, and Lyons, 2000).
1) Increase in production
2) Reducing regional imbalances
3) Unbalanced cropping pattern

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 4) Social imbalances
5) Increase in investment
6) Economic stability

7 Nobel Prize
A Nobel Prize is one of a set of prizes that are awarded each year to people who have done important work
in science, literature, or economics, or for world peace.

7.1 Nobel Prizes for Research in Plant Science

The Nobel Prizes awarded in two appropriate science categories (chemistry as well as physiology or
medicine) and the peace category since 1901 were studied to evaluate the plant science related research that
had received recognition.

Table 1.3: Nobel Prizes for Research on Plant Sciences.

Year Scientist Feild Research Recognition

1995 Richard Willstatter (Germany) Natural product chemistry For his research on plant Pigments,
especially Chlorophyll.
1930 Hans Fischer (Germany) Natural product chemistry For his research on into the constitution
of haemin & Chlorophyll.
1937 Paul Karrer (Swiztzerland) Natural product chemistry For his investigation on carotenoids,
Flavin & vitamin A B.
1938 Richard Kuhn (Austria) Natural product chemistry For his work on carotenoids & vitamin

1943 George de Hevesy (Swedon) Nuclear Chemisrty For his work on the use of isotopes as
tracers in the study of chemical
processs
1945 Arturi Virtanen (Finland) Agricultural Chemisrty For his research & invention in
agricultural & nutrition chemistry,
especially for his fodder preservation
method.
1947 Robert Robinson (UK) Natural product chemistry For his investigation on plant products
of biological importance, especially the
alkaloids.

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1961 Melvin Calvin (USA) Biochemisrty For his work on carbon dioxide
assimilation in plants.
1978 Peter Mitchell (UK) Biochemistry For his contribution to the
understanding of biological energy
transfer through the formulation of the
chemiosmotic theory.
1988 Johann Deisenhofer Biochemistry For the determination of three-
(Germany) dimensional structure of a
photosynthetic reaction Centre.

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8 References
Bates, S. L., Zhao, J. Z., Roush, R. T., Shelton A. M. (2005). Insect resistance management in GM crops:
past, present and future. Nat. Biotechnol. 23:57–62
Beyer, P, Al-Babili S., Ye X, Lucca P, Schaub P, Welsch R, Potrykus I (2002) Golden Rice: Introducing
the beta-carotene biosynthesis pathway into rice endosperm by genetic engineering to defeat
vitamin A deficiency. J. Nutrition 132(3):506-510.
Bruening, G. and Lyons, J. M. (2000). The case of the FLAVR SAVR tomato. California Agriculture
54(4):6-7.
Hammond B. G., Campbell K. W., Pilcher C. D., Degooyer T. A., Robinson A. E., McMillen B. L., et al.
(2004) Lower fumonisin mycotoxin levels in the grain of Bt corn grown in the United States in
2000–2002. J. Agric. Food Chem. 52: 1390–1397
Kikkert, J. R, Vidal, J. R, Reisch, B. I. (2005). Stable transformation of plant cells by particle
bombardment/biolistics. Methods Mol Biol. 286:61-78.
Klein, T. M., Wolf, E. D., Wu R, Sanford, J. C. (1987). Hugh-velocity microprojeectiles for delivering
nucleic acids into living cells. Nature (327):70-3.
Novina, C. D., and Sharp, P. A. (2004) The RNAi revolution, Nature 430: pp.161-164.
Saurabh, S., Vidyarthi, A. S., Prasad, D. (2014). "RNA interference: concept to reality in crop
improvement". Planta. 239 (3): 543–64.
Shahid, J. (1999). Pakistan: Fifty Years of Nationhood, Vanguard Books. Lahore. Page123.
Sommer, A. (1988). New imperatives for an old vitamin (A). J. Nutrition. 119: 96–100.
Tang, G., Galili, G., Zhuang, X., (2007). RNAi and microRNA: breakthrough technologies for the
improvement of plant nutritional value and metabolic engineering. Metabolomics 3(59): 357–369.
Internet sources
 www.naturalstandard.com
 http://www.isaaa.org/resources/publications/pocketk/34/

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