´Production of Transgenic Plants using Viral Genesµ

1)c ©ntroduction:
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Transgenic plants are the unique plants which contain a foreign gene (gene of interest
or transgene) and are produced through the techniques of Biotechnology i.e.
Recombinant DNA Technology and not through Conventional Breeding methodologies
(NCALR©).

With the passage of time man has got unbelievable advancements in every field of life
and life style has become comfortable but still there are some problems that we are
facing such as the following

Œc The increase in the demand of food due to enormous increase in population as

most people of the world especially in Asia and in Africa are in such areas of the
world that are ´Food ©nsecureµ. By 2050 the Global food demand will be
doubled as population will increase up to 9.3 billion, 90% of which will reside in
Asia.

Œc The conversion of arable land into commercial places, recreation centers and
industrial zones.

Œc Resources like water, nutrients and fertilizers are becoming scarce

Œc The people of the developing countries are suffering from malnutrition (852
million)

Œc 55 % Women of the world in the developing countries are suffering from ©ron
deficiency.

Œc 140 million children are having Vitamin A deficiency which leads to Blindness.

Œc The loss of crop yield due to Biotic and Abiotic stresses

(A DECADE OF COMMERC©AL© ED TRANSGEN©C CROPS ²ANALYSES OF THE©R GLOBAL ADOPT©ON, SAFETY AND
BENEF©TS by. M. MANJUNATH)

Œc Our natural ecosystem is also polluted with different industrial wastes, which has
negative impacts on our arable land

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 Œc Not only the above problems but there is also need of effective vaccines,
proteins etc. for curing different human problems

Transgenic technology in crops is one of the most powerful methods and the only and
only solution to these problems is the use of Biotechnology for the production of
Transgenic Plants, which can control these problems, having the following
characteristics.

Œc ©mproved Quality and Yield

Œc Plants enhanced with nutrients and vitamins (Beta Sweet® carrot and
Golden Rice)

Œc Energy plants (Transgenic : duckweed, oil palm, sugarcane, cassava)

Œc Resistance to biotic stresses (©nsects, fungi etc.)

Œc Resistance to abiotic stresses (Drought, salinity etc.)

Œc Biopharmaceutical Farming

Œc ©ndustrial and Environmental applications

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Transgenic plants are produced by using different techniques of biotechnology but here
in this report main concern will be the use of Viral Genes and viruses in the production
of transgenic plants that how we can produce transgenic plants using viral gene for
different purposes such as virus resistant plants and plant edible vaccines. There is also
discussion about different techniques of plant transformation, benefits of transgenic
plants and risks concerned with transgenic plants.

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 2)c Main Body:
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2.1 Transgenic Plants:
Transgenic plants are the unique plants which contain a foreign gene (gene of interest
or transgene) and are produced through the techniques of Biotechnology i.e.
Recombinant DNA Technology and not through Conventional Breeding methodologies
(NCALR©).

The gene of interest may be from another plant or completely different species such as
Humans, animals and microbes etc. Transgenic Bt corn, for example, which produces its
own insecticide, contains a gene from a bacterium Bacillus Thuringiensis. So
Recombinant DNA Technology has made it possible to overcome the phylogenetic
barriers.

Usually a transgenic plant is derived from a single cell and all the cells of a transgenic
plant express the desired gene of interest.

2.2 Production of Transgenic Plants:

Following steps are used in the production of transgenic plants.

1)c ©solation of gene of interest:

A gene of desired characteristic can be isolated from the host organism by using Type ©©
Restriction endonuclease enzymes.

First the donor organism·s DNA is extracted and then its Restriction digestion is
performed using Type ©© Restriction enzymes.

After this, the DNA fragments are separated using Agarose Gel Electrophoresis, Gene of
interested can be identified with the help of markers.

2)c Amplification of Gene of ©nterest:

The desired gene of interest is amplified i.e. more copies are produced using PCR.

3)c Designing Genes for insertion into Host plants:

After the isolation and modification of the gene of interest, certain modification are
done for successful expression in the host plants such like as

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 a)c A promoter must be added to the gene of interest as it·s necessary for the
transcription of the gene. ©n Molecular Biology for plants the most commonly
used promoter is the 35S promoter of Cauliflower mosaic virus, which enhance
transcription to higher degrees
b)c A termination sequence which is required for the termination of transcription of
a gene
c)c Selectable marker gene, which is usually required for the identification of
transformed plant tissues or cells, it may be an antibiotic resistance gene.
But now a day ©PT genes are used along with a Recombinase enzyme gene as
selectable marker, which are then excised due to the expression of recombinase
enzyme and leads to the production of marker free transgenic plants.

MARKER GENE'PROMOTER'TRANSGENE'TERM©NAT©ON SEQUENCE

Simplified representation of a constructed transgene, containing necessary components for successful

integration and expression

4)c Plant Transformation:

Transformation refers to the uptake of Foreign DNA by host organism which is
integrated into its genome producing a stable heritable change.

For transforming plants mostly vectors are used they may be either plasmids or viruses
as they can be integrated into the genome.

Following methods are used for transforming plants:

a)c Agrobecterium mediated transformation in which the modified gene of interest

is inserted into the Ti Plasmid of the bacterium using the same Restriction
Enzymes and DNA ligases. The Agrobacterium having modified Ti plasmid is
used to infect plants especially dicots, as the Ti plasmids can integrate into the
host genome naturally.
b)c Plant Viruses, as most of the plant viruses are RNA Viruses so they can·t be
integrated into the host genome, only Cauliflower mosaic Viruses can be used as
they are somehow good vectors and can carry large pieces of foreign DNA if
helper virus strategy is used. The Gemini Viruses are also DNA viruses but the
undergo rearrangements so cant b used as vectors.
c)c Agroinfection and Gene Transfer, in this process a modified virus (having gene of
interest) infects the host plants as a part of the T DNA of the Ti Plasmid, after

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 infection viral DNA is released to become functional viruses and spreads
systematically, its mainly used for Cereals.
d)c Gene Gun, in this method tiny gold particles are coated with gene of interest
and is bombarded into the host genome using a gene gun, but here there is a
chance of gene silencing due to multiple insertions.
e)c Protoplast electroporation, in this method plant·s protoplast are transformed
using electric shocks
f)c Other methods include Liposomes mediated, Heat shock mediated, PEG
mediated transformation and Vacuum ©
filtratio
.

5)c Selectio
of Tra
sformed Tissues/cells:

©
order to obtai
successfully tra
sformed tissues, Selective MS medium co
tai
i
g
agar plates are used i
which we provide the a
tibiotic whose resista
t ge
s ar usd
as slctabl markrs.

Us tra
sformd tissus/clls as xpla
ts, so th tissus which will grow o
th a
tibiotic
co
tai
i
g MS mdium agar plats will b succssfully tra
sformd.

©f w hav usd ©T as slctabl markr th
thr is
o
d to provid a
tibiotics as
th callus ¶ll appar as xtrm shooty ph
otyp (©T shoots).

Rmov th tra
sformd tissus from th host pla
t a
d us as xpla
ts o
th plats,
th tissus or clls which grow will b succssfully tra
sformd.

6)c Co
formatio
of Tra
sformd Tissus:

Co
firm th g
 of i
trst by xtracti
g th DNA from th tra
sformd clls/tissus
a
d doi
g its CR or by prformi
g Southr
Blotti
g.

7)c Rg
ratio
of Tra
sg
ic la
ts:

Tra
sg
ic la
sts ca
b rg
ratd from th tra
sformd tissus by usi
g th
tch
iqus of la
t tissu Cultur i
which diffr
t hormo
s,
tri
ts, mdia tc. ar
sd to rg
rat pla
ts i
vitro.

(Dpartm
t of Soil a
d Crop Sci
cs at Colorado Stat U
ivrsity + G
 Clo
i
g a
d DNA
a
alysis)

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2.2 Use of Viral Genes and Viral expression systems in producing
transgenic plants:

2.2.1c Use of Viral Genes/ Viruses as Vectors:

a)c ©n Molecular biology the most widely used promoter, for production of
transgenic plants, is the 35S promoter as it is the most powerful bringing
transcription of a gene to higher levels. ©t·s also not influenced by the
environmental conditions and tissue type (which is to be transformed). ©t·s about
400 bp in length. (Gene cloning and DNA analysis)
b)c Using CP genes (cDNA in case of RNA viruses) of a virus can produce virus
resistant transgenic plants such as CP genes expressed in transgenic Tobacco
plants provided resistance against TMV.(Virus resistance mechanisms in transgenic
plants by ChristelleDutilleul, Eric Lainé)
c)c Using Replicase enzyme gene of the virus can produce virus resistant transgenic
plants such as TMV genes encoding replicase enzyme expreesed in tobacco
plants. (Virus resistance mechanisms in transgenic plants by ChristelleDutilleul, Eric
Lainé)
d)c Using cDNA (complementary DNA) of Satellite RNA (Sat RNA) of some viruses
can produce virus resistant transgenic plants such as sat-cDNA expression in
tobacco plants against CMV. (Virus resistance mechanisms in transgenic plants by
ChristelleDutilleul, Eric Lainé)
e)c Using viral genes in transgenic plants from viruses which damage insects such as
baculovirus and entomopoxvirus, can produce insect resistant plants. (Virus-
derived genes for insect-resistant transgenic plants, by Liu S, Li H, Sivakumar S,
Bonning BC. - Department of Entomology, ©owa State University, Ames, ©owa 50011,
USA)
f)c Cauliflower mosaic virus can be used as vector in molecular biology as its size the
same as that of the lambda phage and can carry large segments of foreign DNA
when non-essential parts are deleted from its genome using the helper virus
strategy.(Gene cloning and DNA analysis)

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2.2.2 Viral Expression Systems:
Transgenic plants are used for the production of immunogenic proteins and peptides
and it·s preferred because of the large scale production of edible vccines, no chance of
contamination of animal pathogens and reduced costs.

For this purpose there are two types of RNA based viral expression systems used which
are stated below

a)c Epitope presentation systems, in this system the viral vector is designed in such
a way that short antigenic peptides are fused to the capsid protein (CP), the
antigenic peptides are displayed on the surface of assembled virus particles and
don·t interfere with CP of the modified virus to assemble. This is system is mainly
used for the production of novel vaccines and purification is easy. The following
viruses are mainly used for this type of expression system.
i)c Cowpea mosaic virus
ii)c Tobacco mosaic virus
iii)c Tomato bushy stunt virus
iv)c Plum pox virus
v)c Potato virus X
vi)c Alfalfa mosaic virus

b)c Polypeptide expression systems, in this system a whole gene is introduced

into the viral genome so that unfused polypeptides are produced in the infected
cells and can be supplied to the animals by direct feeding or orally. Following
viruses are used in this expression system.
i)c Tobacco mosaic virus
ii)c Potato virus X
iii)c Plum pox virus
iv)c Tomato bushy stunt virus
v)c Cowpea mosaic virus

(Use of viral vectors for vaccine production in plants by M Carmen Cañizares, Liz Nicholson and George
P Lomonossoff)

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2.3 Benefits of Transgenic Plants:
Following are some of the benefits of transgenic plants

Œc ©ncrease in production of crop yield

Œc ©ncrease in the production of food protein, carbohydrate etc. content
Œc ©mproved shelf-life and organoleptic quality of foods
Œc Production of edible vaccines and drugs i.e. Plant Vaccines
Œc A source of ©ndustrial Raw material
Œc Resistant against abiotic stresses like drought, salinity etc.
Œc Resistant against biotic stresses like insects, fungi, viruses etc.
Œc Phytoremediation
Œc Environmental Protection

(Rakchanok KOTO Srinakarinvirot University)

2.4c Risks concerned with Transgenic Plants:

2.4.1c Health Hazards:

We all are eating transgenic crops to some extent and up to now no such
health effect has been reported but an experiment carried out in Scotland on
Mice which fed on transgenic potatoes containing a lectin gene from
snowdrops caused internal organs damage

2.4.2c Transfer of Antibiotic Resistant Genes:

There may be a chance that antibiotic resistant genes, used as selectable
markers, get transferred to pathogenic bacteria which will become resistant
to antibiotics but now a day marker free transgenic plants are produced by
using ©PT genes are selectable markers

2.4.3c 35s Promoter of CaMV:

There is a chance that 35s Promoter of CaMV may get incorporated into
humans· genome and may bring transcription of human genome to higher
levels and may cause cancer.

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2.4.4c Bt Toxin:
Bt Toxin which is produced in in transgenic crops by a bacterial gene is mostly
effective against Monarch butterfly larvae and also the pest population may
become resistant to Bt Toxin.

2.4.5c Reduced Biodiversity:

As now a day transgenic varieties are replacing traditional varieties,
especially in developing countries, causing loss of biological diversity.
But now a day genes are conserved in Gene Banks so it·s not a big problem.

2.4.6c Super weeds:

There is a risk that transgenic resistant crops may produce super weeds and
its creation has been demonstrated as wild mustards in Canola and jointed
goatgrass in wheat.

(Center for Life Sciences and Department of Soil and Crop Sciences at Colorado State University)

3c Conclusions:
Pakistan is an agricultural country and most of its population is dependent on
agriculture so transgenic plants can do allot in overcoming the needs of the people.

Transgenic plants production is the most powerful technique which can fulfill our needs
by providing us different resistant plants against biotic and abiotic stresses, delayed
ripening fruits, enriched vitamin and nutrients in plant etc.,

The most important output of transgenic plants is the edible vaccines using viral genes.

The production of transgenic plants using viral genes is a best way to help humans
either by producing virus resistant plants or edible plant vaccines.

As far as risks are concerned with Transgenic plants especially using viruses so up till
now there are no such reports about human damages due to use of transgenic plants
because these are fully tested before release and certain authorities like FDA approves
the transgenic plants.

So © think it·s a good way to produce transgenic plants because it can fulfill the needs of
a developing country like our Pakistan and there should be no worries about loss of
biodiversity because of establishment of Gene Banks.

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