Transformation of Arabidopsis plants through Agrobacterium mediated transformation and selective screening of putative transgenic plants through PCR analysis

This lab report focuses on the successful transformation of Arabidopsis plants though the growth of seedlings. It involves screening of the putative Arabidopsis gene where Transformation has taken place due to Agrobacterium mediated transformation. The aim is to identify transformed Arabidopsis plants through Agrobacterium mediated transformation and subsequent screening of such putative transgenic plants through molecular methods.

Running head: GENE CLONING EXPRESSION AND ANALYSIS
Title: Transformation of Arabidopsis plants through Agrobacterium mediated
transformation and selective screening of putative transgenic plants through PCR analysis
Student Name:
Student Number:

1GENE CLONING EXPRESSION AND ANALYSIS
Abstract
Over the years experiments based on plant transformations have been in demand due to the
burden on the food industry. Transformation of desirable genes into a host plant has proved
effective in production for genetically modified crops. Moreover these transfusion
experiments have also helped in the constitution of genetic makeup in the upcoming
generations. The following lab report would focus on the successful transformation of
Arabidopsis plants though the growth of seedlings. This lab report further focuses on the
screening of the putative Arabidopsis gene where Transformation has taken place due to
Agrobacterium mediated transformation. Thus the successful transfer of foreign genes
through plant transformation can prove to be a fruitful method of dealing with the increasing
crop demand and the use of biofuels in the upcoming years.

2GENE CLONING EXPRESSION AND ANALYSIS
1.
2. Aim:
The main aim of the given lab report is to
identify transformed Arabidopsis plants
through Agrobacterium mediated
transformation. It also involves subsequent
screening of such putative transgenic
plants through molecular methods. In the
experiment, seeds represent the T1
generation which have been obtained from
the Arabidopsis plants and will undergo
the process of Agrobacterium mediated
transformation. This will be followed by
extraction of genomic DNA for molecular
analysis from the plantlets which will use
the polymerase chain reaction or PCR for
screening of putative transgenic plants.
3. Introduction:
2.1. General Background:
Plant transformation is defined as a
scientific method of inserting foreign DNA
of superior quality into the genome of the
species of interest .Such plants termed as
transgenic are important for both
agriculture as well as research. Tradition
techniques of plant breeding along with e
consideration of the intragenic
crosses ,chemicals s well as physical
mutagenesis along with different breeding
methods have been non-specific have been
the reason behind transgenic have been
chosen over traditional methods.
Moreover, random mutation of large
segments along with excess time
consumption of backcrosses for the
segregation of the undesired and
unrequired changes involved in the
offspring (Ran, Liang & Gao, 2017 ).
Transgenic technology is also involved in
improving the man characteristics of crops
which are economically important. Traits
involve diseases resistance, abiotic stress
tolerance as well as nutrient imbalance.
Arabidopsis is considered as an ideal
model for transformation procedures.
Development of highly efficient as well as
simple protocols without the
implementation of plant regeneration

3GENE CLONING EXPRESSION AND ANALYSIS
procedures are the reasons behind
Arabidopsis being the primary model for
conducting transformation experiments
(Rivero et al.2014). Zimenowoicz (2014)
states that agrobacterium is the best choice
for controlling such experiments as they
are the most commonly used vectors for
the creation of transgenic plants.
Moreover, this technique helps in the
enhancement of crop productivity,
resistance towards pests especially through
agrobacterium.
2.2. Similar research done in the field:
Similar kind of agrobacterium mediated
transformation has been done in Sorghum
plants where the application of an
improvised protocol has led to large scale
sorghum transformation experiments.
Through the implementation of modified
experiments, the frequency of
transformation have increased to almost
33% .Through the varieties of TX430, 848
independent calluses have been formed.
Quantitative PCR analysis has been done
for molecular analysis followed by
statistical quantitative studies using chi
square tests. Change in medium has
significantly improved transformation
frequencies. Strain and size of DNA have
also been important factors for this
experiment (Wu et.al 2014).
Agrobacterium mediated transformation is
basically a type of transient transformation
and often fluorescent fusion proteins are
used in this method for the transient
transformation for following the in vivo
behavior of the included proteins. Similar
studies of such agrobacterium mediated
transformation has been done in onion
epidermis which have ultimately helped in
determination of the protein subcellular
localization of large scales (Xu et
al.,2014).
2.3. Gap in literature
Transformation has been done before
using various plat species. However,
extraction of genomic DNA from various
plantlets has not been done on wide basis.
Gene analysis like that of TAIR has been
done for the transformation studies

4GENE CLONING EXPRESSION AND ANALYSIS
involved in transformation. Moreover,
molecular techniques used for extraction
of new transformed saplings of
Arabidopsis have not been done. Further
extensive research has to be done in the
creation of transformed plants using
specially designed primers for
identification of the transgenic lines for
transformation using agrobacterium.
2.4. Limitations
According to Mangano et al. (2014),
various efforts conducted at increasing the
frequency of transient transformation
success of Arabidopsis plants have found
that difficulties have been faced regarding
the plant immunogenic responses triggered
by the perception of Agrobacterium.
Moreover, limitations to Agrobacterium
mediated transformation includes the
heterologous system which has been used
to express genes. It might not reflect the
native activity or subcellular distribution
which is seen in the corresponding
proteins. Some of the problems associated
with plant transformation studies include
the ever-increasing expenses in carrying
out such experiments for a bulk amount of
plant material. Moreover, large scale
transformation experiments as well as
screening programs are affected and this
simultaneously affects their ability to
produce useful trans-formants Moreover
the various problems and strategies vary
with the kind of thinking in the
contemporary world along with the
progress in scientific understanding along
with technical development in the various
reliable systems for conducting
experiments on genetic transformations.
2.5. Future implications
Plant transformation holds a
significant role in the future. Increased
investments in plant science has enhanced
the foundational discoveries of various
crop genes which is often determined with
increased use of synthetic biology. It can
be applied in the three important field in
the future. Traditional methods include
application of plant transformation of

5GENE CLONING EXPRESSION AND ANALYSIS
Arabidopsis in the food sector where the
ever-increasing demands of the crops are
met. Genetically modified crops are a
solution to this problem leading to a
number of effective application in the field
of biotechnology. Production of biofuel
would be another factor facilitated by
processes like plant transformation. Plant
tissue and synthetic plant formation along
with the application of transgenic plants
have a pivotal role to play in the field of
therapeutics as well as pharmaceuticals
which use biological molecules like
peptides, antibodies and vaccines (Plant-
ditech.com, 2019). According to Long,
Colon and Zhu (2015), crop yield will
significantly increase with the rise in
photosynthetic crop through genetically
modified crops as photosynthesis and its
rate and a strong influence on the total
yield of plants. Phytohormone engineering
is another domain which can be facilitated
through the incorporation of genes in
transformation of plants. The
amalgamation of phyto-hormones and the
respective roles played by them in plat
development, plant growth as well as
abiotic stress response helps in the
enhancement of the quality and quantity of
food crops (Wani et al.,2016).
2.6. Hypothesis of the
statement
The hypothesis of the given article is the
transformation of Arabidopsis plants
through Agrobacterium mediated
transformation. Moreover the hypothesis
involves screening of transgenic plants
through molecular processes like PCR.
Furthermore, the genetic analysis of the
transgene is also considered though
various software tools and the sequence
similarity of the template gene of the host
and the foreign gene against which the
given primers have been used is compared
investigating the constitutional makeup of
thee transgene.

6GENE CLONING EXPRESSION AND ANALYSIS
3. Materials and methods:
The materials required for
conducing the experiment include the use
of variety of containers like petri plates of
either 10 cm or 15 cm of diameter,
Magenta boxes as well as culture tubes
depending on the purpose for conducting
the experiment. 1x MS medium has been
used for the growth of the Arabidopsis
saplings. For the following experiment the
growth of Arabidopsis under sterile
conditions is required for the experimental
settings involving selection of transformed
as well as drug resistant plates,
examination of the type of shoot as well as
root phenotypes which is followed by
identification of the various homozygous
lethal mutants.
The whole practical have been
divided into various parts. The first
practical involves the growth of
Arabidopsis among sterile conditions
which have been grown on solid media.
The culture medium used here is
Murashige and Skoog medium with
addition of 1g sucrose alongside 0.05 g of
2-(N-Morpholino) ethane sulfonic
acid( MES) to beakers containing 80 ml of
water. The solution is diluted by adding
distilled water to make it 100 ml. The
following steps include adjustment of pH
to 5.7 inclusive of 1M KOH. After
autoclaving, the proper cooling of the
medium is required to 5 degree Celsius.
The media is poured into labelled petri
plates and they are cooled for allowing
them to solidify at room temperature.
The second practical involves the
process of sterilization of the seeds. For
helping the seeds to plant, one seed has to
be adhered to the tip and the seeds are then
released at the desired location. The water
accumulated at the top should be allowed
to dry .The whole set up should be sealed
with Micropore paper for prevention of
desiccation and the plates should be kept at
4 degree Celsius for 1 day for
stratification. Line germinate synchronizes
at 4 degree Celsius . The plates should be
transferred to proper growth environments.

7GENE CLONING EXPRESSION AND ANALYSIS
Suitable growth conditions are inclusive of
illumination at continuous light at 120-150
mol/m2 sec and a temperature of
approximately 22-23 Degree Celsius.
The third practical involves the
process of DNA extraction. The methods
have been done from Arabidopsis
seedlings using ISOLATE II Plant DNA
Kit. The aliquot of DNA buffer have been
first warmed to 65 degree Celsius after
heating the water bath to the required
temperature conditions. Removal of one or
two leaves is done from the Arabidopsis
seedlings where it is placed in a weigh bat
and cut into various small pieces. A 1.5
ml tube is first measured on a weighing
balance and then the plant material is put
on the balance and weighed. Addition of
40 microliter of buffers PA1 as well as 10
microliter of RNAase A of stock solution
has been done. For disruption of the
tissues, eppendorf pestle is often used
which is followed by incubation of the
mixture for a time period of 10 minutes at
65 degree Celsius. The whole mixture has
to be mixed thoroughly at least 2 to 3
times during the period of incubation and
then the tube is to be inverted following
the process of centrifugation of the mixture
at 14,000 rpm for 5 minutes. The
ISOLATE II is Filter is placed into 2 ml
collection tube and then the lysate has
been loaded into the column. Only the
supernatant should be loaded into the filter
tube. This process is again followed by a
period of centrifugation at 14,000 rotations
per minute for a minimum of two minutes.
The clear flow through has to be collected
and the ISOLATE II filter has to be
discarded to obtain the original isolate.
If visibility of the flow through is seen
then the clear supernatant should be
transferred without disturbing the
formation of the pellet to a fresh 1.5 mL
micro centrifuge tube. The 450 micro liter
of the binding buffer is added and then the
solution is mixed thoroughly through
pipetting up and down or by vortexing for
proper mixing for atleast 4 to 5 times. The
ISOLATE II is then transferred to a 2mL

8GENE CLONING EXPRESSION AND ANALYSIS
collecting tube and then the sample is
loaded with maximum amount of 700 μL.
This step is followed by centrifugation of
the solution sample at 11,000 rpm for
minimum 1 min and the flow through is
discarded. 400 μ and 700μL of Wash
buffer PAW1 and PAW2 is added
respectively which is followed by
centrifugation for 1 minute in both the
cases. Then again another amount of
200μL is added and then centrifugation is
done at 11,000 rpm for at least 2 minutes.
Removal from the wash buffer is done and
then it is transferred to dry silica
membrane completely. The ISOLATEII
Plant Dna Spin Column is then transferred
to proper new labelled 1.5 ml micro
centrifuge tubes.Then the elution buffer
PG at 65 degree Celsius which has already
been preheated is added at an amount of
50μ and placed in the center of the
membrane. Then incubation is done for
minimum 5 minutes at 5 degree Celsius.
Finally centrifugation is done at 11, 0000
g.
For carrying out the Polymerase
Chain reaction (PCR), standard conditions
have been used. Two sets of primers that is
the forward and the reverse primers have
been used. Among the samples usually
three samples are taken comprising of the
positive and the negative samples along
with the control where there is absence of
the template. Thermo cycling conditions
are normally taken at 95 ºC for 2 minutes,
followed by 35 cycles of 94ºC for 15
seconds and 62ºC for 3 minutes which is
followed by72 ºC for at least 2 minutes as
the final extension step.
4. Results:

9GENE CLONING EXPRESSION AND ANALYSIS
Fig 1. Growth of transgenic plants after
plant transformation.
Fig 2. Gel electrophoresis of the multiplex
PCR amplification products.
Lane 1: Primer set 1 – A Lane 2: Primer
set 1 –B Lane 3- +ve control Lane 4 - -ve
control Lane 5 –Water Lane 7 500 bp
Ladder Lane 8- Primer set 2-A Lane 9
Primer set 3- B Lane 10- +ve control Lane
11-Negative control Lane 13-Water
For gel electrophoresis:
TBE buffer – 100 mL
Agarose gel – 2g
2 g Agarose + 100 L TBE buffer (2% agar
in 100 ml )
For designing primers against the target
gene:
Table 2: Primers used in the PCR

10GENE CLONING EXPRESSION AND ANALYSIS
Table 1: Master mix for PCR
Template
DNA
Primer set
1
Primer set
2
Samples Tubes 1 to
4
Tubes 1 to
4
Positive
control
Tube 5 Tube 5
Negative
control
Tube 6 Tube 6
No template
control
Tube 7 Tube 7
Table 3: PCR tubes to set up
Table 4: Composition of TBE for gel
electrophoresis
Compound Amount
Tris Base 10.8g
Boric acid 5.5 g
EDTA 0.584g
Distilled Water 1 L
Table 5: Percentage of gels
Expecte
d
product
size
Agarose
concentratio
n
TBE
concentratio
n
< 150
bp 3%
1X
150-300
bp 2%
1X
Reagents Starting
concentration
Final
concentration
Quantity for 1
reaction
(PCR cocktail)
Quantity for 8
Reactions
(Master mix for each primer set)
Redmix(RM
)
2X 1X
Forward
primer(F)
10 μM 0.2 M
Reverse
Primer (R)
10 μM 0.2 M
Template
dna (DNA)
- - 1 μL -
Distilled
water
- -
Total - - 25 μL 120 μL
Forwa
rd
prime
rs
5’-
AAGAACTCGTCAAGAAG
GCGATA-3’
Rever
se
prime
rs
5’-
ATGGGGATTGAACAAGA
TGGATT-3’

11GENE CLONING EXPRESSION AND ANALYSIS
>300 bp
1%
0.5X
Table 6: Agarose concentration used in the
PCR
Componen
t
Final
concentratio
n
Quantit
y
1X TBE - 100 ml
Agarose 2% -
5. Discussion:
Picture 1 is a result of the plantlets
which has been grown after transformation
through Agrobacterium. The seeds which
has been plated after the process have
grown in the given medium and the
specified culture conditions. Thus there
has been successful transformation of the
target gene of interest into the Arabidopsis
seedlings through the transformation using
agrobacterium as a medium of transfer.
Since it is an agrobacterium mediated
transfer, the genetic material that is the Ti
plasmid have been transferred into the
plant cell. Thus the growth of the plants is
shown in the MS medium.
The Picture 2 is a result of the
electrophoresis of the genomic DNA
which have been extracted from the
plantlet for the screening of the transgenic
plants.

12GENE CLONING EXPRESSION AND ANALYSIS
1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16
The given picture is a result of the
Polymerase Chain Reaction of the
transgenic gene which has been used to
differentiate and screen the putative
transgenic plants. The designed target gene
which has been inserted which contains a
forward and a reverse primer necessary for
transformation. The bands have been
clearly shown in band 9and band of the
next gel both of which contain the set for
reverse primer 2. The gene which has been
amplified have shown the band against the
primer set 2 confirming that the foreign
target gene is present in the transgenic
plant and thus transformation has been
successfully carried out.
For the identification of the
reporter genes in the given plant, the target
can be tagged with a florescent gene and
ten transformation can be done. Thus the
use of fluorescent tagged proteins are the
best way for assessing the identification of
the target gene for checking whether the
transformation has been successful or not.
forward primers 5’-
AAGAACTCGTCAAGAAGGCGATA-3’
and
reverse primers 5’-
ATGGGGATTGAACAAGATGGATT-3’
as
The following forward as well as
reverse primers have been considered for
designing against the np II gene which is
used for transformation and introduced
into the host plant cell using agrobacterium
mediated transformation. Neomycin
phosphotransferase genes are the most
common marker genes which are used as
marker genes for plant transformation.

13GENE CLONING EXPRESSION AND ANALYSIS
These sequences have been chosen for the
forward and reverse markers as these
designed against the given marker gene
would help in identification of the
transgene. Moreover, only these sequences
have been taken due to their easy
availability and commonly available
sequences for designing.
6. Conclusion:
Thus from the following
paragraphs, it can be observed that the
forward as well as reverse primers which
has been designed against the target gene
have been successful in being expressed in
the transformation process through
Agrobacterium. This can be concluded
from the following observations. Firstly
the seeds of the T1 generation in
Arabidopsis plants where transformation
has taken place has been grown in
Moorashig Skoog medium under favorable
conditions. After few days young plantings
have come out containing the transgene.
Secondly molecular analysis studies have
been done which shows that the primers
which have been used for designing the
gene have been successfully incorporated
into the transgene as bands have been
received for only the second set of primers.
Thus, the presence of the gene shows that
transformation have been successful and
the sequence of the primers of the set 2 are
the desired set of putative transgenic
plants. The genes from the template of the
Arabidopsis plant and the designed
sequence of the primers can be compared
through bioinformatics software tools like
BLAST for the determination of the
transgenic line. Moreover, the unknown
transgene showing the presence of the
primer set can be found using FASTA.
The identification of successful transgenes
through Agrobacterium mediated
transformation can be useful for the
production in the field of genetically
modified crops and the enhanced use in
Biofuels production as well. Thus, the
screening of the putative transgenes can be
used in the food and pharmaceutical
industries and this application will be used

14GENE CLONING EXPRESSION AND ANALYSIS
for lucrative production of the food and
pharmaceutical industries.
Based on the protocol of this
experiment, further experiments can be
conducted using variety of target genes for
the determination of the sequence of the
transgene. Moreover, this information can
be useful for the determination of the
genetic makeup of the T1 generation
received and can subsequently help in
studying the change in the genetic pattern
of the T1 generation and the reflection of
the genetic pattern changes in the
genotypic as well as phenotypic
constitution of the future progenies.

15GENE CLONING EXPRESSION AND ANALYSIS
References
Altpeter, F., Springer, N.M., Bartley, L.E.,
Blechl, A.E., Brutnell, T.P., Citovsky, V.,
Conrad, L.J., Gelvin, S.B., Jackson, D.P.,
Kausch, A.P. and Lemaux, P.G., 2016.
Advancing crop transformation in the era
of genome editing. The Plant Cell, 28(7),
pp.1510-1520.
Long, S.P., Marshall-Colon, A. and Zhu,
X.G., 2015. Meeting the global food
demand of the future by engineering crop
photosynthesis and yield
potential. Cell, 161(1), pp.56-66.
Mangano, S., Gonzalez, C.D. and
Petruccelli, S., 2014. Agrobacterium
tumefaciens-mediated transient
transformation of Arabidopsis thaliana
leaves. In Arabidopsis Protocols (pp. 165-
173). Humana Press, Totowa, NJ.
Plant-ditech.com. 2019. [online] Available
at:
http://www.plant-ditech.com/images/pdf/
Moshelion%20and%20Altman
%20(2015)%20Trends%20in
%20Biotechnology
%2033,%20337%E2%80%93342.pdf
[Accessed 7 Mar. 2019].
Puchta, H. and Fauser, F., 2014. Synthetic
nucleases for genome engineering in
plants: prospects for a bright future. The
Plant Journal, 78(5), pp.727-741.
Ran, Y., Liang, Z. and Gao, C., 2017.
Current and future editing reagent delivery
systems for plant genome editing. Science
China Life Sciences, 60(5), pp.490-505.
Rivero, L., Scholl, R., Holomuzki, N.,
Crist, D., Grotewold, E. and Brkljacic, J.,
2014. Handling Arabidopsis plants:
growth, preservation of seeds,
transformation, and genetic crosses.
In Arabidopsis protocols (pp. 3-25).
Humana Press, Totowa, NJ.
Rivero, L., Scholl, R., Holomuzki, N.,
Crist, D., Grotewold, E. and Brkljacic, J.,
2014. Handling Arabidopsis plants:
growth, preservation of seeds,
transformation, and genetic crosses.

16GENE CLONING EXPRESSION AND ANALYSIS
In Arabidopsis protocols (pp. 3-25).
Humana Press, Totowa, NJ.
Wani, S.H., Kumar, V., Shriram, V. and
Sah, S.K., 2016. Phytohormones and their
metabolic engineering for abiotic stress
tolerance in crop plants. The Crop
Journal, 4(3), pp.162-176.
Wu, E., Lenderts, B., Glassman, K.,
Berezowska-Kaniewska, M., Christensen,
H., Asmus, T., Zhen, S., Chu, U., Cho,
M.J. and Zhao, Z.Y., 2014. Optimized
Agrobacterium-mediated sorghum
transformation protocol and molecular data
of transgenic sorghum plants. In Vitro
Cellular & Developmental Biology-
Plant, 50(1), pp.9-18.
Wu, H.Y., Liu, K.H., Wang, Y.C., Wu,
J.F., Chiu, W.L., Chen, C.Y., Wu, S.H.,
Sheen, J. and Lai, E.M., 2014.
AGROBEST: an efficient Agrobacterium-
mediated transient expression method for
versatile gene function analyses in
Arabidopsis seedlings. Plant
methods, 10(1), p.19.
Xu, K., Huang, X., Wu, M., Wang, Y.,
Chang, Y., Liu, K., Zhang, J., Zhang, Y.,
Zhang, F., Yi, L. and Li, T., 2014. A rapid,
highly efficient and economical method of
Agrobacterium-mediated in planta
transient transformation in living onion
epidermis. PLoS One, 9(1), p.e83556.
Ziemienowicz, A., 2014. Agrobacterium-
mediated plant transformation: factors,
applications and recent
advances. Biocatalysis and Agricultural
Biotechnology, 3(4), pp.95-102.