UNIVERSITY OF AGRICULTURE, FAISALABAD

Centre of Agricultural Biochemistry and Biotechnology (CABB)

(Synopsis for MPhil Biotechnology)

TITLE: Agrobacterium-mediated Transformation of EPSPS and PR10 in Triticum

aestivum

Name of student: Muhammad Rizwan

Registration No. : 2013-ag-3467
Supervisor: Dr. Iqrar Ahmad Rana

Abstract
Wheat (Triticum aestivum L.) belongs to gramineae family and is one of the
most privileged staple food/cash crops. It exhibit high nutrition value and grows
commonly in temperate and tropics but at a narrow range of temperature. It is highly
sensitive to abiotic and biotic stresses which ultimately result in heavy yield losses.
Development of transgenic wheat is the only purpose of this study for acquiring
resistant as well as tolerant traits against such stresses at the first place. Epsps and
PR10 genes have been selected as desired genes to be transformed into AARI and
galaxy, two native wheat genotypes by cloning into T-DNA region of p7i-UG
vector. Embryo of the seeds will be targeted as explant for the transformation
process. Screening of transformed plants will be done by leaf paint assay via Basta
herbicide solution. Basta resistant plants will be declared as putative transgenics.
For their molecular characterization, their DNA will be isolated and PCR will be
performed.
 UNIVERSITY OF AGRICULTURE FAISALABAD
Centre of Agricultural Biochemistry and Biotechnology
(Synopsis for M.Phil. Biotechnology)

TITLE: Agrobacterium-mediated Transformation of EPSPS and PR10 in Triticum

aestivum

Date of Admission: 27-09-2017

Date of Initiation: 27-09-2017
Probable Duration: 2 years (four semesters)
PERSONNEL
i) Name of the student: Muhammad Rizwan
ii) Name of the supervisor: Dr. Iqrar Ahmad Rana
iii) Registration No.: 2013-ag-3467

SUPERVISORY COMMITTEE
i) Dr. Iqrar Ahmad Rana (Chairman)
ii) Dr. Muhammad Adnan Khan Niazi (Member)
iii) Dr. Farooq Ahmad Khan (Member)

Introduction
Triticum aestivum (Bread Wheat) lies among the most widely grown and used crops
worldwide. Almost 95% of wheat grown in the world is bread wheat which is far greater than all
the other cereal crops. It was grown at the first in Asia as a domestic crop and then cultivated over
rest of the world in prehistoric period. Modern genotypes of wheat exhibit short-stems which are
due to expression of Rht genes that are responsible for dwarfism and leads to shortening of plants.
These genes were introduced in early 1960s as a result of work done by Norman
Borlaug upon Norin 10 wheat cultivars cultivated in Japan. Short-stems are way better and
significant as compared to the longer stems because chemical fertilizers leads to their collapse.
These height of stems are beneficial while implementing latest methods of harvesting over wheat
as they are easily manageable (Cheng et al., 2001).
Bread wheat is the third biggest cereal crop of world which was reported to be having
drastic increase in yield of almost 713 million tons in year 2013 only while that of the previous
year was from 560 million tons. Wheat is a cereal crop which belong to C3 group. The largest
producer of wheat crop has been China since year 1991 mainly due to its highest demand and
population in the world. Twenty percent of calories for human body can be easily met by taking
up wheat (Vasil, 2007). There are many essential vitamins and minerals present within this crop.
A survey by IFPRI (International Food Policy Research Institute) has shown that demand for wheat
is going to rise up to 841 million tons by the year 2020. There are certain great challenges for
wheat production which involve exponential increase in population of world, limited arable land
and climate change.
The availability of food to the continuously increasing population for feeding purpose is a
big challenge for approximately 9 billion around the globe in upcoming years. Wheat is of the
major staple food which can help us meet this increasing demand. There has been least
improvement by genetic engineering as well as biotechnology in this crop mainly due to its larger
and complex genome. Additionally, there are many repetitive DNA sequences within this
hexaploid genome and there is a very low regeneration reported after transformation of any gene(s)
(Křenek et al., 2015). There are no new commercial varieties of wheat which are developed using
conventional methods since the emergence of biotechnology as they seem to be more costly and
time-taking. Major reason in this regard was lack of reference genome sequence in past while
genetic transformation of wheat was quite difficult as well. Transformation of plants with novel
genes like EPSPS and PR10 can be achieved using agrobacterium-mediated methods and putative
transgenics can be obtained by collecting their germplasm which can later on be used or
regenerated in future. A novel method named as: PureWheat was introduced by a Japanese
company for the enhancement of efficacy of transformation significantly. This method has
increased the hope for scientist to manipulate its genes with greater efficacy and also in a diverse
manner. This also opens the gateway for novel genome editing approaches which can be used for
genetic improvement of wheat.
The two commonly equipped techniques in order to induce genetic transformation include:
Agrobacterium-mediated as well as microparticle bombardment methods. These techniques have
been used for many years for transformation, firstly within a dicot crop and then in other monocot
crops like barley, rice and wheat as well (Tingay et al., 1997). Agrobacterium mediated
transformation is a simple transformation approach which is used commonly because of its ability
to allow the stable gene integration of desired fragment of DNA within plant genome. It also gives
the lower copy number of integrated gene within host. There are least variations or complications
along with an enhanced gene expression stability in upcoming generations as compared to that of
the direct DNA delivery techniques (Dai et al., 2001).
The other most common method for transformation is the micro-particle bombardment or
biolistics (Zhang et al., 2000). The first ever successful transformation in wheat by agrobacterium
method was reported for the first time in the year 1997 (Cheng et al., 1997). This was achieved by
using embryogenic callus and immature embryos to generate fertile transgenic plants. The
frequency of transformation recorded was almost 1-4%, however the results obtained were quite
reserved upto a minor or lab-scale while the screening was done by selection on basis of neomycin
phosphotransferase II (nptII) gene (Amoah et al., 2001).
The two genes which are chosen for this project includes: EPSPS and PR10. EPSPS (5-
Enolpyruvylshikimate-3-phosphate synthase) which is the second last enzyme in shikimate
biosynthesis pathway and its main role is to develop herbicide resistance in plant system. However,
this gene has not been well-characterized in wheat till now. Moreover, PR10 gene (Pathogenesis
related) has been reported to respond against both biotic and abiotic stresses. These both genes can
help the wheat crop to withstand stresses and weeds (Kumar et al., 2014).
Researchers are trying to use new technologies of latest era to develop a stable method of
agrobacterium mediated transformation in wheat. Now they are moving towards implementation
of advanced biotechnological approaches i.e. genomics and genetic transformation as they have
most promising applications (Razzaq et al., 2012). This current project will focus on utilizing the
agrobacterium method for transformation of herbicide/stress resistant genes like EPSPS and PR10.
These genes will be introduced in crop under controlled conditions and will then be identified for
the transformants.
Review of Literature:
The first ever transgene introduced into wheat was done by biolistic particle method in year
1992. High velocity micro-projectile bombardment method was used for the successful
transformation of Bar gene. This is when the transformation of wheat was all started. This method
was greatly used for the transformation of many genes later on. Some of these genes includes:
Yr10, TaNAC2, TapIMP1, r19PR1 and TaCPK respectively. There are many reports which shows
the tests being performed for genetically enhanced wheat lines that are way more resistant to both
biotic and abiotic stresses. Though the biolistic transformation method has a limitation of a lower
efficacy of transformation.
There are two plasmids in Agrobacterium tumefaciens (Ti and Ri Plasmid) and there is a
T-DNA present on them. Agrobacterium infection is used to transfer the T-DNA into the genome
of plant. The first ever use of agrobacterium mediated transformation on wheat was done in 1997
and since than it has become a better option for transformation in wheat (Cheng et al., 1997). Large
segments of DNA can be transferred with least rearrangement using this method rather than
biolistic one which has limited amount of DNA that can be transferred. There method has also a
lower transgene copy number and a far more stable gene expression in the transgenic progeny.
There are many Chinese researchers which have shown many cases linked with
agrobacterium mediated transformation of wheat. Some of the factors that can affect the efficiency
of this method includes: genotype, size and type of explant, vector and strains of Agrobacterium,
selectable marker, the concentration of bacteria, co-culture conditions (temperature, time, light,
medium composition) and surfactant (Wu et al., 2003; Cheng et al., 2004).
Huang et al. (2002) has shown the comparison of anther culture, immature embryos, pre-
cultured immature embryos and young spikes for the transformation of wheat explant using
agrobacterium method. He found that the immature embryos which were pre-cultured for almost
15 days show the best result on regeneration and transient expression. Another report suggested
the use of spikelets as explants which raised the transformation efficiency from 2 to 3.2%
respectively (Hu et al., 2003).
Ye et al. (2005) performed his research on 35 genotypes of wheat to check their sensitivity
againt C58C1 strain of agrobacterium in China. Infection was done with this strain and final
efficacy of transformation was also checked. He found that six of these cultivars (P187, Yangmai
158, PM97034, Xinchun 9, Kefeng 6 and Ba1401051) were shown to be more sensitive to this
strain. The successfully obtained transgenic lines were from PM97034, Bobwhite and Xinchun 9
with 2.0, 1.1 and 3.3% frequency of transformation respectively. He also showed that by using the
shoot apical meristem for explant has shown the efficacy of transformation to raise up to 9.82%
respectively (Zhao et al., 2006).
 Wang et al., (2009) developed a transformation method of agrobacterium for wheat using
explants which were obtained from mature embryos and he obtained the efficacy for
transformation of about 1.79%. On the other hand, another scientist optimized the conditions for
the agrobacterium transformation method in mature embryos and the results were quite good.
These results showed that with the addition of picloram, the regeneration rate was reached 27.1%
for elite wheat Emai 12 variety (Ding et al., 2009). The best suitable combination was a mature
embryo which was pre-cultured for 14 days and later it was immersed in suspension MS media
with full strength of slants in dark conditions at temperature of 23 to 25°C for almost 3 hours. Co-
culturing was done later on at 23 to 24°C for almost 2 to 3 days in dark desiccation condition.
Another efficient method for wheat using agrobacterium was developed by He et al.
(2010). This method shows that by increasing the concentration of AS (Acetosyringone) to 400
mmol has led to an increased rate of GUS transient expression and increased delivery efficacy of
T-DNA. This increased the transformation efficacy from 4.7 to 6.3% respectively. He also show
that by increasing the concentration of pocloram from 2mg to 10 mg led to the enhanced efficacy
of transformation from 2.8 to 6.3% respectively.
Liu et al. (2011) reported the use of immature callus embryos for Luoyang 8716 and
Mianyang 19 wheat cultivars as explants. From his experiment he found that the best protocol of
co-culture was agrobacterium with a cellular density of 600=0.8 (OD) with an incubation period
of 30 minutes for EHA105 strain and cell density of 600-1.0 (OD) with an incubation period of 30
minutes for LBA4404 strain. Regeneration of wheat immature embryos which were infected with
agrobacterium tumefaciens was improved by Tao et al. (2011). Another report later on compared
the agrobacterium transformation of wheat tillering node and apical meristem as explants. These
results proved that the apical meristem as explants has a high efficacy of transformation of about
13.79% rather them tillering node which has about 3.25% respectively (Hu et al., 2013).
Hu et al. (2012) isolated a cDNA for PEPC (phosphoenolpyruvate carboxylase) gene from
another crop belonging to C4 group (Zea mays) in an attempt to increase the photosynthetic rate.
He introduced this gene intro wheat (C3 crop) using agrobacterium mediated transformation
method. He found that the seed weight for per spike was 0.23 grams and thousand grain weight
was 1.21 grams higher then control.
Zhang et al. (2014) reported the introduction of both cDNA of PEPC and PPDK from
maize into wheat to produce transgenic lines of wheat which has either cDNA of PEPC, (PC lines)
PPDK (PK lines), or both (PKC lines). These lines showed that the maximum rate of
photosynthesis was raised by 26.4% for PKC, 13.3% for PC, and 4.5% for PK lines respectively.
The grain weight was also increased as compared to the control.
Zhao et al. (2013) successfully integrated and expressd a NR (Nitrate Reductase) gene from
tobacco in transgenic wheat. The overexpression of this gene also improved the T1 foliar NR
activity and significantly augmented T2 seed protein content and thousand grain weight in 63.8
and 68.1% of T1 offspring, respectively.
Materials and Methods
This experiment will be performed within Transformation laboratory at Center for
Advanced studies in agriculture and food security, University of Agricultural Faisalabad.
Plant Material Collection
Seeds of two different wheat genotypes i.e. AARI and galaxy will be taken from a
government organization, Ayub agricultural research institute (AARI) Faisalabad, Pakistan for
carrying out the proposed experiment.
Transformation Procedure
Seeds of two wheat genotypes i.e. AARI and galaxy will be sterilized at the first and then
transformed by causing injury through puncturing with needle. Culture inoculum of
Agrobacterium containing PR10 or EPSPS gene within p7i-UG vector will be inoculated into seeds
along with some induction media which will enhance the transformation efficiency. The inoculum
will be prepared by transforming desired genes and plasmid into agrobacterium strain by heat
shock method and confirmation of process will be done on the basis of existing selectable marker
i.e. antibiotic resistance (spectinomycin+rifampicin). Seeds will be grown in growth room under
controlled conditions within germination trays containing compost and will be watered at regular
intervals. Plantlets after attaining the 4-5 leaves stage, will be screened under basta, herbicide,
selection pressure in order to get the successfully transformed identified. The DNA of resistant
plants will be isolated in lab and molecular characterization will be done to get confirmation of
gene integration.
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SIGNATURES

Student (Muhammad Rizwan) ――――――――――――――

SUPERVISORY COMMITTEE:

Chairman (Dr. Iqrar Ahmad Rana) ――――――――――――――

Member (Dr. Muhammad Adnan khan Niazi) ――――――――――――――
Member (Dr. Farooq Ahmad Khan) ――――――――――――――

FORWARDED
――――――――――――――
Director,
Centre of Agricultural Biochemistry
and Biotechnology (CABB)
University of Agriculture, Faisalabad

FACULTY SCRUTINY MEMBER:

――――――――――――――
(Dr. Faiz Ahmad Joyia)

REVIEWED AND WITNESSED:

――――――――――――――
Dean,
Faculty of Agriculture,
University of Agriculture, Faisalabad