1. Agrobacterium-mediated transformation on
maize
Maria Tharumalingam
York Mills CI, 490 York Mills Rd, Toronto, ON M3B 1W6
Abstract:
Bacteria have been observed, time and again, to be extremely useful to not only humans, but
many other organisms and the environment. Some bacteria however are known to be pathogens
and parasites to animals and plants. A specific genus of bacteria known as Agrobacterium
tumefaciens has different strains that cause mutations and diseases such as galls and tumors in
plants [4]. The interesting aspect of this is that the T-DNA found in this species, is transferred
into the genome of the plant cell. Only after the genes become incorporated in the plant genome
do they become active and cause diseases. Agrobacterium-mediated transformation is the process
of removing these virulent genes and instead, using this natural process, transfer non-virulent,
advantageous genes to the plant [5]. This journal will analyze the possibility of agrobacterium-
mediated transformation leading to increased growth/yield of maize.
Introduction:
Every habitat on earth is home to the well-studied microbes commonly known as bacteria.
Bacteria occupy habitats such as soil, rock, oceans, and arctic soil; in addition, some live on
other organisms such as plants and animals. In fact, there are more bacterial cells in the human
body than there are somatic cells [5]. This not only describes the importance of bacteria, it also
gives an indication as to how extensive and progressive the evolution of bacteria has become.
Scientists use taxonomy to classify all living organisms- the end result is that all organisms can
be defined as a distinct species. Due to the diversity and complexity of bacteria, it is given its
own domain according to taxonomic ranks [6]. These single celled microbes have an extremely
simplistic cell structure- they have no nucleus, or other membrane bound organelles. Their
genetic information is contained in a single loop of DNA. Some bacteria may contain additional
genetic information in the form of plasmid, an extra circle of genetic information that may give
some bacteria an advantage over others [6]. Most bacteria are incredibly useful and have many
different functions. Some of these functions include the cycling of nutrients and the production
of fermented foods such as soy sauce. [7]However, some bacteria are parasites and pathogens
that cause harm to humans and plants. Naturally, many individuals are fully aware of the virulent
bacteria that cause harm to humans such as Salmonella and certain strains of E.coli bacteria. One
sophisticated bacterium that has been observed to cause severe tumors in plants, is known as
Agrobacterium tumefaciens. The genus Agrobacterium, has been divided into multiple species
which reflect the disease symptomology and host range [4]. The most important fact is that the
sophistication and complexity of the bacterial genes being transferred into the plant to cause
tumors, is a process that occurs naturally. A well-defined segment of DNA from the bacterial
tumor inducing (Ti) plasmid is transferred to the genome of the host cell. The genes transferred
are activated and expressed only after being transferred to the plant [5]. This leads to the
production of opines and amino acid derivatives. The production of opines and amino acid
derivatives provide a source of carbon and nitrogen that the Agrobacterium tumefaciens can use,

2. allowing them to thrive [4]. This process has been observed and studied and scientists have
found a way to use it to their advantage. Manipulating the genes in the plasmid before being
transferred into the plant has opened up many doors in the growing field of biotechnology. This
type of genetic engineering is more commonly known as Agrobacterium- mediated
transformation. To summarize the process, the virulent genes on the plasmid are removed, and
scientists manipulate the remaining genes that will be transferred into the plant, so that they will
not cause harm to the plant, but rather be beneficial [3]. The objective of this article is to show
how the genetic engineering of A.tumefaciens can affect the growth of genetically modified crops
such as maize. If A.tumefaciens is genetically engineered so that when T-DNA is transferred into
plants, the genes that are expressed induce plant growth, then there should be further investment
into biotechnology because it will have a positive economic impact in society. A major
advantage of Agrobacterium-mediated transformation is that a small number of copies of
relatively large segments of T-DNA with defined ends are integrated into the plant genome with
minimal rearrangement [4]. The expected outcome of this proposal is that more individuals
become aware of how advantageous biotechnology is to our society and why more money should
be invested into this research. The increase in the growth of genetically modified crops can help
boost the economy. Investing money for research and experiments on A. tumefaciens can have a
positive impact in the future. Possible ideas that can stem from this proposal is the effect of this
same proposed experiment on other genetically modified crops such as barley, soy bean,
wheat… etc. Or, ways to simplify agrobacterium-mediated transformation to reduce costs. The
objective is that more ideas, opinions and questions arise as result of reading this proposal.
Rationale:
Molecular biology is a very fast growing field and it is extremely important that we continue to
invest money into the research that can be conducted with it. Society can progress and evolve
when we take time to understand the significance of genetics. As a high school student,
molecular genetics was by far the most interesting unit that was covered. Not only was it a
fascinating subject, being educated on it was very beneficial. For example, one major concept
that applies to healthcare right now is antibiotic resistance. By understanding why antibiotics are
important and the significance of antibiotic resistance, students can go around educating others
about its importance. Molecular biology is also a very diverse field. Through studying it, it is
possible to explore astrobiology which is the application of molecular biology on extraterrestrial
planets in the universe. For example, analyzing certain bacteria and its capability to thrive within
the soil on Mars may be the start to inhabiting Mars and creating a viable ecosystem.
Furthermore, the general concept of using A.tumefaciens and genetically engineering it improve
plant health, as opposed to destroy it by causing tumors, is a sophisticated and complicated
process. The more research put into it, the more that can be learned from it. This might inspire
others to come up with more complicated research topics that could lead to major breakthroughs
in this field.

3. Methodology and Materials:
Extensive amounts of research will be conducted to determine the most efficient way to carry out
this experiment. Many academic journals will be referred to when considering the materials and
equipment that should be used for the experiment. Some of the materials needed would be the A.
tumefaciens strain carrying appropriate T-DNA-containing vectors (for plant growth) and the
maize seeds of inbreds. The equipment needed would be a scalpel blade, surgical tape made of
unwoven fabric, parafilm (Pechiney Plastic Packaging), one 270 mm pot (270 mm in diameter
and 270 mm in height) for maize cultivation and, one 230 mm pot (230 mm in diameter and 190
mm in height) for maize cultivation [2,3].
Transgenic maize plants were first obtained from protoplasts by an electroporation method. For
the last two decades, dicot plants were transformed using the soil phytopathogen A.
tumefaciens. A. tumefaciens is first transformed with the DNA construct of interest (T-DNA);
this modified bacterial strain is then used to introduce the T-DNA into plants. For the successful
production of transgenic plants in any species, foreign genes must be delivered to
undifferentiated, dedifferentiated or dedifferentiating cells that are actively dividing or about to
divide and that are capable of regenerating plants. In maize, the material of choice is immature
embryos. When the Agrobacterium strain with the appropriate vectors is transferred to the maize
embryos, the primary determinants of a successful transformation are the response of immature
embryos in tissue culture, the types of cells that grow from immature embryos and characteristics
in growth and regeneration [1]. Agrobacterium strain AGL1 is used together with appropriate
pBract vectors which contain the hpt gene. The pBRACT vectors are based on pGreen, a small,
versatile vector designed for easy manipulation in E. coli with a high copy number. To enable the
small size of pGreen, the pSa origin of replication required for replication in Agrobacterium, is
separated into its‟ two distinct functions. The replication origin is present on pGreen, and the
trans-acting replicase gene is present on an additional vector, named pSoup. Both vectors are
required in Agrobacterium for pGreen to replicate. Three different basic plant tissue culture
media are used during the transformation and regeneration process; callus induction, transition
and regeneration media. After the Agrobacterium strain is prepared, the culture is used to
inoculate the barley embryos. The plates containing the barley embryos treated with the
Agrobacterium strain are sealed with MicroporeTM surgical tape and incubated at 23-24 degrees
Celsius for three days. After three days co-cultivation, the embryos are transferred to fresh callus
induction plates containing hygromycin as the selective agent and timentin to remove the
Agrobacterium from the cultures. The embryos are transferred to selection plates a total of three
times with a two week time interval in-between each selection. After six weeks selection on
callus induction medium, the embryo derived callus is transferred to transition medium, again
containing hygromycin and timentin, for two weeks at 24 degrees Celsius under low light. After
the two weeks on transition medium, the embryo derived material is transferred one final time to
regeneration medium, in deep Petri dishes, without any growth regulators but still with the same
levels of hygromycin and Timentin. Once plants have grown 2-3 cm in length and roots are
formed, they are transferred correctly into soil where leaf samples can be collected for further
analysis to confirm the presence of the introduced genes [3].

4. Expected outcome / Discussion:
The transformation experiment on Maize [1] suggests that the mechanism for the transformation
still requires improvement. There was a high frequency of transformation in some of the
genotypes within the maize culture; however, the other genotypes were not expected to be
transformed efficiently [1]. –The barley transformation experiment [3] displayed positive results.
The genes were expressed within the barley after the embryos were allowed to grow. This shows
that the results from that experiment were conclusive and that the transformation occurred
effectively and immediately without complications. The gene that was expressed was the gus
gene in leaf samples [3]. Overall the concept that was learned from these results is that two
different mechanisms of Agrobacterium-mediated transformation were used; one method was
extremely effective and the other was not. If this proposal was funded and the experiment was
carried out, the results would be positive. Immediate growth of the maize would occur which
would show that Agrobacterium-mediated transformation should be funded further. If this
proposal is funded, the money would be used to ensure that all materials are present and that all
the crops grown are put in ideal conditions so that the data is accurate, and reliable. The main
objective is to prove that Agrobacterium-mediated transformation will result in the increased
growth rate of maize. Biotechnology based approaches targeting increased yield and better crop
production need to be extended to more food crops. The core idea in this proposal can be
extrapolated to other crops and the approach described herein has potential to be a valuable
alternate approach in crop biotechnology.

5. References:
1. Ishida, Y., Hiei, Y., and Komari, T. (2007) Agrobacterium-mediated transformation of
maize. Nat Protoc Nature Protocols 2, 1614–1621.
2. Tzfira, T., and Citovsky, V. (2006) Agrobacterium-mediated genetic transformation of
plants: biology and biotechnology. Current Opinion in Biotechnology 17, 147–154.
3. Harwood*, W. A., Bartlett, J. G., Alves, S. C., Perry, M., Smedley, M. A., Leyl, N., and
Snape, J. W. (2008) Barley Transformation Using Agrobacterium-Mediated Techniques.
Methods in Molecular Biology™ Transgenic Wheat, Barley and Oats 137–147.
4. Gelvin, S. B. (2003) Agrobacterium-Mediated Plant Transformation: the Biology behind
the "Gene-Jockeying" Tool. Microbiology and Molecular Biology Reviews 67, 16–37.
5. Analyzing Plant Gene Expression with Transgenic Plants. Analyzing Plant Gene
Expression with Transgenic Plants.
6. Wang, Q., Garrity, G. M., Tiedje, J. M., and Cole, J. R. (2007) Naive Bayesian Classifier
for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy. Applied
and Environmental Microbiology 73, 5261–5267.
7. Ireland, K. (2015) Good Uses for Bacteria. LIVESTRONG.COM. LIVESTRONG.COM.
8. Wusirika, R. Genetics, genomics and breeding of maize.