PAPER OF BIOTECHNOLOGY

“PROCESS INSERTED GENES TO AGROBACTERUM AS VECTOR

AND HOW TO ITS PROLIFERATION”

Arranged by:
Endang Sri Istanti Putri
Dinda Ilona M.
Nita Nathalia Fildzah
Rima Rahma
Muh. Fiqriansyah W. S.
Citra Dara Puspita
: 185040200111049
: 185040200111153
: 185040200111210
: 185040201111066
: 185040201111189
: 185040207111058

STUDY PROGRAM OF AGROECOTECHNOLOGY

FACULTY OF AGRICULTURE
BRAWIJAYA UNIVERSITY
MALANG
2019
 CHAPTER I

INTRODUCTION

1.1 Background
Agrobacterium is known naturally to have the ability to transfer DNA
between kingdoms. In plants, Agrobacterium is usually used as a genetic
engineering agent to produce transgenic plants. Besides Agrobacterium can also
be used for gene transformation between living cells, for example from
prokaryotic cells into fungal cells or human cells. The ability of Agrobacterium to
carry out genetic transformations into host cells has been studied for decades
(Tzfira, 2004).
According to Gelvin,2003 Agrobacterium has been divided into several
type and reflected to the most of Agrobacterium species. Thus are A. radiobacter
is an “avirulent” species, A. tumefaciens causes crown gall disease, A. rhizogenes
causes hairy root disease, and A. rubi causes cane gall disease. More recently, a
new species has been proposed, A. vitis, which causes galls on grape and a few
other plant species. Agrobacterium tumefaciens in nature causes Crown-gall
disease, which is swelling that occurs due to polyperation like tumors that occur in
most plants 2 dicots. According to Matthyyse, 2006 Agrobacterium Tumefaciens
also causes call-grown in most dicots, some monocots, and some gymnosperms.
During tumor induction Agrobacterium attaches to plant cells and then transfers
part of its DNA to some of these cells. The transferred DNA (T-DNA) which
places on a large Ti (tumor inducing) plasmid, is processed in the bacterium and
exported to the plant where it becomes integrated into the plant genome. Proteins
encoded by the virulence (vir) region of the Ti plasmid regulate T-DNA
processing and transfer. Phenolic compounds derived from a wounded plant cell
wall induce expression of the vir region genes. (Ziemienowicz, 2017)
Broadly speaking, there have been two successful gene transfer
techniques: direct gene transfer (for example with the chemical compound
polyethylene glycol (PEG), electroporator or DNA shooter) and indirectly using
Agrobacterium tumefaciens soil bacteria. Each technique has advantages and
disadvantages. Direct DNA transfer techniques have a tendency to insert DNA
with a large number of copies, so the Agrobacterium transformation technique
becomes an alternative choice. The greater number of copies of genes inserted can
cause unstable gene expression. This is due to the silencing of genes and the
process of rearrangement (rearrangement) is increasingly high. The application of
gene transformation technology enables the insertion of important genes, so that it
is hoped that they will not change other traits. Gene transformation in plants is an
attempt to improve the genetic traits of plants. (Rahmawati, 2006).
Genetic transformation is usually used for genetic engineering and has
been carried out on several crops including agricultural crops. An efficient genetic
transformation procedure can be used to facilitate the study of molecular biology
and produce superior varieties of plants, for example on corn plants. Genetic
transformation of corn has been successfully carried out by three methods, namely
using a biolistic shooter, electroporation and Agrobacterium. Transformation
using Agrobacterium is more favorable than biolistic and electroporation shooters
because it produces greater expression transgenter with a lower number of loci.
The efficiency of genetic transformation using Agrobacterium is influenced by the
strain of Agrobacterium used. The difference between Agrobacterium strains is
determined by the presence of chromosome type and Ti-plasmid type. The type of
chromosomes in Agrobacterium bacterial cells consists of octopine, nopaline, and
3chrysopine; while the Ti-plasmid type consists of octopine, nopaline, chrysopine,
and succinamopine (Utomo, 2004).
The advantages of transformation techniques using Agrobacterium include
1). The efficiency of transformation with a single gene copy is higher, 2). Can be
done with simple laboratory equipment. Genes with a single copy are easier to
analyze and usually segregate following Mendel inheritance patterns.
1.2 The Purposes
The purpose of making papers on how to insert genes into Agrobacterium
is so that students can understand more clearly what is meant by Agrobacterium
and how or how to insert genes into Agrobacterium for plants
 CHAPTER II

DISCUSSION
2.1 Insertion of Genes in Agrobacterium into Plants
Genetic transformation of agricultural crops is a routine procedure to get
superior plant varieties. There are two soil bacteria that are usually used in plant
transformation, namely Agrobacterium tumefaciens and Agrobacterium
rhizogenes which cause tumors and hairy roots in dicotyledonous plants.
Although the disease caused by the two bacteria above is different, but the process
or mechanism of infection is the same. Agrobacterium has the ability to transfer
T-DNA from a plasmid known as Ri Plasmid (root inducing plasmid) into plant
cells through injury (Walden, 1991).
The process of genetic transfer from Agrobacterium in plant cells
The process of genetic transfer from Agrobacterium in plant cells through several
stages, namely:
1) Bacterial colonization
It is an important initial step to induce tumor formation. This stage
plays a role when Agrobacterium attaches to the surface of plant cells.
Polysaccharides found on the surface of Agrobacterium cells play an
important role in the process of colonization.
2) Induction of a virulent bacterial system
T-DNA transfer is indicated by a product encoded by 30-40 kb of
Vir (virulence protein) region on the Ti Plasmid. This area consists of at
least six essential operons (Vir A, Vir B, Vir C, Vir D, Vir E and Vir G)
and non-essential operons (Vir F and Vir H). The number of genes in each
operon is different, Vir A, Vir G and Vir F consist of only one gene; Vir
C, Vir E and Vir H consist of two genes; whereas Vir D and Vir B have
four and seven genes, respectively. Vir A-Vir G is a two component
system that activates transcription in other Vir genes. Activated Vir A has
the capacity to transfer phosphate to aspartate residue that corresponds to
the cytoplasmic DNA binding protein Vir G. Vir G function is as a
transcription factor that regulates the regulation of Vir gene expression
when phosphorylated by Vir A. The C-terminal region is responsible for
 DNA binding, whereas N-terminal is a phosphorylation domain that shows
homology with the Vir sensor domain A. Activation of the Vir system also
depends on external factors such as temperature and pH. At temperatures
over 32 ° C, the Vir gene will not be expressed because it changes the
folding conformation of Vir A which induces inactivation. The effect of
temperature on Vir A is suppressed in the form of mutant Vir G (Vir Go),
which activates the expression of the Vir gene.
3) The formation of a complex generation of T-DNA
Activation of the Vir gene results in the generation of single strand
molecules (ss) that represent copies of T-DNA strands. DNA located
between the T-DNA boundaries will be transferred into plant cells as
ssDNA, and then integrated into the plant genome. Vir D1 and Vir D2
proteins are proteins that play an important role at this stage, by
recognizing the T-DNA boundary sequence and nicking (endonuclease
activity) at the lower strand at each boundary. In the nick region it is
assumed to be the place of initiation and termination for the recovery of
the T strand. After cutting endonucleotides, Vir D2 will covalently attach
to the 5 'end of the ss T strand important actor in the T-DNA transfer
complex.
4) T-DNA transfer includes two models for the translocation of T-DNA
complexes
The means of transfer into the plant nucleus is the ssT-DNA
protein complex. This must be translocated into the plant nucleus through
three membranes, plant cell walls and cellular space. Based on the most
accepted model is the ss T-DNA Vir D2 complex which is covered by 69
kDa Vir E2 protein, ssDNA binding protein. This association prevents
nuclei and the addition of the T-DNA ss-strand extension thereby reducing
the complex diameter by about 2 nm. This results in easier translocation
through the membrane. However, the association cannot stabilize the T-
DNA complex in Agrobacterium. Vir E2 consists of two plant signals,
NLS (nuclear location signal) and Vir D2 consisting of one NLS. This
fact shows that both proteins seem to play an important role in plant cells
 as intermediaries for the transfer of T-DNA complexes into the nucleus.
Vir 5E1 is needed to export Vir E2 into plant cells, although other specific
functions are not yet known. Another alternative model is that the transfer
complex is covalenteric ssDNA at the end of 5 with Vir D2, but without
being covered by Vir E2 protein. The independent export of Vir E2 into
plant cells is a natural process, and once the VirD2 ssT-DNA complex is
entered into plant cells, it will be covered by Vir E2 protein. This also
allows the process to occur as an alternative when the condition is
infected.
5) Integration of T-DNA in plant genomes
In plant cells, the ssT-DNA complex is a nucleus target to pass
through the nucleus membrane. Two Vir proteins have been identified as
important at this stage, namely Vir D2 and Vir E2 are the most important,
and Vir F is likely to play a minor role in this process. The NLS signals
from Vir D2 and Vir E2 play an important role as nucleus targets in
delivering the ssT-DNA complex as an initial description. Vir D2 has a
functional NLS. The ss T-DNA complex is a large nucleoprotein complex
of about above 20 kb consisting of only one end of the 5 covalently
attached to the Vir D2 protein per complex. Each complex is surrounded
by a large Vir E2 molecule of around 600 per 20 kb T-DNA, and each
consists of 2 NLS. These two NLS of Vir E2 have been considered as
important for the continuation of the ss-TDNA impotence, the possibility
of keeping both nuclei of the nucleus open simultaneously. Nucleus
imports may be mediated by specific NLS binding proteins, which are
present in the cytoplasm of plants.
`The final stage of T-DNA transfer is the integration of T-DNA
into the plant genome. Based on the above model, a small base pair known
as micro-homology is needed for the pre-annealing stage between the T-
DNA strand pairing with Vir D2 and plant DNA. This homology is very
small and has little specificity in the recombination process by positioning
Vir D2 for ligation. At the 3 'end or adjacent sequences on the T-DNA
there is some slight homology to the plant DNA which produces initial
 contact (synapses) between the T strand and plant DNA and forms a gap in
the 3'-5'DNA strand of the plant. The removal of plant DNA will cut off at
the end 3 'in the gap by the endonuclease, and the first nucleotide at 5'
attaches to the Vir D2 pair with the nucleotide at the end (5'-3 ') of the
plant DNA strand. At 3' overhangs, T-DNA along with plant DNA
removal is a digestive event either by endonuclease or 3'-5' exonuclease.
Then, 5 'sticks to the Vir D2 suffix and the other 3' end on stand T (paired
with plant DNA during the early stages of the integration process) just
below the DNA strand of the plant. This is the introduction of the T strand
in the 3'-5 'strand of perfect plant DNA, a twist followed by a nick on the
opposite strand of the plant DNA is produced. This situation activates
repair mechanisms in plant cells and complementary strands synthesized
through the initial insertion of T-DNA strands as molds (Gustafo, et al.,
1998).

Picture 1. Agrobacterium T-DNA transfer process in plant cells

Plant response to Agrobacterium infection results in several influences on
plant tissues such as necrosis in grapes, apoptotic response in maize. The response
is tissue necrosis and DNA cutting. The nucleus becomes an oligonucleosome
fragment by the endogenous enzyme please characterize the caspase-protease
reaction (Gelvin, 2003). Agrobacterium tumefaciens is only able to infect
dicotyledonous plants. In line with the success of the research that has been done,
Agrobacterium transformation can also be carried out on monocotyledonous
plants. Rice (monocot groups) is one of the agricultural crops that has been done
to obtain superior varieties of rice. Some genes that have been successfully
inserted in rice plants, and are being evaluated for expression and function, are
genes for resistance to yellow stem borer pests, fungal diseases, and drought
tolerance (Rahmawati, 2006). Another monocotyledonous plant, the sugar cane
plant (Saccharum officinarum L.), has also been successfully transformed through
Agrobacterium, although it has been constrained for several years. Sugarcane is a
raw material for sugar production, so its production is very high, especially in the
tropics and subtropics. The use of genetic transformation methods by introducing
recalcitrant genes into plant genomes has a large impact on the production of
sugarcane and other agricultural crops. Agrobacterium transformation which
introduces a number of external DNA copies is an important procedure and is
needed to increase production of plants that are recalcitrant. Plants such as cereals,
legumes and woody ones are very difficult to transform. The existence of genetic
engineering with Agrobacterium greatly helps the development and advancement
of technology for the production of agricultural crops such as rice which is the
main food ingredient in Indonesia.
2.2 Process of Proliferation
Cell Proliferation on Agrobacterium
According to Money (2013) cell proliferation is defined by the cycling
behavior of the cells (i.e., how quickly cells pass through the four phases of the
cell cycle) and the numbers of cells that are actively cycling, which is known as
the growth fraction. Agrobacterium that has been inserted with certain genes
needs to be duplicated so that genes that have been inserted will multiply so that
the gene will be easier to insert into the callus. According to Lewin (2008)
Replication of agrobacterium is triggered at a single origin when the cell mass
increase past a threshold level, and the segregation of the daughter chromosomes
is accomplished by ensuring that they find themselves on opposite sides of the
septum that grows to divide the bacterium into two. The agrobacterium growth
can be described in terms of the unit cell, an entity 1.7 μm long. A bacterium
contains one nucleus per unit cell, a rapidly growing cell with two nucleus is 1.7
to 3.4 μm long. The rate of bacterial growth is assessed by the doubling time, the
period required for the number of cell to double. The replication cycle can be
defined in terms of two constants.
a) C is the fixed times of 40 minutes required to replicate the entire bacterial
chromosome (at normal temperature).
b) D is fixed time of 20 minutes that elapses between the completion of a
round of replication and the cell division with which it is connected. This
period may represent the time required to assemble the component needed
for division

Picture 2. Growing cell alternates between cell division of a mother cell into two daughter and
growth back to the original size (left) and replication initiates at the bacterial origin when a cell
passes a critical threshold of size (right)
The division of a bacterium into two daughter cells is accomplished by the
formation of a septum, a structure that forms in the center of the cell as an
invagination from the surrounding envelope. The septum forms an impenetrable
barrier between two parts of the cell and provides the site at which the two
daughter cells eventually separate entirely. The annulus starts at a central position
in a new cell. As the cell grows, two event occur: a septum forms at the middle
cell position defined by the annulus, and new annuli from on either side of the
initial annulus. These new annuli are displaced from the center and move along
the cell positions at one quarter and three quarters of the cell length. These will
become mid-cell positions after the next division. The displacement of the
periseptal annulus to the correct position may be the crucial event that ensures the
division of the cell into daughter equal size.
 The behavior of the periseptal annulus suggest that the mechanism for
measuring position is associated with the cell envelope. It is possible to suppose
that the envelope could also be used to ensure segregation f the chromosomes. A
direct link between DNA and the membrane could account for segregation. If the
daughter chromosomes are attached to the membrane they could be physically
separated when the septum forms.

Picture 3. Attachment of bacterial DNA to membrane could provide mechanism for segregation
Plant Proliferation
According to Zeng (2013) cell division could be studied in plant tissue
materials in a proliferative state such as meristematic and zygotic tissues, in
undifferentiated cell clusters, protoplast suspensions and embryogenic cells.
Tumor cell proliferation mediated by Agrobacterium tumefaciens
andrhizogenes causes the development of a crown gall structure that could be
used to better understand mechanism underlying the cell cycle. In a similar way,
Rhizobium nodule and arbuscular mycorhizal infection site-derived symbiotic
association are also used as target candidates to investigate how extracellular
proteins might influence cell proliferation.
In both systems, stem cells and cancer or symbiotic cells share an
ability to divide continuously without undergoing senescence. In such
experimental systems, the environment is controllable and the population of target
cells should be as homogeneous as possible. Since these requirements are
difficult to meet when using intact plants as experimental systems, in vitro
plant cell cultures might be an alternative to meet such requirements. Within
available tissue culture systems, embryogenic cells were shown to be the most
suitable target material for plant genetic manipulation and gene transfer
technology. In fact, plant somatic cells have the ability to undergo sustained
divisions and give rise to an entire organism. This remarkable feature is called
plant cell totipotency. Somatic embryo is a notable illustration of plant totipotency
and involves reprogramming of development in somatic cells toward the
embryogenic pathway. Hence, embryogenic cell suspensions appear to provide a
valuable experimental tool for studying how cells acquire totipotency.
Agrobacterium tumefaciens causes crown gall disease on various plant
species by introducing its T-DNA into the genome. Therefore, Agrobacterium has
been extensively studied both as a pathogen and an important biotechnological
tool. The infection process involves the transfer of T-DNA and virulence proteins
into the plant cell. At that time the gene expression patterns of host plants differ
depending on the Agrobacterium strain, plant species and cell-type used. Later on,
integration of the T-DNA into the plant host genome, expression of the encoded
genes, and increase in phyto hormone levels induce a fundamental reprogramming
of the transformed cells. This results in their proliferation and finally formation of
plant tumors. The process of reprogramming is accompanied by altered gene
expression, morphology and metabolism.
Agrobacterium tumefaciens causes crown gall disease on a wide range of
host species by transferring and integrating a part of its own DNA, the T-DNA,
into the plant genome (Chilton et al., 1977). This unique mode of action has also
made the bac-terium an important tool in plant breeding. After attachment of
Agrobacterium to plant cells and expression of multiple viru-lence (vir) genes,
several effector proteins, together with T-DNA, are transported into the plant cell
by a type-IV-secretion system (Thompson et al., 1988; Ward et al., 1988, 2002;
Kuldau et al., 1990; Shirasu et al., 1990; Beijersbergen et al., 1994). Plant factors
assist with T-DNA integration into the plant genome (Gelvin, 2000; Mysore et al.,
2000; Tzfira et al., 2004; Magori and Citovsky, 2012). After integration,
expression of the T-DNA-encoded oncogenes iaaH, iaaM, and ipt induces
biosynthesis of auxin and cytokinin (Morris, 1986; Binns and Costantino, 1998).
Increased levels of these phytohormones result in enhanced proliferation and for-
mation of crown galls.
According to Gohike (2014) Agrobacterium tumefaciens causes crown
gall disease on a wide range of host species by transferring and integrating a part
ofits own DNA, the T-DNA, into the plant genom. This unique mode of action
has also made the bacterium an important tool in plant breeding. After attachment
of Agrobacterium to plant cells and expression of multiple virulence (vir) genes,
several effector proteins, together with T-DNA,are transported into the plant cell
by a type-IV-secretion system. Plant factors assist with T-DNA integration into
the plant genome. After integration, expression of the T-DNA-encoded oncogenes
iaaH, iaaM, and ipt induces biosynthesis of auxin and cytokinin. Increased levels
ofthese phytohormones result in enhanced proliferation and for-mation of crown
galls.
Earlier studies have shown that pre-treatment of explants with either auxin
alone or both auxin and cytokinin increase T-DNA transfer efficiency and stable
transformation (Krens et al., 1996; Chateau et al., 2000) as well as crown gall
growth (Gafni et al., 1995). In this respect, Agrobacterium produced
phytohormones play a role at very early time points of infection (Figure 1A),
before T-DNA-encoded enzymes catalyze synthesis of cytokinin and auxin in the
transformed host cell. Concerning the mecha-nism causing an increase in
susceptibility it was speculated that phytohormones induce plant cell division and
that the cell cycle phase influences agrobacterial attachment and stable
transformation. It seems likely that phytohormone-mediated modification of the
physiological state of the cell increases competence for T-DNA transformation
and integration. More recent investigations addressed the question about the
molecular mechanism and the signaling pathways by which these phytohormones
influence host cell susceptibility.
Through the traditional and novel experimental approaches, it was shown
that extracellular and intracellular signaling molecules play a critical role in the
regulation of cell division. Phytohormones are intracellular regulators, whose
significance for proliferation is of no doubt. It was found that phytohormones are
able to either directly determine the entry of cells into the cell cycle or operate
through a variety of regulatory proteins. As positive regulators of cell division,
auxins and cytokinins were studied in most details.
Like in all other eukaryotes, plant cell division includes the phases of
DNA replication and segregation: S phase (S) and mitosis (M). Between them
there are two gaps, G1 and G2: G1 is the interval time between M and S, whereas
G2 – between S and M. Inorder each daughter cell received similar set of the
inherited material, G1/S and G2/M transitions should be controlled. The main
regulators of these transitions are Ser/Thr cycling dependent proteinkinases
(CDK), which are activated by binding to the regulatory proteins cyclins (CYC).
In Arabidopsis, seven classes of CDK are distinguished (from A to G) on the basis
of sequence similarities in domains of CYC binding. In all eukaryotes, CDKA are
the main regulators of G1/S andG2/M transitions. CDKB kinases, detected only in
plants, bind CYC with aPPTA/TLRE motif and operate almost exclusively during
G2/M transition.
 CHAPTER III
CLOSING
3.1 Conclusion
Gene transformation with Agrobacterium is an efficient method for
obtaining transgenic plants. The advantages of transformation techniques using
Agrobacterium include 1). The efficiency of transformation with a single gene
copy is higher, 2). Can be done with simple laboratory equipment. The method of
transformation through Agrobacterium is different for each type of plant. This can
be done through the optimization of the method used by considering two things:
first, optimization of Agrobacterium and plant interactions in competent cells
from different tissues. Second, through the development of appropriate methods
for cell regeneration that play a role in transformation. The transformation of
Agrobacterium will continue to open up opportunities to increase crop production
both in terms of resistance to disease pests, drought, herbicides, and to increase
antioxidant content. cell division could be studied in plant tissue materials in
a proliferative state such as meristematic and zygotic tissues, in undifferentiated
cell clusters, protoplast suspensions and embryogenic cells. Tumor cell
proliferation mediated by Agrobacterium tumefaciens andrhizogenes causes
the development of a crown gall structure that could be used to better
understand mechanism underlying the cell cycle. In a similar way, Rhizobium
nodule and arbuscular mycorhizal infection site-derived symbiotic association
are also used as target candidates to investigate how extracellular proteins might
influence cell proliferation. In both systems, stem cells and cancer or
symbiotic cells share an ability to divide continuously without undergoing
senescence.
 REFERENCES
A.de la Riva, G., Joel, G.C., Roberto, V.P., Camilo, A.P. 1998. Agrobacterium
tumefaciens: a natural tool for plant transformation. Journal of
Biotechnology.Vol 1. No.3.
Gelvin, S.B. 2003. Agrobacterium-Mediated Plant Transformation: the
Biologybehind the “Gene-Jockeying” Tool. Rev.Microbiol and Mol
Biol. Vol 67.1:16-37.
Jochen Gohlke and Rosalia Deeken.2014.Plant responses to Agrobacterium
tumefaciens and crown gall development.Plant Science. 5(155):1-
11
Lewin, Benjamin.2008.Genes XI. Sudbury:Jones and Bartlett Publishers
Matthysse, Ann. 2006. The Genus Agrobacterium. 10.1007/0-387-30745-1_5.
Mooney, S.M., P.J.Lein, M.W.Miller. 2013. Neural Circuit Development and
Function in the Brain.Cambridge:Academic Press
Rahmawati, S.2006.Status Perkembangan Perbaikan Sifat Genetik Padi.Jurnal
AgroBiogen.vol 2.No1.
Tzfira T., Jianxiong L., Benoit L., and Vitaly C. 2004. Agrobacterium T-DNA
Integration: molecules and models. Rev. Trends in Genetic. Vol.20:
No.8.
Utomo, S. D. 2004. Pengaruh Strain Agrobacterium Terhadap Efisiensi
Transformasi Genetik Jagung Genotipe Hibrida HiII. Jurnal Ilmu
Pertanian. Vol 11 No. 2:1-10.
Walden, R., and Jeff, S. 1991.Tissue Culture and the Use of Transgenic Plants to
Study Plant Development. In Vitro Cell. Def. Biol. 27:1-10
Zeng, X., C. Zhang. 2013. Cell Proliferation: Processes, Regulation and
Disorders.Hauppauge: Nova Science
Ziemienowicz, Alicja. 2001. Odyssey of agrobacterium T-DNA. Acta biochimica
Polonica. 48. 623-35