Biotransformation by Dr. Jayarama Reddy, St. Joseph's College, Bengaluru-27

Agrobacterium Mediated
Biotransformation
13-01-2022
Dr. Jayarama Reddy, Professor, St. Joseph's College, Bengaluru-27.
1
Dr. Jayarama Reddy,
Professor,
St. Joseph's College,
Bengaluru-27.

Biotransformation
13-01-2022
Dr. Jayarama Reddy, Professor, St. Joseph's College, Bengaluru-27.
2

Methods of delivering DNA into plant cells
•Biological
•Agrobacterium
•Other bacteria
•Viruses
•Physical
•Particle bombardment
•Electroporation
•Silicon carbide whiskers
•Carbon nanofibers
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Dr. Jayarama Reddy, Professor, St. Joseph's College, Bengaluru-
27.
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Transformation overview
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Biotransformation
Agrobacterium-mediated transformation (AMT)
heavily relies on the capability of bacterial
pathogen Agrobacterium tumefaciens in
transferring foreign genes into a wide variety of
host plants. Currently, AMT is the most
commonly used method for generating
transgenic plants.
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Biotransformation
Agrobacterium tumefaciens is a soil phytopathogen that
naturally infects plant wound sites and causes crown gall
disease via delivery of transferred (T)-DNA from bacterial
cells into host plant cells through a bacterial type IV
secretion system (T4SS). Through the advancement and
innovation of molecular biology technology during the
past few decades, various important bacterial and plant
genes involved in tumorigenesis were identified.
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Biotransformation
With the help of more comprehensive
knowledge of how A. tumefaciens interacts with
host cells, A. tumefaciens has become the most
popular plant transformation tool to date. Any
gene of interest can now easily be used to
replace the oncogenes in the T-DNA region of
various types of binary vectors to perform plant
genetic transformation with A. tumefaciens.
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Binary vector system
• The binary cloning vector contains either the E. coli and A. tumefaciens origins of DNA
replication or a single broadhost range origin of DNA replication.
• All the cloning steps are carried out in E. coli before the vector is introduced into A.
tumefaciens.
• The recipient A. tumefaciens strain carries a modified (defective or disarmed) Ti plasmid that
contains a complete set of vir genes but lacks the T-DNA region, so that this T-DNA cannot be
transferred.
• With this system, the defective Ti plasmid synthesizes the vir gene products and acts as a
helper plasmid.
• This enables the T-DNA from the binary cloning vector to be inserted into the plant
chromosomal DNA.
• Since transfer of the T-DNA is initiated from the right border, the selectable marker, is usually
placed next to the left border.
• A few binary vectors have been designed to include two plant selectable markers, one
adjacent to the right border and the other adjacent to the left border.
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Binary vector system
A binary vector is a standard tool in the
transformation of higher plants mediated by
Agrobacterium tumefaciens. It is composed of the
borders of T-DNA, multiple cloning sites,
replication functions for Escherichia coli and A.
tumefaciens, selectable marker genes, reporter
genes, and other accessory elements that can
improve the efficiency of and/or give further
capability to the system.
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Co-integrate vector system
• The cointegrate vector has a plant selectable marker gene,
the target gene, the right border, an E. coli origin of DNA
replication, and a bacterial selectable marker gene.
• The cointegrate vector recombines with a modified
(disarmed) Ti plasmid that lacks both the tumor-producing
genes and the right border of the T-DNA within A.
tumefaciens.
• The entire cloning vector becomes integrated into the
disarmed Ti plasmid to form a recombinant Ti plasmid.
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Domain: Bacteria
Phylum: Proteobacteria
Class: Alphaproteobacteria
Order: Hyphomicrobiales
Family: Rhizobiaceae
Genus: Agrobacterium
Species: A. radiobacter
Bacillus radiobacter Beijerinck and van Delden 1902
Bacterium tumefaciens Smith and Townsend 1907
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A. tumefaciens Causal Agent of Crown Gall Disease
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A. tumefaciens.
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Discovery
Marc Van Montagu and Jeff Schell, (University
of Ghent and Plant Genetic
Systems, Belgium) discovered the gene
transfer mechanism in 1977.
Between Agrobacterium and plants resulted in
the development of methods to alter the
bacterium into an efficient delivery system
for genetic engineering in plants.
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Marc Van Montagu Jeff Schell
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Agrobacterium tumefaciens
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Agrobacterium tumefaciens (also sometimes known
as Rhizobium radiobacter ) is an aerobic, gram-
negative, motile, rod-shaped bacterium with one to
six flagella. It is about 1.5-3.0 x 0.6-1.0 µm in size and
causes the formation of galls on plant hosts.
This bacterium is closely related to nitrogen-fixing
bacteria that can be found on root nodules in
legumes. Rhizobium radiobacter has also been used
for the genetic modification of different organisms
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The first symptom plants commonly exhibit when
infected by Agrobacterium tumefaciens is the
presence of tumorous overgrowths (galls) on the
roots, stem (trunk), and crown at or below the soil
surface. Initially, these galls are round, white or flesh
colored, and soft and spongy. In later stages, the
emerging gall develops into a woody interior with an
irregular, rough, corky surface. This outer tissue
darkens (8). The gall color will range from light brown
to black depending on age and host.
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Agrobacterium tumefaciens (Gram –ve) is
a soil bacterium that has the ability to infect
plant cells and transfer a defined sequence of
their DNA to the plant cell by infection and a
causative agent of crown gall disease.
Agrobacterium tumefaciens cell contains a
plasmid known as the Ti (tumor-inducing)
plasmid (140–235 kb).
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Agrobacterium tumefaciens incites plant
tumors that produce nutrients called opines,
which are utilized by the bacteria during host
colonization. Various opines provide sources of
carbon, nitrogen, and phosphorous, but virtually
nothing was previously known about how A.
tumefaciens acquires sulfur during colonization.
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Key steps from natural Agrobacterium to “useful”
Agrobacterium
•Some vir genes deleted--disarmed
•Opines not going to be produced
•Deleting tumorogenesis function
•Choosing strains that transfer DNA in lab
•Clone in genes of interest, antibiotic resistance
genes, etc.
•Binary system-- two plasmids are better than one Ti
plasmid
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A tumour inducing
(Ti) plasmid is a
plasmid found in
pathogenic species
of Agrobacterium
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A tumour inducing (Ti) plasmid is a plasmid found in
pathogenic species of Agrobacterium, including A.
tumefaciens, A. rhizogenes, A. rubi and A. vitis.
Evolutionarily, the Ti plasmid is part of a family of
plasmids carried by many species
of Alphaproteobacteria. Members of this plasmid
family are defined by the presence of a conserved DNA
region known as the repABC gene cassette, which
mediates the replication of the plasmid, the
partitioning of the plasmid into daughter cells
during cell division as well as the maintenance of the
plasmid at low copy numbers in a cell
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The Ti plasmids themselves are sorted into different
categories based on the type of molecule, or opine,
they allow the bacteria to break down as an energy
source. The presence of this Ti plasmid is essential
for the bacteria to cause crown gall disease in
plants. This is facilitated via certain crucial regions in
the Ti plasmid, including the vir region, which
encodes for virulence genes, and the transfer
DNA (T-DNA) region, which is a section of the Ti
plasmid that is transferred via conjugation into host
plant cells after an injury site is sensed by the
bacteria.
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These regions have features that allow the delivery
of T-DNA into host plant cells, and can modify the
host plant cell to cause the synthesis of molecules
like plant hormones (e.g. auxins, cytokinins) and
opines and the formation of crown gall tumours.
Because the T-DNA region of the Ti plasmid can be
transferred from bacteria to plant cells, it
represented an exciting avenue for the transfer of
DNA between kingdoms and spurred large amounts
of research on the Ti plasmid and its possible uses in
bioengineering.
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Agrobacterium contains a tumour-
inducing (Ti) plasmid, which includes
virulence (vir) genes and a transferred-
DNA (T-DNA) region. Genes of interest
can be inserted into the T-DNA.
Wounded plant cells produce phenolic
defence compounds, which can trigger
the expression of the Agrobacterium vir
genes. The encoded virulence (Vir)
proteins process the T-DNA region from
the Ti-plasmid, producing a 'T-strand'.
After the bacterium attaches to a plant
cell, the T-strand and several types of Vir
proteins are transferred to the plant
through a transport channel. Inside the
plant cell, the Vir proteins interact with
the T-strand, forming a T-complex. This
complex targets the nucleus, allowing
the T-DNA to integrate into the plant
genome and express the encoded genes.
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Agroinfiltration—forcing Agrobacterium with
transgenes into leaves
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Agroinfiltration--tobacco
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Stable transformation using Agrobacterium
•Floral dip transformation of Arabidopsis
•Seems to transform ovule
•Not easily conducive for other species
•Most species: using organogenesis or
embryogenesis-based tissue culture
methods to regenerate transgenic plants
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Floral dip: a simplified method for
Agrobacterium-mediated
transformation of Arabidopsis
thaliana. Floral dip method has
been used in genetic engineering
strategies as it directly produces
genetically modified seeds
bypassing the laborious tissue
culturing procedures. In addition,
tissue culture could generate
transformed plants harboring
undesirable mutations from
somaclonal variation
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Limitations as routine Ti plasmid vectors
• The production of phytohormones by transformed cells prevents them
from being regenerated into mature plants.
• A gene encoding opine synthesis is not useful to a transgenic plant and
may lower the final plant yield
• Ti plasmids are large (approximately 200 to 800 kb).
• Ti plasmid does not replicate in Escherichia coli, therefore it cannot be
cloned in E. coli.
• Transfer of the T-DNA, which begins from the right border, does not
always end at the left border. Rather, vector DNA sequences past the left
border are often transferred.
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Ti plasmid-based vectors: Components
• A selectable marker gene, such as neomycin phosphotransferase, put
under the control of plant (eukaryotic) transcriptional regulation signals,
including both a promoter and a termination–polyadenylation sequence.
• An origin of DNA replication that allows the plasmid to replicate in E. coli .
• The right border sequence of the T-DNA region. Most cloning vectors
include both a right and a left border sequence.
• A polylinker to facilitate insertion of the cloned gene into the region
between T-DNA border sequences.
• A “killer” gene encoding a toxin downstream from the left border to
prevent unwanted vector DNA past the left border from being
incorporated into transgenic plants. If this incorporation occurs, and the
killer gene is present, the transformed cells will not survive.
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A. rhizogenes, the causative agent of hairy root
syndrome, is a common soil bacterium (Gram negative)
capable of entering a plant through a wound and causing
a proliferation of secondary roots. The underlying
mechanism of hairy root formation is the transfer of
several bacterial genes to the plant genome. The observed
morphogenic effects in the plants after infection have
been attributed to the transfer of part of a large plasmid
known as the Ri (root-inducing) plasmid. The symptoms
observed with A. rhizogenes are suggestive of auxin
effects resulting from an increase in cellular auxin
sensitivity rather than auxin production.
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Ri plasmids
Ri plasmids are large (200 to greater than 800 kb) and contain one or
two regions of T-DNA and a vir (virulence) region, all of which are
necessary for tumorgenesis. The Ri-plasmids are grouped into two
main classes according to the opines synthesized by hairy roots. First,
agropine-type strains induce roots to synthesise agropine, mannopine
and the related acids. Second, mannopine-type strains induce roots to
produce mannopine and the corresponding acids. The agropine-type
Ri-plasmids are very similar as a group and a quite distinct group from
the mannopine-type plasmids. Perhaps the most studied Ri-plasmids
are agropine-type strains, which are considered to be the most
virulent and therefore more often used in the establishment of hairy
root cultures.
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Ri plasmids Genes
The T-DNA of the agropine-type Ri-plasmid consists of two
separate T-DNA regions designed the TL-DNA and TR-DNA. Each
of the T-DNA fragments spans a 15 - 20 kb region, and they are
separated from each other by at least 15 kb of non-integrated
plasmid DNA. These two fragments can be transferred
independently during the infection process. The genes encoding
auxin synthesis (tms1 and tms2) and agropine synthesis (ags)
have been localised on the TR-DNA of the agropine type Ri-
plasmid. The mannopine type Ri-plasmids contain only one T-
DNA that shares considerable DNA sequence homology with TL of
the agropine-type plasmids.
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Mutation analysis of the TL-DNA has led to
identification of four genetic loci, designed
locus rolA, rolB, rolC, and rolD, which affect
hairy root induction. The complete
nucleotide sequence of the TL-region
revealed the presence of 18 open-reading
frames (ORFs), 4 of which, ORFs 10, 11, 12
and 15, respectively, correspond to the rolA,
rolB, rolC, and rolD loci.
Mutations in Ri plasmids Genes
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Mechanism of Action
One of the earliest stages in the interaction between
Agrobacterium and a plant is the attachment of the
bacterium to the surface of the plant cell. A plant cell
becomes susceptible to Agrobacterium when it is
wounded. The wounded cells release phenolic
compounds, such as acetosyringone, that activate the
vir-region of the bacterial plasmid. It has been shown
that the Agrobacterium plasmid carries three genetic
components that are required for plant cell
transformation.
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It has been shown that the Agrobacterium plasmid carries three
genetic components that are required for plant cell
transformation. The first component, the T-DNA that is integrated
into the plant cells, is a mobile DNA element. The second one is
the virulence area (vir), which contains several vir genes. These
genes do not enter the plant cell but, together with the
chromosomal DNA (two loci), cause the transfer of T-DNA. The
third component, the so-called border sequences (25 bp), resides
in the Agrobacterium chromosome. The mobility of T-DNA is
largely determined by these sequences, and they are the only cis
elements necessary for direct T-DNA processing.
Mechanism of Action
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Mechanism of Action
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Hairy roots are fast growing and laterally highly branched, and are
able to grow in hormone-free medium. Moreover, these organs are not
susceptible to geotropism anymore. They are genetically stable and
produce high contents of secondary metabolites characteristic to the host
plant. The secondary metabolite production of hairy roots is stable
compared to other types of plant cell culture. The alkaloid production of
hairy roots cultures has been reported to remain stable for years. The
secondary metabolite production of hairy roots is highly linked to cell
differentiation. Alkaloid production decreased clearly when roots were
induced to form callus, and reappeared when the roots were allowed to
redifferentiate. An interesting characteristic of some hairy roots is their
ability to occasionally excrete the secondary metabolites into the growth
medium. However, the extent of secondary product release in hairy root
cultures varies among plant species.
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The average growth rate of hairy roots varies from 0.1 to 2.0 g dry
weight/liter/day. This growth rate exceeds that of virtually all-conventional
roots and is comparable with that of suspension cultures. However, the
greatest advantage of hairy roots compared to conventional roots is their
ability to form several new growing points and, consequently, lateral
branches. The growth rate of hairy roots may vary greatly between species,
but differences are also observed between different root clones of the
same species. The pattern of growth and secondary metabolite production
of hairy root cultures can also vary. Secondary production of the hairy
roots of Nicotiana rustica L. was strictly related to the growth, whereas
hairy roots of Beta vulgaris L. exhibited non-growth-related product
accumulation. In the case of the hairy roots of Scopolia japonica Jacq. and
H. muticus, the secondary products only started to accumulate after
growth had ceased. Secondary metabolite synthesis dissociated from
growth would be desirable for commercial production, as it would allow
the use of continuous systems.
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1. Induction of hairy roots via A. rhizogenes Infection:
Surface-sterilized cotyledons are wounded and infected with A.
rhizogenes strains. The inoculated cotyledons are co-cultivated with A.
rhizogenes strains for 2 days at 25C with a 16 hours photoperiod. The
experiment is designed to be completely randomized with four
replicates. Forty explants are used for each population. After co-
cultivation, explants are transferred to hormone-free growth
mediums (High salt media such as MS favors hairy root formation in
some plants. Low salt media such as B5 favor excessive bacterial
multiplication in the medium and therefore the explant needs to be
transferred several times to fresh antibiotic containing medium
before incubation.), semi-solid MS (Murashige and Skoog) medium
solidified with 0.8% agar, and contained 3% sucrose, plus 0.4g/l
augmentin to kill the bacteria (pH: 5.7) at a density of 10 explants per
plate (9 cm petri dish), and cultured at 25C, with a 16 hr photoperiod.
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Frequency of hairy root formation for each treatment is
scored 30 days after co-cultivation. Individual roots will
emerge from the wound sites, they are excised and sub-
cultured onto the same medium. Forty days after co-
cultivation, hairy roots are weighed out and transferred to
50 ml of MS liquid medium (pH= 5.7) containing 3%
sucrose, and shaken in an orbital shaker at 120 rev/min at
25℃ in the dark. The roots are then sub-cultured onto the
same medium every 4 weeks. After 4 months in liquid
culture, hairy roots from each explant are weighed out
and the mean weight for each treatment iss calculated.
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Culture Conditions
1). The susceptibility of plant species to Agrobacterium
strains varies greatly. Significant differences were
observed between the transformation ability of different
strains of Agrobacterium.
2).The age and differentiation status of plant tissue can
affect the chances of successful transformation.
3). The level of tissue differentiation determines the ability
to give rise to transformed roots after A. rhizogenes
inoculation. In this case, successful infection of some
species can be achieved by the addition of acetosyringone.
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Detection of Transformation
1) Determination of Anthraquinone Contents; how much opine
produced in hairy roots.
Hairy roots were dried in the dark at 60℃ for 2 days. Dried roots
were powdered by mortar and pestle, and 50 mg of this fine powder
was then soaked in 50 ml of distilled water for 16 h. This suspension
was heated in water bath at 70℃ for 1 h. After the suspension was
cooled, 50 ml of 50% methanol (MeOH) was added and then filtrated.
The clear solution was measured by spectrophotometer (Shimadzu
UV-160A) at a wave length of 450 nm and compared with a standard
solution containing 1mg/100ml alizarin and 1 mg/100ml purpurin
with the absorption-maximum 450 nm.
However there is a disadvantage that opines production can be
unstable in hairy roots and may disappear after a few passages.
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2) T-DNA detection by southern blot hybridization
Genomic DNA from transformed and non-transformed soil-grown plants
were extracted using the CTAB extraction method. Approximately 10 mg of
DNA from each sample were digested with HindIII, BamHI, EcoRI,
respectively, non-transformed plant was digested with EcoRI, then
separated by electrophoresis 0.8% (w/v) agarose gel, transferred from the
agarose gel to Hybond+ nylon membrane and cross-linked to the
membrane by UV light for 3 min. Probe were labeled with a 32P labeled
probe specific to the coding sequence of the introduced rolB, or rolA, or
rolC gene for Southern hybridization. Filters were pre-hybridized in 5 ¡ֱ SSC,
5 ¡ֱ Denhardt's solution, 0.5% SDS, 20 mg/ml denatured salmon sperm DNA
at 65oC and subsequently hybridized overnight with labeled probe. After
stringent washing (0.1 ¡ֱSSC, 0.1% SDS, 65oC) filters were autoradiographed
at -70oC for 3 days with an intensifying screen.
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3) Bacterial gene detection by PCR
The polymerase chain reaction was used to confirm the
presence of rolB, or rolA, or rolC gene in roots by their primers.
The PCR reactions were carried out in a total volume of 30 ml:
1 ml samples of the transformed plant genomic DNA, 20 pmol
of each primer, 200 m M each dNTP, 0.5 units Taq DNA
polymerase and 3 ml 10 ¡ֱ PCR buffer. Cycling conditions were:
denaturation at 94oC for 1 min, annealing at 55 oC for 1 min
and extension at 72 oC for 3 min. Samples were subjected to
30 cycles. Amplification products were analyzed by
electrophoresis on 0.8% agarose gels and detected straining
with ethidium bromide.
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Advantages of Hairy root Cultures
Hairy roots also offer a valuable source of root derived
phytochemicals that are useful as pharmaceuticals, cosmetics,
and food additives. These roots can also synthesize more than a
single metabolite and therefore prove economical for commercial
production purposes. Transformed roots of many plant species
have been widely studied for the in vitro production of secondary
metabolites. Transformed root lines can be a promising source for
the constant and standardized production of secondary
metabolites. Hairy root cultures produce secondary metabolites
over successive generations without losing genetic or
biosynthetic stability. This property can be utilized by genetic
manipulations to increase biosynthetic capacity.
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Biotransformation by Dr. Jayarama Reddy, St. Joseph's College, Bengaluru-27

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