Principles behind the procedures

PCR

PCR involves of exponential amplification of a DNA fragment, based on the mechanism of

DNA replication in vivo: dsDNA is denatured to ssDNA, duplicated, and this process is
repeated. It may involve a single or several copies of DNA across several orders of
magnitude, generating thousands to millions of copies of a particular DNA sequence. It is a
chain reaction. One DNA molecule used to produce two copies then four, then eight and so
forth. This continuous doubling is accomplished by specific proteins known as polymerases,
enzymes that are able to string together individual DNA building blocks to form long
molecular strands. Polymerases requires a supply of bases viz, A,T,G and C. Also need
fragments of DNA known as primers to which polymerase attach the building blocks as well
as a longer DNA molecule to as a template for constructing a new strand. The three major
steps involved in the PCR technique are denaturation, annealing and extension. During the
denaturation step, the dsDNA melts opening up to ssDNA, and all enzymatic reactions stop.
To DNA denaturation, the temperature is usually raised to 93-96 C, breaking the Hydrogen bonds and thus increasing the number of non-paired bases. The temperature at which half
of the dsDNA is single-stranded is known as the melting temperature, Tm. The concentration
of G/C and T/A can also change the Tm value. G/C-rich DNA sequences have higher Tm values
compared to those T/A-rich. The second phase, i.e. annealing of primers to ssDNA, takes
place at temperatures closer to their Tm. and is named as temperature of annealing. The
oligonucleotides used as primers typically consist of relatively short sequences (15-25 nt)
complementary to recognition sites, flanking the segment of target DNA to be amplified.
Once the temperature is reduced, the two complementary ssDNA chains tend to rehybridise
into a dsDNA molecule. In this phase, ionic bonds are constantly formed and broken
between the single-stranded primer and the single-stranded template. If primers adequately
anneal to the template, the ionic bond is strong enough between the template and the
primer to stabilise the nascent double stranded structure and allow the polymerase to
attach and begin copying the template. The extension phase is carried out across the target
sequence by using a heat-stable DNA polymerase in the presence of dNTPs and MgCl2,
resulting in a duplication of the starting target material. This enzyme has 5' to 3' DNA
polymerase activity, it adds dNTPs from 5' to 3' , reading the template from 3' to 5' . When
the primers have been extended a few bases, they possess a stronger ionic attraction to the
template, which reduces the probability of unbinding. The duration of the extension step
can be increased if the region of DNA to be amplified is long. After each cycle, the newly
synthesised DNA strands can serve as template in the next cycle. The major product of this
exponential reaction is a segment of ds-DNA whose termini are defined by the 5' termini of
the 2 primers and whose length is defined by the distance between the primers. The
products of a successful first round of amplification are heterogeneously sized DNA
molecules, whose lengths may exceed the distance between the binding sites of the two
primers. In the second round, these molecules generate DNA strands of defined length that
will accumulate in an exponential fashion in later rounds of amplification and will form the
dominant products of the reaction.

Cold stratification
Pre-treating seeds is a simple measure to break seed dormancy causing the seed to be more
ready to germinate. By subjecting the seeds to pre-treatment is in general providing natural
winter condition.
Plasmid isolation (alkali method)
Alkaline lysis is a methd used in molecular biology to isolate proteins by breaking the cell
open. Solution I ( Resuspension buffer) contains EDTA, Tris HCl and Glucose. Glucose is
required to make thesolution isotonic. EDTA chelates the divalent cations which are
released upon bacterial lysis. Divalent cations are required for many enzymatic reactions.
Tris HCl acts as a buffering agent. Lysis buffer contains SDS and NAOH (solution II). The
detergent cleaves the phospholipid bilayer of membrane and the alkali denatures the
protein invoved in maintaining the structure of cell membranes. Solution III (neutralization
buffer) contains potassium acetate which decreases the alkalinity of the solution. Under
these conditions the hydrogen bonding between the bases of the single stranded DNA can
be re-established, so the ssDNA can re-nature to dsDNA. This is the selective part. While it is
easy for the the small circular plasmid DNA to re-nature it is impossible to properly anneal
those huge gDNA stretches. This is why it's important to be gentle during the lysis step
because vigorous mixing or vortexing will shear the gDNA producing shorter stretches
that can re-anneal and contaminate your plasmid prep.
While the double-stranded plasmid can dissolve easily in solution, the single stranded
genomic DNA, the SDS and the denatured cellular proteins stick together through
hydrophobic interactions to form a white precipitate. The precipitate can easily be
separated from the plasmid DNA solution by centrifugation.
Now your plasmid DNA has been separated from the majority of the cell debris but is in a
solution containing lots of salt, EDTA, RNase and residual cellular proteins and debris, so it's
not much use for downstream applications. The next step is to clean up the solution and
concentrate the plasmid DNA.
There are several ways to do this including phenol/chloroform extraction followed by
ethanol precipitation.
Plasmids general theory
Plasmid is a double stranded, circular extra chromosomal DNA of bacterium. It is used in
recombinant DNA experiments to clone genes from other organisms and make large
quantities of their DNA. Plasmid can be transferred between same species or between
different species. Size of plasmids range from 1-1000 kilo base pairs. Plasmids are part of
mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages
and are associated with conjugation. Even the largest plasmids are considerably smaller
than the chromosomal DNA of the bacterium, which can contain several million base pairs.
The term 'plasmid' was introduced by an American molecular biologist Joshua Lederberg.
Plasmids are considered as transferrable genetic elements or 'replicons'. They are actually

naked DNA. Plasmids are important tools in genetic and biotechnology labs where they are
commonly used to multiply or express particular genes. Plasmids are also used to make
large amounts of proteins.
Based on function plasmids can be of five types:

F/Fertility plasmid for conjugation.

R/Resistant plasmid which contains genes that provides resistance to antibiotics. It

also helps bacteria in producing pilus.

Col plasmid which contain genes that code for bacteriocin (toxins produced by
bacteria to inhibit the growth of similar or closely related bacterial strains)

Degradative plasmid which help in the digestion of unusual substances like toluene.

Virulence plasmid which is responsible for pathogenicity.

Plasmid DNA may appear in one of the five conformations, which run at different speeds in
a gel during electrophoresis. The different plasmid conformations are listed below in the
order of electrophoretic mobility .

1. Nicked Open-Circular DNA ,which has one strand cut.

2. Relaxed Circular DNA is fully intact with both strands uncut, but has been enzymatically
relaxed.

3. Linear DNA has free ends, either because both strands have been cut, or because
the DNA was linear in vivo.
4. Super coiled (or Covalently Closed-Circular) DNA is fully intact with both strands
uncut, and with a twist built in, resulting in a compact form.
5. Super coiled Denatured DNA is like super coiled DNA, but has unpaired regions that
make it slightly less compact; this can result from excessive alkalinity during plasmid
preparation.

Restriction Digestion
Restriction enzymes are Nucleases which can cleave the sugar-phosphate backbone of
DNA, found in bacteria. As they cut within the molecule, they are commonly called
restriction endonucleases. They specifically cleave the nucleic acids at specific nucleotide
sequence called Restriction sites to generate a set of smaller fragments .Restriction
enzymes form part of the restriction-modification system of bacterial cells that provides
protection against invasion of the cell by foreign DNA especially bacteriophage DNA.
But the cells own DNA is not cleaved by these Restriction enzymes. This self protection is
achieved by the help of the specific DNA methyltransferase enzyme which will
methylates the specific DNA sequence for its respective restriction enzymes by
transferring methyl groups to adenine or cytosine residues to produce N6methyladenine or 5-methylcytosine. An interesting feature of restriction endonuclease

is that they commonly recognize recognition sequences that are mostly palindromes they shows the same forward (5' to 3' on the top strand) and backward (5' to 3' on the
bottom strand) sequences. In other words, they are nucleotide sequences or
complimentary strands that read the same in opposite direction.
Type I enzymes :- Type I restriction enzymes exhibit both restriction and DNA
modification activities.They require the cofactors such as Mg2+ ions, Sadenosylmethionine (SAM) and ATP for their activity. The recognition sequences are
quite long with no recognizable features such as symmetry. Type I restriction endo
nucleases cleaves DNA at nonspecific sites and that can be 1000 base pair or more from
recognition sequence. However, because the methylation reaction is performed by the
same enzyme which mediates cleavage, the target DNA may be modied before it is cut.
Because of these features, the type I systems are of little value for gene manipulation.
Eg EcoKI R

Type II enzymes :- Type II enzymes and their corresponding modification

methyltransferases act as separate proteins. They have a number of advantages over
type I and III systems. First, restriction and modication are mediated by separate
enzymes so it is possible to cleave DNA in the absence of modication. Secondly, the
restriction activities do not require cofactors such as ATP or S-adenosylmethionine,
making them easier to use. They require only Mg2+ ions as cofactors. These enzymes are
site-specific as they hydrolyze specific phosphodiester bonds in both DNA strands. Class
II restriction endonucleases are generally used as the key material in molecular biology
and recombinant DNA techniques, including genome mapping, RFLP analysis, DNA
sequencing, and cloning.
Eg. HindIII, HhaI, NotI, EcoRI, PstI ,BamhI, bglii, smai, xbha I, kpn I

Type III enzymes :- Like Class I enzymes, Type III enzymes possess both restriction and
modification activities.They recognize specific sequences and cleave 25 - 27 base
pairs outside of the recognition sequence, in a 3 direction. They require Mg2+ ions for
their activity. Eg - EcoPI

Type IIs enzymes, have similar cofactors and macromolecular structure to those of type
II systems, the fact that restriction occurs at a distance from the recognition site limits
their usefulness.
Eg FokI , AlwI
Class II restriction enzymes generate three types of DNA ends, all possessing 5-phosphate and 3-hydroxyl groups:
a) Cohesive 5 ends:- For example, ends generated by EcoR I

b) Cohesive 3 ends:- For example, ends generated by PstI

c) Blunt ends:- For example, ends generated by Hae III
Type iv
Type IV enzymes recognize modified, typically methylated DNA and are exemplified by
the McrBC and Mrr systems of E. coli
Eg
Type V
Type V restriction enzymes utilize guide RNAs to target specific non-palindromic sequences
found on invading organisms. They can cut DNA of variable length provided that a suitable
guide RNA is provided. The flexibility and ease of use of these enzymes make them
promising for future genetic engineering applications
Eg Cas9 in CRISPRS

Sticky ends (Blunt ends) are produced by cutting the DNA in a staggered manner within the
recognition site producing single stranded DNA ends. These ends have identical nucleotide
sequence and are sticky because they can bind to complementary tails of other
DNA fragments cut by the same Restriction enzyme.
Isoschizomers and neoschizomers: Different restriction enzymes, isolated from different
organisms can have identical recognition sequences, such enzymes are called isoschizomers.
Neoshizomers are Isoschizomeric enzymes but it cleaves at different recognition site.
Restriction enzymes are powerful tools of molecular genetics used to:
Map DNA molecules
Analyze population polymorphisms
Rearrange DNA molecules
Prepare molecular probes
Create mutants
Factors affecting Restriction Enzyme Activity:
Temperature: Most digestions are carried out at 37C. However, there are a few
exceptions e.g., digestion with Sma I is carried out at lower temperatures (~25C), while
with Taq I at higher temperature i.e., 65C.
Buffer Systems: Tris-HCl is the most commonly used buffering agent in incubation
mixtures, which is temperature dependent. Most restriction enzymes are active in the
pH range 7.0-8.0.

Ionic Conditions: Mg2+ is an absolute requirement for all restriction endonucleases, but
the requirement of other ions (Na+/K+) varies with different enzymes.
Methylation of DNA: Methylation of specific adenine or cytidine residues within the
recognition sequence of the restriction enzyme affects the digestion of DNA.
Star Activity is an alteration of the specificity of restriction enzyme mediated cleavage of
DNA that can occur under some non standard conditions that differ from the optimum
for the enzyme. This alteration leads to the cleavage at non specific sites.
Nonstandard conditions include:
1. High pH (>8.0).
2. Glycerol concentrations >5% (important, because enzymes are usually delivered as
concentrated stock in 50% glycerol).
3. High concentration of enzyme (>100 U/g of DNA).
4. Prolonged incubation time with enzyme.
5. Presence of organic solvents in the reaction (e.g., phenol, chloroform,
ethanol,DMSO).
6. Incorrect cofactor (i.e., Mn2+,Hg2+or Co2+instead of Mg2+)
To avoid star activity, always use the optimal buffer system and enzyme amount
recommended. Make sure that the DNA preparation is free of organic solvents and
contaminating salts.
Gateway cloning
Gateway recombination cloning technology circumvents traditional restriction enzyme
based cloning limitations, enabling you to access virtually any expression system in just a
few simple steps. From protein expression to functional analysis, Gateway cloning
technology is applicable for a variety of research areas, for truly multidisciplinary scientific
studies
Gateway cloning essentials are
Entry clones and vectors
Donor vectors
Recombination enzymes
Destination vectors
Multisite cloning
An Entry clone contains your gene of interest flanked by attL sequences, which are then
used to recombine with attR sequences to create your desired expression clone. There are
three methods you can use to produce an Entry clone: BP cloning, restriction enzyme and

ligase cloning, and TOPO cloning into a Gateway Entry vector, which is the most common
method. Single gateway refers to the method to produce an entry clone using BP cloning.
As explained, the core of Gateway cloning is the Entry vector. Once the Entry clone is ready,
the gene of interested is easily shuttled to a secondary plasmid, the Destination vector. This
reaction is mediated by a robust enzyme mixture called LR Clonase, which contains the
necessary protein activity to excise the gene of interest from the Entry clone and integrate it
into the Destination vector, which then becomes your expression clone. Reversing this
reaction simply entails performing a BP reaction with BP Clonase enzyme mix. Both LR and
BP Clonase enzyme mixtures are easy-to-use master mix formats ensuring consistency and
reliability from reaction to reaction.
Types of Clonase Enzymes
Gateway BP Clonase Enzyme Mixtures
Gateway BP Clonase enzyme contains both Int (Integrase) and IHF (Integration Host Factor)
proteins that catalyze the in vitro recombination of PCR products or DNA segments from
clones (containing attB sites) and a Donor vector (containing attP sites) to generate Entry
clones.
Gateway LR Clonase Enzyme Mixes

Gateway LR Clonase enzyme mix contains a proprietary blend of Int (Integrase), IHF
(Integration Host Factor) and Xis (Excisionase) enzymes that catalyze the in vitro
recombination between an Entry clone (containing a gene of interest flanked by attL sites)
and a Destination vector (containing attR sites) to generate your expression clone.
Multi Gateway and Multi Gateway Pro are extensions of the Gateway site-specific
recombinational cloning technology, which is based on the recombination properties of
bacteriophage lambda . MultiSite Gateway allows cloning of exactly three DNA fragments
into pDEST , while MultiSite Gateway Pro allows cloning of two, three, or four DNA
fragments in a defined order and orientation into any pDEST vector containing attR1 and
attR2 sites. Both systems provide a rapid and efficient way to recombine DNA elements into
vector systems for functional analysis and protein expression. The LR recombination
reaction occurs between two specific attachment sites (attL and attR) on the entry clones
and the destination vector, allowing the recombination of fragments into the destination
vector. The reaction is mediated by Gateway LR Clonase II Plus enzyme mix.

Transformation by heat shock

Bacterial transformation is a widely used method where foreign DNA is introduced into a
bacterium, which can then amplify, or clone the DNA. Cells that have the ability to readily
take up this DNA are called competent cells. Although transformation is naturally occurring
in many types of bacteria, scientists have found ways to artificially induce and enhance a
bacterial cells competency. Transformation can occur in nature in certain types of bacteria.
In molecular biology, transformation is artificially reproduced in the lab via the creation of
pores in bacterial cell membranes. Bacterial cells that are able to take up DNA from the
environment are called competent cells. In the laboratory, bacterial cells can be made
competent and DNA subsequently introduced by a procedure called the heat shock method.
Heat shock transformation uses a calcium rich environment provided by calcium chloride to
counteract the electrostatic repulsion between the plasmid DNA and bacterial cellular
membrane. A sudden increase in temperature creates pores in the plasma membrane of the
bacteria and allows for plasmid DNA to enter the bacterial cell. Placing the cells on ice after
the shock closes the pores and prevent the plasmid to escape.
The negative charges of the incoming DNA, however, are repelled by the negatively charged
portions of the macromolecules on the bacteriums outer surface. The addition of
CaCl2 serves to neutralize the unfavorable interactions between the DNA and the polyanions
of the outer layer. The DNA and competent cells are further incubated on ice for thirty
minutes to stabilize the lipid membrane and allow for increased interactions between
calcium ions and the negative components of the cell. The reaction mixture is then exposed
to a brief period of heat-shock at 42oC. The change in temperature alters the fluidity of the
semi-crystalline membrane state achieved at 0oC thus allowing the DNA molecule to enter
the cell through the zone of adhesion
DH5 strain of E.Coli
DH5a is the most frequently used E.Coli strain for routine cloning applications. In addition to
supporting blue/white screening recA1 and endA1 mutations in DH5a increase insert
stability and improve the quality of plasmid DNA prepared from minipreps.
Application:
Highest transformation efficiency
General cloning
Blue-white selection
Plasmid isolation
Genotype:
F- 80dlacZ M15 (lacZYA-argF) U169 recA1 endA1hsdR17(rk-, mk+) phoAsupE44 -thi-1 gyrA96
relA1
Transformation by electrophoresis

Electroporation makes use of a specialized device called an electroporator. Typically cells

are placed into an electroporation cuvette, which has electrodes on each side that make
electrical contact with the machine once inserted. Bacterial cells mixed with DNA are loaded
into the electroporation cuvette and an electric field on the order a 1000 to 10,000 volts per
centimeter is applied for a few milliseconds. This causes the voltage across the membrane
to reach 0.5-1 volts, which is believed to lead to a rearrangement of the phospolipid bilayer
that comprises the cell membrane such that pores will form. In this state plasmid DNA will
pass through the membrane and when pulsing is complete the bilayer will repair itself.
Having taken up the plasmid, bacteria can then grow on agar plates containing antibiotic.
Bacteria that are prepared for electroporation are referred to as electrocompetent cells.
Unsuccessful pulsing causes an electrical discharge, which is observable as a visible spark
and audible pop. This discharge, referred to as arcing, can be the result of having too much
salt in your competent cells or DNA. The success of your transformation can be predicted by
noting the time constant, which is the duration it takes for the voltage to decay after
applying the pulse. When salt is present and the electroporation solution is very conductive,
the decay happens rapidly, causing the discharge, and thereby killing many of your cells. For
bacteria, good time constants range from 5-10 milliseconds.
C58 strain of agarobacterium
A. tumefaciens is an unusual bacteria because is one of the few that has both a linear and a
circular chromosome. Its genome has a total of 5.7 million base-pairs, with 2.8 million
residing on its circular chromosome and 2.1 million residing on its linear chromosome. Most
of the genes essential for its survival are located on the circular chromosome, although
through evolution some essential genes have migrated to the linear chromosome. Based on
sequence analysis, it was determined that the linear chromosome was derived from a
plasmid that was transformed into the bacteria a long time ago
The ends of the linear chromosomes are protected by a telomere that forms a covalently
closed hairpin, like in other bacteria which contain a linear chromosome . In addition to the
two chromosomes, strain C58 also contain two plasmids, pTiC58 (generically called Ti) and
pAtC58 (also called the "cryptic plasmid"). pTiC58 contains genes necessary for its
pathogenicity against plants , including the T-DNA which is injected into the plant and
causes it to produce opines, along with accessory proteins which helps the T-DNA enter and
transform the plant cell into a tumor cell. It is believed that pAtC58 contains genes essential
for opine catabolism , or its ability to use opines as an energy source, which is important for
its lifestyle as a pathogen.
pCAMBIA Vector
The pCAMBIA vector backbone is derived from the pPZP vectors. While not perfect and
having technical and IP limitations, pCAMBIA vectors offer:
high copy number in E.coli for high DNA yields
pVS1 replicon for high stability in Agrobacterium

small size, 7-12kb depending on which plasmid

restriction sites designed for modular plasmid modifications and small but adequate polylinkers for introducing your DNA of interest
bacterial selection with chloramphenicol or kanamycin
plant selection with hygromycin B or kanamycin
simple means to construct translational fusions to gusA reporter genes
Agaro bacterium mediated Plant transformation

The general infection mechanism in nature begins with Agrobacterium sensing small
sugars and plant metabolites that are often leached from a plant wound.
Agrobacterium responds by expressing several genes. The genes are harbored on a
special plasmid called a Ti plasmid, also known as a tumor-inducing plasmid. The
extrachromosomal plasmid genes encode for proteins that help in transferring bacterial
DNA to the plant. The transfer DNA (T-DNA) itself codes for genes responsible for
inducing the plant to manufacture plant hormones, or phytohormones. Thus, the T-DNA
is indeed transferred to the plant cell nucleus. The hormones include auxins and
cytokinins and result in a gall (or tumor) around the infection site. The fact that some
genes cause a tumor has led researchers to refer to them as oncogenes. Agrobacterium
has been thought to take up residency within the gall caused by oncogene products.
Other metabolites are produced in the plant including pines, which can serve as a
nitrogen, carbon, and energy source for Agrobacterium.
Plant biotechnologists have taken advantage of Agrobacterium, after years
of studying how the T-DNA moves from bacterium to host.7
The Ti plasmid is a rather large plasmid at more than 200,000 bases and plays a central
role in facilitating the DNA transfer. Some of the genes on the Ti plasmid encode for a
small molecular syringe that helps deliver the T-DNA. What plant molecular biologists
figured out was not only that the T-DNA delivered causes the crown gall, but that parts
of the T-DNA could be removed and replaced with other DNA that did not cause tumors.
The biological origin of the newly inserted DNA did not matter as much as the placement
of the T-DNA on the Ti plasmid. That is to say, the T-DNA always needed to be put in the
same position on the Ti plasmid in order to be successfully transferred to the plant.
Researchers later learned that the T-DNA on the Ti plasmid is flanked by inverted
nucleotide base repeats. Agrobacterium virulence proteins responsible for helping to
excise the T-DNA recognize the inverted sequence as cut sites. No matter what DNA
sequence was inserted in between the inverted sequences, the T-DNA region was always
excised and transferred.
For decreasing the size of plasmid the inverted repeats could be placed onto a second,
smaller plasmid, and one other than the Ti plasmid. They termed the second plasmid a

binary plasmid. The plasmid also came to be known as a binary vector since it was
involved in harboring foreign DNA and would also be present in Agrobacterium with the
Ti plasmid. Although the modified Ti plasmid lacked the inverted repeats and the tumorcausing genes responsible for the phytohormone production, the crucial virulence genes
for foreign DNA delivery were retained. This is why the Ti plasmid is now also known
as a "helper" plasmid. Subsequently, Agrobacterium strains used in labs are now said to
be "disarmed" since the modified Ti plasmid lacks tumor-inducing capabilities.
Disarmed Agrobacterium was then able to be transformed with binary vectors carrying
new pieces of DNA. Crucial to selecting transgenic plants that received a copy of the TDNA was the need to also transform the plant with a selectable marker, a gene whose
protein product confers a selective advantage in the presence of a selective agent (e.g.
herbicide or antibiotic). Thus, in tandem with a gene of interest on the binary vector is a
selectable marker.
1. Binary vector: the vir genes required for mobilization and transfer to the plant reside
on a modified pTi.
2. consists of the right and left border sequences, a selectable marker (kanomycin
resistance) and a polylinker for insertion of a foreign gene.

Floral dip method

Of worthwhile mention is a second indirect transfer method of plant genetic
modification called the floral dip. In this method, immature seeds (ovules) residing in
the ovary of the flower are directly exposed to Agrobacterium carrying a binary vector
with a gene of interest. The preparation of Agrobacterium cells involves overnight
culture , centrifugation of cells, and resuspending cells to an appropriate density in a
sucrose solution. A small amount of detergent (Tween20) is also added to help the
Agrobacterium cells make contact with the ovules. Once agrobacterium is prepared and

the correct density is obtained, the plant is literally dipped into the Agrobacterium
suspension.
After dipping, excess Agrobacterium solution is removed by gently shaking the plant.
The plant is then placed in a plastic bag or other container to keep humidity high, which
is thought to extend the life of Agrobacterium and thus, increase potential ovule
infections. After no more than 24 hours, the plants are removed and allowed to recover
under normal growing conditions. The fruits are allowed to ripen and the mature seeds
are subsequently harvested, sterilized with a series of ethanol washes, and sown on
selective media. The plates are given a cold treatment (~5C) in the dark for 48-72 hours,
which is called stratification. The subsequent removal from cold treatment and
placement under continuous light encourages all seeds to germinate at the same time.
While nearly all seeds germinate, only those with the selective marker persist and
develop green leaves with long roots. The main advantage of the floral dip rests with the
ability to allow Agrobacterium to deliver foreign DNA directly to the ovules. Such a
technique allows one to completely avoid the time consuming and meticulous work that
tissue culture requires. Additionally, although the method is called "floral dip" one does
not have to completely submerge the flowers. One can also use a spray solution, a
syringe, or pipette to apply Agrobacterium cells. Similarly, entire plants do not have to
be placed in a plastic bag, only the flowers where Agrobacterium was applied.