Principles Behind the Procedures

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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 rehybridiseinto 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.

Underthese 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 beseparated 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 ofDNA, 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 N6-

methyladenine 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 DNAmodification activities.They require the cofactors such as Mg2+ ions, S-

adenosylmethionine (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 modified 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 modification are mediated by separate

enzymes so it is possible to cleave DNA in the absence of modification. 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 biologyand 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´-phos-

phate 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 bythe 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 37°C. However, there are a few

exceptions e.g., digestion with Sma I is carried out at lower temperatures (~25°C), while

with Taq I at higher temperature i.e., 65°C.

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/µg 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 arethree 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 thenecessary 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 abacterial cell’s 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 afterthe 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 bacterium’s 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 0o

C thus allowing the DNA molecule to enterthe cell through the zone of adhesion

DH5α 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 afterapplying 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 poly-

linkers for introducing your DNA of interest

bacterial selection with chloramphenicol or kanamycinplant 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 aspecial 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 tumor-

causing 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 T-

DNA 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 ovuleinfections. 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 (~5°C) 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 atechnique 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.

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Principles Behind the Procedures