4

4 Binary vectors for

plant
transformation

Introduction
Vectors for plant transformation have to be created in the same way as any other
plasmid-derived cloning vector—by using molecular biology techniques. It is
therefore useful to look at some of the basic features that are necessary or
desirable in any vector. Such features are described here because the usefulness
of vectors often equates with the ease with which they can be manipulated, both
in standard laboratory hosts such as Escherichia coli and in vitro, before being
used to transform plants. Later in this chapter we will see that many of the
recent advances in the design of plant transformation vectors are aimed at
improving the ease with which they can be manipulated in the laboratory, rather
than their ease of use for plant transformation per se.
In addition, this chapter will describe the basic features of vectors used for
plant transformation, their development into systems allowing the routine
transformation of a range of plant species and some of the problems associated
with vector design and transgene expression.

Desirable features of any plasmid vector

Ideally, as well as the ability to replicate independently from the chromosome
(which depends on the presence of an origin of replication), cloning vectors
should have the following properties:
1. Be of a small size (low molecular weight). Small size has several important
advantages. Small plasmids are easier to manipulate in vitro as they are less
liable to damage by shearing. Small plasmids are also usually present in
higher copy number and therefore plasmid yields are higher. There is also
less chance of the vector having other sites for restriction endonucleases,
making the design and integration of a multiple cloning site (MCS) simpler.
2. Confer a selectable phenotype on the host cells so that transformed cells can
be selected for. Most plasmid cloning vectors therefore carry genes that
confer resistance to an antibiotic, very often ampicillin (a penicillin
derivative).
3. Contain single sites for a large number of restriction enzymes to enable the
efficient production of recombinant vectors.
4. Enable the identification of bacterial colonies containing recombinant
plasmids. This is usually achieved by clustering the unique restriction sites in
one small area, the multiple cloning site, which is situated in the lacZa
sequence that encodes the N-terminal part of E. coli (3-galactosidase. In
suitable E. coli hosts, complementation results in the production of active (3-
galactosidase, which is detected by the bacterial colony turning blue due to
hydrolysis of a chromogenic substrate. Cloning DNA into the MCS
effectively results in the insertional mutagenesis of lacZa. After
transformation with these recombinant plasmids, E. coli will produce white
colonies as no complementation can occur. Although not 100% reliable
(both false-positives and false-negatives can be generated), such a system
drastically reduces the number of colonies that need to be screened in order
to identify those harbouring recombinant plasmids.

These features are illustrated in Figure 4.1, which shows a typical cloning
vector widely used in molecular biology.

Development of plant transformation vectors

As the process of Agrobacterium-mtdiated plant transformation was elucidated
(see Chapter 3) important information came to light that made the development
of efficient plant transformation vectors possible. Chief amongst these were the
following discoveries: that the only features of the T-DNA necessary for
integration into the host plant genome were the short border sequences; the
discovery that the removal of the oncogene sequences enabled plants to be
regenerated from transformed plant tissue by manipulating the plant hormone
composition of the medium; and the fact that the vir genes function in trans.

Basic features of vectors for plant transformation

Some of the basic factors that contribute to the design of a successful vector
have already been considered. However, when considering the design of plant
transformation vectors, several additional degrees of complication become
apparent:
1. The plasmid (if it is to be used in Agrobacterium-mediated transformation)
needs to be able to replicate not only in E. coli (so that routine manipulations
can be carried out) but also in Agrobacterium.
2. Additional selectable markers need to be included so that the successfully
transformed plants can be identified.
3. Border sequences need to be incorporated into the design of plasmid vectors
for Agrobacterium-mediated transformation to ensure integration of the
genes of interest into the host plant genome.
4. The genes (particularly if they are from prokaryotes or non-plant eukaryotes)
that are to be integrated into the genome of the host plant may need to be
made ‘plant-like’. This includes the use of appropriate promoters and
terminators to ensure that expression of the genes occurs. These features are
often incorporated into the basic vector.

Promoters and terminators

An obvious requirement for any genes that are to be expressed as transgenes in
plants is that they are expressed correctly (or at least in a predictable fashion). It
is known that the major determinant of gene expression (level, location and
timing) is the region upstream of the coding region. This region, termed ‘the
promoter’, is therefore of vital importance (see Chapter 1). Any genes that are
to be expressed in the transformed plant have to possess a promoter that will
function in plants. This is an important consideration as many of the genes that
are to be expressed in plants, particularly reporter genes and selectable marker
genes, are bacterial in origin. They therefore have to be supplied with a
promoter that will drive their expression in plants.
Transgenes also need to have suitable terminator sequences at their 3'
terminus to ensure that transcription ceases at the correct position. Failure to
stop transcription can lead to the production of aberrant transcripts and can
result in a range of deleterious effects, including inactivation of gene products
and increased gene silencing.
In addition to the basic need for the promoter to be capable of driving
expression of the gene in plants, there are other considerations that need to be
taken into account, such as promoter strength, tissue specificity and
developmental regulation.
Agrobacterium-derived promoter and terminator sequences
The genes from the Ti plasmid of Agrobacterium that code for opine synthesis,
and in particular the nopaline synthase (nos) gene, are widely used as a source
of both promoters and terminators in plant transformation vectors. Although
derived from bacterial genes, their presence on the T-DNA means they are
adapted to function in plants. The nos promoter is usually considered to be
constitutive.
The 35S promoter
The most widely used promoter used to drive expression of genes in plant
transformation vectors is the promoter of the cauliflower mosaic virus 35S RNA
gene (35S promoter). This promoter is considered to be expressed in all tissues
of transgenic plants (though not necessarily in all cell types). In dicots it drives
expression at high levels, although in monocots the level of expression is not as
high. This makes the 35S promoter ideal for driving the expression of selectable
marker genes, and in some cases of reporter genes, as expression is more or less
guaranteed. The activity of the 35S promoter can be further increased by the
inclusion of one or more copies of the enhancer region (Figure 4.2 shows the
structure of the 35S promoter). High-level constitutive

Binary vector system for genetic engineering:

Vir genes and T-DNA can be on separate plasmids.
Only left and right borders are required for T-DNA to be transformed. Cloning
sites are mentioned after LB. The cointegration vector is shown below:

Antisense Vector:

RNAi vector:
Viruses as cloning vectors:
Modified versions of lambda and M13 bacteriophages are important cloning
vectors for E. coli. Most plants are subject to viral infection, Viruses are
convenient to use than other types of vector, because with many viruses
transformation can be achieved simply by rubbing the virus DNA onto the
surface of a leaf. The natural infection process then spreads the virus throughout
the plant. One problem is that the vast majority of plant viruses have genomes
not of DNA but of RNA. RNA viruses are not so useful as potential cloning
vectors because manipulations with RNA are more difficult to carry out. Only
two classes of DNA virus are known to infect higher plants, the caulimoviruses
and geminiviruses.

Caulimoviruses:
Although one of the first successful plant genetic engineering experiments, back
in 1984, used a caulimovirus vector to clone a new gene into turnip plants, two
general difficulties with these viruses have limited their usefulness.The first is
that the total size of a caulimovirus genome is, like that of l, constrained by the
need to package it into its protein coat. Even after deletion of non-essential
sections of the virus genome, the capacity for carrying inserted DNA is still
very limited.Recent research has shown that it might be possible to circumvent
this problem by adopting a helper virus strategy, similar to that used with
phagemids. In this strategy, the cloning vector is a cauliflower mosaic virus
(CaMV) genome that lacks several of the essential genes, which means that it
can carry a large DNA insert but cannot by itself direct infection.
Plants are inoculated with the vector DNA along with a normal CaMV genome.
The normal viral genome provides the genes needed for the cloning vector to be
packaged into virus proteins and spread through the plant. This approach has
considerable potential, but does not solve the second problem, which is the
extremely narrow host range of Caulimoviruses. This restricts cloning
experiments to just a few plants, mainly brassicas such as turnips, cabbages, and
cauliflowers. Caulimoviruses have, however, been important in genetic
engineering as the source of highly active promoters that work in all plants and
that are used to obtain expression of genes introduced by Ti plasmid cloning or
direct gene transfer.
Figure 4.1 The eight coding regions are shown by the thick grey arrows, and
the different reading frames are indicated by the radial positions of the boxes.
The thin lines in the centre indicate the (plus and minus) DNA strands with the
three discontinuities. The major transcripts, 19S and 35S, are shown around the
outside.
Geminivirus vectors:
Geminiviruses are particularly interesting because their natural hosts include
plants such as maize and wheat, and they could therefore be potential vectors
for these and other monocots. But geminiviruses have presented their own set of
difficulties, one problem being that during the infection cycle the genomes of
some geminiviruses undergo rearrangements and deletions, which would
scramble up any additional DNA that has been inserted, an obvious
disadvantage for a cloning vector. Geminiviruses are beginning to find some
specialist applications in plant gene cloning. One of these is in virus induced
gene silencing (VIGS), a technique used to investigate the functions of
individual plant genes. This method exploits one of the natural defence
mechanisms that plants use to protect themselves against viral attack. This
method, called RNA silencing, results in degradation of viral mRNAs. If one of
the viral RNAs is transcribed from a cloned gene contained within a
geminivirus genome, then not only the viral transcripts but also the cellular
mRNAs derived from the plant’s copy of the gene are degraded. The plant gene
therefore becomes silenced and the effect of its inactivation on the phenotype of
the plant can be studied.

Gateway cloning:
The gatewat Cloning Technology is based on the site-specific recombination
system used by phage lambda to integrate its DNA in the E. coli chromosome.
Both organisms have specific recombination sites called attP in phage
lambda site and attB in E. coli. The integration process (lysogeny) is catalyzed
by 2 enzymes: the phage lambda encoded protein Int (Integrase) and the E.
coli protein IHF (Integration Host Factor). Upon integration, the recombination
between attB (25 nt) and attP (243 nt) sites generate attL (100 nt) and attR (168
nt) sites that flank the integrated phage lambda DNA.The process is reversible
and the excision is again catalyzed Int and IHF in combination with the
phage lambda protein Xis. The attL and attR sites surrounding the inserted
phage DNA recombine site-specifically during the excision event to reform
the attP site in phage lambda and the attB site in the E. colichromosome.
Figure 4.2 Cell killing by CcdB is accompanied by filamentation, defects in chromosome
and plasmid segregation, defects in cell division, formation of anucleate cells, decreased
DNA synthesis and plasmid loss. Interferes with the activity of DNA gyrase, inducing it to
form a covalent GyrA-DNA complex that cannot be resolved, thus promoting breakage of
plasmid and chromosomal DNA.

Figure 4.3 ccdB codes for the toxic protein (CcdB) that acts as a DNA gyrase poison,
locking up DNA gyrase with broken double stranded DNA and ultimately causing cell death.
Promoters & Terminators
Types of promoters include Constitutive promoters, tissue specific promoters,
inducible promoters
Constitutive promoters:
Nos (code for opine synthesis, also used as terminators).35S (promoter of the
cauliflower mosaic virus 35S RNA gene, high expression level in dicots,
expressed in all tissues, add one or more copies of the enhancer region to
increase activity). Maize ubiquitin 1 or rice actin promoters (high expression in
monocots)
Expression pattern of AtEXPA1. Expression of the GUS reporter gene under the
control of the AtEXPA1 promoter in plants. a GUS activity was restricted to
guard cells. b Gus activity in a transgenic young leaf. c Quantitative real-time
PCR analysis of AtEXPA1 expression under H2O, 50 mM KCl (pH 6.1),
50 mM KCl (pH 5), 50 mM KCl (pH 7), 100 μM ABA and light treatment. The
data represent the means of three independent assays. The error bars represent
the standard deviation (SD).

Tissue specific promoters

Drive expression in specific tissues. Useful for production of harmful
substances limited to tissues not consumed by humans & animals. Genes
involved in specific process limited certain tissues. Careful consideration since
cis-acting regulatory sequences are found in regions other than promoter.
Promoter::GUS activity shows CHX expression in pollen and in vegetative
tissues of transgenic Arabidopsis plants. AtCHX08 :: GUS (A) and AtCHX23 ::
GUS (B) expression in pollen grains. C, AtCHX17 :: GUS expression in
epidermal and cortical cells of root. D to F, AtCHX13 :: GUS expression in
reproductive organs. D, Whole flower; E, transverse section of anthers; F,
longitudinal section of stigma showing growing pollen tubes expressing
AtCHX13 :: GUS . G to I, AtCHX14 :: GUS expression in flowers and
vegetative tissues. G. anthers and pollen, H. leaf trichomes, I. root vascular
tissues

Inducible promoters:
controls the timing of gene expression. Includes three categories: non plant
derived systems, Plant derived system that respond to environment signals.
Plant derived systems based on developmental control of gene expression.

Advantages and Disadvantes of inducible promoters

Non plant derived system: independent of plant process but require inducers,
Plant derived system:dependent on the normal plant process thus simpler to use
for agriculture application, Chances of leaky effect

Non plant derived Systems

Examples include Tetracycline, Alcohol (ethanol), Steroid inducible, Copper
inducible. Non-plant-derived systems require certain desirable features in any
inducible expression system that depends on the application of an exogenous
chemical. (1) there should be no expression of the transgene in the absence of
the inducer. (2) The system should respond to only one inducer or one class of
inducer, ensuring specificity. (3) The induction of gene expression should be
rapid following the application of the inducer. (4) gene expression should cease
rapidly once the inducer is withdrawn. (5) the inducer should be non-toxic (6)
should not cause non-specific changes in gene expression.

Tetracycline
Tetracycline is an antibiotic that can be used to either de-repress or inactivate
gene expression. In bacteria, the tetracycline repressor (TetR) binds to the tet
operator and negatively regulates its expression. When TetR binds to
tetracycline, it is modified and released from the operator. This system has been
adapted for use in plants, where TetR is constitutively overexpressed from a
35S promoter. The transgene is controlled by a chimeric promoter consisting of
a core 35S promoter and multiple copies of the tet operator. Normally, TetR
represses transgene expression, but when tetracycline is applied, TetR is
released from the operators in the transgene promoter, leading to transgene
expression. This system was one of the earliest developed but has drawbacks,
including its inconsistent functionality across plant species and the need for
continuous tetracycline application due to its short half-life.Tetracycline can
also be used to inactivate transgene expression through a similar promoter
system. In this case, TetR is modified into a tetracycline transactivator (tTA),
which binds to the operators and induces gene expression in the absence of
tetracycline. When tetracycline is introduced, tTA is released, and transgene
expression ceases.

Alcohol Inducible system

In the alcohol (ethanol) inducible system, a chimeric promoter is used to drive
transgene expression. This promoter includes a core 35S promoter region along
with binding sites for the AlcR transcription factor, which is derived from
Aspergillus nidulans and is constitutively expressed. When ethanol is applied to
the system, AlcR binds to its specific binding sites within the promoter, leading
to the activation of transcription.

Steroid-inducible systems
regulate transgene expression using a modified transcriptional activator that
binds to a steroid hormone or its analogue. The binding sites for this
transcription factor are incorporated into a chimeric promoter, which controls
transgene expression. In the absence of an inducer, the transcription factor
remains inactive. However, when the inducer is applied, it binds to the promoter
and activates gene expression. Various steroidal inducers have been developed,
including glucocorticoids (such as dexamethasone), estrogen, and ecdysone (an
insect hormone). The dexamethasone-based system has certain drawbacks that
may limit its utility. However, the ecdysone-based system is particularly
promising because it also responds to RH5992 (tebufenozide), a non-steroidal
ecdysone agonist. Since RH5992 is a widely used agrochemical, it presents
potential for field applications of this system.

Copper Inducible
Copper-inducible systems utilize the yeast metallothionein regulatory
mechanism to control transgene expression. A transcription factor is
constitutively expressed in the system. When copper binds to this transcription
factor, it enables the factor to interact with elements in a chimeric promoter,
leading to the activation of gene expression.

Examples of plant derived systems based on environmental signals:

Wound inducible: Wound inducible promoters can be used to drive the
expression of pest-resistance genes after insect damage
Heat shock inducible: Sequence elements, termed “HSEs” (heat shock
elements) mediate the heat shock inducibility of genes

Examples of plant derived systems based on developmental control of gene

expression
Types include Senescence specific gene expression, Abscissic acid inducible
gene expression, Auxin inducible gene expression
1. Senescence-Specific Gene Expression: Senescence-inducible promoters,
such as SAG12 and SAG13 from Arabidopsis, drive transgene expression
during senescence. These promoters have been used in genetic engineering,
for instance, to regulate the ipt gene from Agrobacterium, which is involved
in cytokinin production. When controlled by the SAG12 promoter, this
system can delay senescence in tobacco.
2. Abscisic Acid (ABA) Inducible Gene Expression: ABA-responsive
sequence elements have been identified and can be combined with a core
35S promoter in a chimeric promoter system. This combination enables
ABA-induced transgene expression, allowing for hormone-regulated gene
activation.
3. Auxin Inducible Gene Expression: Auxin-responsive elements (AuxREs)
have been discovered and can be incorporated into chimeric promoters with
a core 35S promoter. This configuration facilitates auxin-inducible transgene
 expression, enabling gene activation in response to auxin or its inactive
analogs.
Example of promotor used in plant transformation include CaMV35S for
Cauliflower mosaic virus with constitutive activity. Example of promoter
induced by oxidative stress include HSP 1&2 gene inducer Thermal shock for
Arabidopsis Thaliana. Example of promoter induced by biotic stress CaPrx
promoter, inducer Nematode infection for Coffea arabica
Selectable Markers
Plant transformation is a low frequency Event. Selection is based on the
inclusion of substance toxic to the plant into the culture media Antibiotics,
herbicides and other examples
Antibiotic resistance
Most common npt11 (neomycin transferase)– Source E.coli– Selective agent
kanamycin and genticin. hpt (hygromycin phosphotransferase) – Source E.coli–
Selective agent hygromycin dhfr (dihydrofolate synthase)– Source mouse–
Selective agent methotre
nptII gene
The nptII gene encodes Aminoglycoside 3'-phosphotransferase II [APH(3')-II or
NPTII], which is the most widely used selectable marker for plant
transformations. The enzyme is encoded by the gene nptII (or neo) gene,
derived from the transposon Tn5, and inactivates by phosphorylation a number
of aminoglycoside antibiotics such as kanamycin, neomycin, geneticin, and
paromomycin (interference with protein biosynthesis). Kanamycin is mostly
used as the selective agent, normally in concentrations ranging from 50 to 500
mg/L. Geneticin is generally more toxic than kanamycin, but useful for several
legumes and gramineae, where kanamycin is ineffective as a selection marker.
Many promoters have been fused to the nptII gene to generate useful selectable
markers. Some vectors contain a mutated form of the nptII gene that encodes a
protein with reduced enzymatic activity. The nptII gene can be used not only as
a selection marker but also as a reporter gene to study gene expression and
regulation. An important advantage of NPTII in this respect is that N-terminal
fusions can be constructed that retain enzymatic activity. The safety for human
consumption of the NPTII protein has been assessed without finding any
concerns.
Hpt gene
Hygromycin B is an aminocyclitol antibiotic that interferes with protein
synthesis. A hygromycin phosphotransferase gene (hpt), originally derived from
E. coli, it was modified for expression in plant cells and has since found wide
application as a resistance gene
amino acid residues near the carboxy-terminus of the enzyme were deleted or
substituted, which increased the level of resistance in tobacco protoplasts, the
hydrophilic carboxy-terminal end itself was preserved and may be essential for
strong HPT activity. Hygromycin B is usually more toxic than kanamycin and
kills sensitive cells more quickly: selection in vitro is applied at concentrations
ranging from 20 mg/L for Arabidopsis thaliana to 200 mg/L for tall fescue
(Festuca arundinacea).
acc(3) gene
Three genes encoding aminoglycoside-3-N-acetyltransferases (ACC(3)) have
been used successfully as selectable markers for plant transformation in
combination with gentamicin selection: acc(3)-I, acc(3)-III, and acc(3)-IV. The
ACC(3)-III and ACC(3)-IV enzymes have a broad substrate specificity in vitro,
modifying gentamycin, neomycin, and kanamycin amongst others. The
ACC(3)-IV marker was shown to confer low levels of kanamycin resistance in
Petunia transformed by Agrobacterium tumefaciens. The ACC(3)-I enzyme, on
the contrary, only modifies gentamycin and some close derivatives and might,
therefore, be more useful if one wishes to combine it with other selection
markers.
Genes conferring resistance to bleomycin and phleomycin
Plant cells are sensitive to the glycopeptide antibiotics bleomycin and
phleomycin, which produce single-stranded and double-stranded breaks in
DNA. Two genes encoding bleomycin-binding proteins have been used to
construct dominant bleomycin or phleomycin resistance markers for plant cell
transformation: a bleomycin resistance gene from Tn5 and a gene derived from
Streptoalloteichus hindustanus actinomycete fungi.
Spt i aadA genes
Streptomycin and spectinomycin resistance markers differ from the above-
described markers in that they allow differentiation by color rather than by
killing. Under appropriate conditions, sensitive plant cells bleach but do not die,
whereas resistant cells remain green. Two dominant resistance genes have been
described for plant transformation work: the streptomycin phosphotransferase
(SPT) gene from Tn5 that provides resistance to streptomycin and the
aminoglycoside-3'-adanyltransferase gene (aadA) conferring resistance to both
streptomycin and spectinomycin. The SPT marker, which provides a cell-
autonomous resistance phenotype, has been successfully applied to monitor
transposon excision. The aadA gene has not only been used as a marker for
nuclear genome transformation, but was also shown to be an excellent marker
for plastid transformation. This is probably due to its dominant nature, in
contrast to a previously used spectinomycin resistance ribosomal RNA gene,
which is a recessive marker.
dhfr genes
Methotrexate (MTX), an analogue of folic acid, is an antimetabolite that inhibits
the enzyme dihydrofolate reductase (DHFR) and thus interferes with DNA
synthesis.
Many plant species are sensitive to methotrexate at low levels: Genes coding for
MTX-insensitive DHFR have been isolated; such dhfr genes can be transferred
into plants to confer resistance against methotrexate. A dhfr gene from E. coli
plasmid R67, which encodes an enzyme practically insensitive to MTX, is able
to confer MTX resistance to tobacco and wheat. Recommended MTX
concentration in the selection media:0.1 mg/L in liquid media 0.5 mg/L in solid
media (in combination with 0.1% active charcoal to prevent deleterious effects
of the MTX selection).A mutant mouse dhfr gene encodes an enzyme with very
low affinity to methotrexate.Fusion of this gene to the CaMV 35S promoter
yielded a methotrexate resistance marker used for transforming different plant
species (e.g., tobacco and Arabidopsis): In Arabidopsis, transformation with
dhfr followed by selection on MTX yielded a lower background of
nontransformed escapes than other selection systems. Methotrexate should be
handled carefully because of its high toxicity for humans.
Other selectable markers manA/pmi(mannose-6-phosphate)– Source: E.coli–
Selective agent: alternative carbon source, mannose. lysC (lysine-threonine
aspartokinase)– Source: E.coli– Selective agent: lysine and threonine. lec(leafy
cotyledon)– Source: maize– Selective agent: none, based on growth

Herbicide Resistance
als(acetolactate synthase)– Source: Arabidopsis,maize,tobacco– Selective agent:
sulphonylureas. aroA/epsps (enolpyruvylshikimate phosphate synthase)–
Source: Petunia hybrida, Agrobacterium spp– Selective agent: glyphosate.
Bar/pat(phosphinothricin acetyltransferase)– Source: Streptomyces hygcopicus/
S.viridochromogenes– Selective agent: bialophos glufosinate
Reporter genes
Used for Assessing gene expression, Easily scored indicators, Preferable non
destructive assay. Examples include, ß glucuronidase (gus), Green fluorescent
protein (gfp), Luciferase (lux or luc), Chloramphenicol acetyl transferase (cat)
ß glucuronidase
 Commonly used reporter gene. Extremely
sensitive and easy to use. It is used to obtain both - quantitative data. substrate
(4-methylumbelliferryl-ß-D-glucuronide) – hyrolysed to 4MU- qualitative data.
Localise in tissue or specific cell type by histochemical assay using Xgluc (5
bromo-4chloro-3 indolyl ß-D-glucuronide) as substrate.

Green fluorescent protein

Nowadays, gfp commonly used GFP can use where GUS cannot, GFP gene was
isolated from jellyfish AequoreaVictoria To efficiently work in plant. Remove a
cryptic intron (to prevent mis-splice)-- Make codon usage more plant like
(transability was improved by introducing AACA sequence upstream of the
translation initiation codon.
A cool protein
Give off the green light in the presence of UV light, Marker of the twenty-first
century, Nobel Prize in Chemistry 2008, Osamu Shimomura, Martin Chalfie,
Roger Y. Tsien
Scientific Impact of GFP
Nontoxic – No significant physiological effects. Can be used to Watch process
that was previously invisible How cancer cell spreads Alzheimer’s disease.
Common use of GFP includes monitoring changes in a cell.
Luciferase
Obtained from firefly (Photinus pyralis), luciferin + ATP → luciferyl adenylate
+ Pyrophosphate, luciferyl adenylate +O2 → oxyluciferin + AMP + light, Not
widely used; useful for detection of low level or highly localised expression

Bioluminescence
(living light) is ability of living organisms to produce light through a chemical
reaction. There are two ways an organism can bioluminescence- Luciferin
which produces the light generically and Luciferase that drives or catalyzes the
reaction. Wide range of applications includeAnimal Sciences Environmental
sciences, Plant sciences and Entomology.
Animal sciences
Imaging cells In vivo-Two different luciferases along with different substrates
for each and their kinetics can be studied in vivo in the tissue
In Gene therapies- Luminescent markers play a critical role in gene therapy to
achieve controlled and effective delivery of genes to target cells avoiding
ectopic expression
Example- Transgenic mice that express the fluc gene under the control of the
nuclear factor kB (NF-kB) promoter. real-time imaging of the NF-B promoter
Mouse model for retinoblastoma suppressor gene-dependent pituitary cancer
development with coexpression of fluc genes enabling long term
bioluminescence imaging, quantification of tumour and assessment of
chemotherapeutic response activity.

Environmental Science
Luminous systems are used as biosensors to monitor environmental toxicity.
Fishes are transformed with luciferases to monitor the level of tetracycline in
their environment. Some bacteria are transformed with luciferases to monitor
the toxic gases in mines.
Plant Science
Tobacco plants are cloned with luciferase gene to express the enzyme and glow
when dipped in luciferin. Transformed Arabidopsis plants with Phytochrome A
(phy A) gene promoter fused with luciferase reporter to study the rhythms in
plants associated with the gene. Luciferin when sprayed into an adult plant
responds very late than a plant that is wounded. This demonstrates that there are
some barriers that restrict the luciferin entry in adult plants, which may cause
unavailability of luciferin and a limiting factor for non-invasive luciferase
assays. Luciferin when sprayed into an adult plant responds very late than a
plant that is wounded. This demonstrates that there are some barriers that
restrict the luciferin entry in adult plants, which may cause unavailability of
luciferin and a limiting factor for non-invasive luciferase assays.
Chloramphenicol acetyl transferase
is widely used in mammalian cells and have limited use in plant system, they
require radioactive assay.

Chloremphenicol Acetyl Transferase Gene

The gene first isolated from E. coli codes for an enzyme (CAT), which is absent
in mammals and higher plants, so that whenever transferred in a gene construct,
its presence can be detected by enzyme assay. Rarely screening for this enzyme
mayalso be used for selection of transformed regenerants, although no selection
pressure can be applied.The enzyme uses acetyl CoA + chloramphenicol (32p)
as substrates and helps in the transfer of acetyl group to chloramphenicol. The
presence of acetyl chloramphenicol is detected through autoradiography. The
gene cat has also been used for identification of a number of regulatory
sequences.
Clean gene technology
Between the loxP sites there are two fragments: (1) XbaI fragment containing
35S:NPT:nos3', (2) SacI fragment containing HSP:Cre:nos3'. Outside is a
HindIII 35S::nos3' fragment with a unique cloning site, MluI. Plasmids pNS13
and pNS14 are used for cloning, where pNS13 is for cloning the full gene
(promoter:gene:terminator) into the HindIII site, and pNS14 is for cloning
cDNA into the MluI site to be expressed by the 35S promoter. Common
features include a binary vector backbone (pPZP200) with spectinomycin
selection, plant selection via the NPT II gene (Kanamycin or Geneticin), an
excision system based on Cre-lox recombination, and an autoexcision system
utilizing heat-inducible Cre-lox recombination. The method of generating
marker-free transgenic plants involves several steps: cloning the gene or cDNA
into pNS13 or pNS14, carrying out Agrobacterium-mediated transformation,
identifying single-copy transgenic plants using PCR or Southern analysis,
treating young plants in tubes or magenta boxes with 42°C heat for 3 hours,
testing marker gene excision three days later by performing PCR on tissues
from different parts of the plant, repeating the heat treatment if necessary,
allowing the plant to grow until maturity, and in the case of sexually propagated
plant species, collecting seeds and testing for the presence of a marker-free
locus by PCR.