Chapter 2: Techniques in Plant Biotechnology

Plant Genetic Engineering

Cloning Vectors
 Vectors for the development of transgenic plant
Promoters and enhancers
Gene Transfer Systems for Plants
Plant Transformation Techniques
 indirect transformation techniques
Direct transformation techniques
Selection and Regeneration of transgenic plants
 Selectable marker Genes

 molecular Analysis of Transgenic Plants

Greenhouse field growth of transgenic plants
 What is genetic engineering???
 It is artificial manipulation or alteration of genes.
 It breaks the species barrier
 It involves „cut and paste’ process.
Some important terms!!!
 Recombinant DNA: the altered DNA

 transgene: transferred gene

 Transgenic: organism that develop after a successful gene transfer .

 transient expression: Transferred gene expressed for short period

 Stable transformation: expression occurs in regenerated plant and

inherited in subsequent generations.
 Basic Tools Used in Genetic Engineering
1. Restriction Enzymes:
 used to cut the DNA at specific places.
 d/t enzymes cut at specific DNA sequences sit (recognition site).

2. DNA Ligase:
 it acts as a glue to stick foreign DNA to DNA of the cloning vector.
 It only work if DNA from the two DNA sources has been cut with the
same restriction enzyme
 i.e. sticky ends of cut DNA will be complementary to each other.
 Cloning Vector
What is cloning Vector???
 is a DNA molecule can replicate in a suitable host organism

 Vectors need to have the following characteristics:

 origin of replication (ori): for autonomous replication

 recognition sites: where foreign DNA fragment can introduced.

 convenient markers: for identifying transformed host.

 the vector should be also easily introducible into the host organism
where it has to replicate
 Types of cloning Vectors
1 plasmids
 Plasmids are circular, double-stranded DNA that are
independent from a cell‟s chromosomal DNA.
 They commonly occur naturally in bacteria

 plasmids provide some benefit to the host cell,

 for example, encode enzymes that inactivate antibiotics.

 Plasmids range from a few thousand bps to more than 100kb.

The plasmids most frequently used in recombinant DNA

technology are derived from and replicate in E. coli
2 Bacteriophages
 Bacteriophages are viruses that infect bacteria.

 one of the best studied phages is bacteriophage λ (lambda)

 It consists of a head containing the 48.5 kb & a long flexible tail.

 During infection, the phage binds receptors on the membrane of E.

coli and subsequently injects its genome into the host cell

This mechanism of the phage adapted for the development of

phage cloning vector
3. Cosmids
 λ phage & plasmid vectors are useful for cloning only relatively
small DNA fragments.

 The common method for cloning large fragments developed from

both plasmid & λ-phage cloning called cosmid
 It can insert up to 45 kb efficiently
4 . Yeast artificial chromosome (yac)
 It constructed by ligating the components required for replication

 It can insert very large fragments of target DNA (1 mb in length)

 In addition to all vector basic components, it contains two telomeric

sequences (Tel) & one centromere region.

5. Bacterial artificial chromosomes (Bac)

 They can contain up to 300–350 kb of insert sequence.

 they are stable and easily introduce into their host cell by
transformation,
 Vectors for the production of transgenic plants
Ti plasmid
 It is the most common vector in the production of a transgenic plant.
 It has an estimated size ranging between 200 and 800 kbp depending
on classes of Ti plasmid.
 Ti plasmid is divided into three main regions
 The T-DNA region that
is transferred into the plant genome
This region is bordered by repeat sequences on each end (left
border and right border).
 The virulence region
 It is responsible for encoding the vir genes, which aids in the
transfer of the T-DNA.
 opine and phytohormones (auxin and cytokinin) biosynthesis
catabolism
 Promoters
 The promoter sequence is the key regulatory region of a gene

 They play an important role in the regulation of gene;

 at different locations & stage of growth or

 in response to internal and external stimuli.

 Investigating the precise function of promoter components reveal

new possibilities of genetic engineering.

 it is practical to study expression genes in an organism by

combining with a promoter of choice

 This approach can use to study the expression of endogenous

genes or to introduce foreign genes

 promoters have a huge influence in molecular research & the

development of Gmos.
 Types of promoters
 promoters divided into different classes according to their
function:
1. constitutive promoters.
 They direct express a gene in all cells / tissues of an organism.

 The genes controlled by such promoters are “housekeeping

genes”,
i.e. genes whose products are constantly needed by the cell
 They are insensitive to environmental influences. So,

They show high sequence conservation between species,

they are active across species & across kingdoms.

E.g CamV 35S promoter, which is frequently used to

drive transgene expression in transgenic plants.
2. tissue-specific promoters.
 They direct the expression of a gene in a specific tissue or cell type
of an organism or during certain stages of development.

Thus, the gene product is only found in those cells or tissues

 Frequently, such promoters rely on the presence or absence of

endogenous factors to function,
3.
Inducible promoters
 they are very important to GE because:
 their performance is dependent on endogenous / external factors

 gene expression can be controlled by the experimenter by simply

adding a certain substance to the cell culture/the organism

 usually only the genes that have been specifically introduced

within the class of inducible promoters can be expressed.

 one can find promoters controlled by abiotic factors such as:

 light, oxygen level, heat, cold and wounding, while others are
controlled by certain chemicals or metabolites.
 once a promoter that responds to a certain compound has been
identified it can be used to GE
Enhancers & silencer
 Enhancers: short (50–1500 bp) regions in a gene that can be
recognized and bound by activator proteins/transcription factors
 Silencers: function as the direct opposite of enhancers.
 Silencers are binding sites for transcription factors known as the
repressors.
 These repressors are known to downregulate the transcription of a
gene.
Any question???
 Chapter 2 part 2
Gene Transfer Systems for Plants
Plant Transformation Techniques
 indirect transformation techniques
Direct transformation techniques
Selection and Regeneration of transgenic plants
 Selectable marker Genes
 molecular Analysis of Transgenic Plants
Greenhouse field growth of transgenic
plants
Plant Genetic Transformation
 There are two main types of plant transformation techniques
I. Indirect methods
1. agrobacterium-mediated plant transformation
2. Virus-mediated plant transformation/transduction
II. Direct methods
1. Micro projectile bombardmet
2. protoplast transformation techniques
2.1. chemical techniques
2.2. Electroporation
2.3. Micro injection
 1. agrobacterium-mediated plant transformation
 A. tumefaciens are soil bacteria that infect plant cells & transfer its
DNA into plant cell.
 Upon integration of the bacterial DNA into a plant chromosome,
 it directs synthesis of several proteins using the plant cell
 Agrobacterium infections result in crown gall disease

 In addition to its chromosomal genomic DNA, an A. tumefaciens cell

contains tumour-inducing (Ti) plasmid.

 The Ti plasmid contains four main parts:

T- DNA
Origin of replication
Virulence (Vir) genes containing part
Opines catabolism region
 Agrobacterium tumefaciens Genetic Engineering History
 it is Plant natural Genetic Engineer
 it has been naturally genetically engineering plants for centuries
A: Agrobacterium
tumefaciens
B: Agrobacterium genome
C: Ti Plasmid :
a: T-DNA
b: Vir genes
c: Replication origin
d: Opine catabolism gene
D: Plant cell
E: Mitochondria
F: Chloroplast
G: Nucleus
  Agrobacterium tumefaciens infection process
 Steps 1 & 2: Bacterial cell
weakly attaches itself to the
plant cell. It then produces
cellulose fibrils to anchor it to the
plant cell (infection).

 Step 3: When the bacterium

detects certain chemical signals
(Acetosyringone) produced by
the plant in response to bacterial
Ti plasmid infection, vir start producing
various compounds.

 Step 4: One vir gene complex

cuts the T-DNA (a) from the Ti
plasmid (C).
Agrobacterium tumefaciens infection process…
 Step 5: In the meantime, other
vir genes produce compounds
that coat the T-DNA to help
export it into the recipient plant
cell
 Step 6: Other vir genes make
T-DNA
the nucleus of the plant cell
T-DNA receiving the T-DNA more
receptive.
 Step 7: T-DNA is integrated
T-DNA into the host genome.

 T-DNA contains genes that

will force the plant to
produce special amino acids
called opines, which the
bacteria can metabolize as
its food source!
How A. tumefaciens adapted for transgenic plant development?
 To harness A. tumefaciens and the ti-plasmid as a transgene vector,
 T-DNA is removed but T-DNA border & vir genes are retained

 desired transgene is inserted b/n T-DNA border regions

 the transgene DNA is transferred to the plant cell and

integrated into the plant’s chromosomes

 To achieve transformation,
Agrobacterium carrying Ti plasmid with transgene co
culture with leaf/root explants to allow T-DNA transfer

explants are subsequently processed through various tissue

culture steps to select & produce transformed plants.
 2. Virus-mediated plant transformation/transduction
 These vectors are derived from plant viruses, e.g. TmV.
 Initial delivery of the virus-based vector achieved by Agrobacterium
 the vector is encoded in the T-DNA, which is transferred to plant.

 Now this method is applicable to whole plants, by the process

of agroinfiltration to avoiding intensive tissue-culture process.

 b/se within plant cell virus-based vectors autonomously

replicated, spread cell to cell & direct expression of transgene
 Advantages:
 applicability to whole plants & much faster outcome
 high-level expression within a short time.
 Disadvantage:
 the process is transient (the expression level decreases over time)
 II. Direct Methods
1. Micro projectile bombardment
 It uses high velocity particles that are coated with DNA & deliver
exogenous genetic material into the target cell or tissue.
 The particles are:
 Either tungsten or gold, are small (0.5-5 µm)

 Big enough to be sufficiently accelerated, penetrate cell wall &

carrying the coated DNA on their surface.

Gold particles are chemically inert/inactive, although rather

costly, and show a high uniformity.

Tungsten particles, showing mild phytotoxicity and being

more variable in size but adequate for most studies.
 Once DNA coated the particles it can be shoot & expression happened
after reaching the nucleus of the plant genome.
steps involved in the generation of genetically transformed plants
using either the Agrobacterium tumefaciens (A. tumefaciens) or
microprojectile bombardment approaches
 2. protoplast transformation techniques
 protoplasts are plant cells in which the cell wall has been removed
 Removal of the cell wall is achieved by cell wall-destroying enzymes;
 pectinases,
cellulases,
 hemicellulases
 approaches exist for the delivery of transgene DNA into protoplasts.
chemical treatments,
 electroporation and
 micro-injection techniques
2.1. Chemical techniques
 Polyethylene Glycol (PEG) treatment is the most widely used
technique, employing solutions of 10-15 percent PEG in
combination with high calcium content and a high pH.

 After mixing the isolated DNA and the protoplasts, followed by

different washes, the DNA may be taken up by the protoplast.

 The role of PEG is to alter the plasma membrane properties,

causing a reversible membrane permeabilization, thus enabling
exogenous macromolecules to enter the cell cytoplasm.
 2.2. Electroporation
 Electrical pulses applied to DNA-protoplast mixture, aggravating
an increase in protoplast membrane permeability to DNA.

 This technique is much simpler than the chemical method,

providing satisfying results.

 However, the electrical pulses must be carefully controlled as cell

death can occur above a certain threshold.

 The pulses induce the transient formation of micropores in the

membrane lipid bilayer which persist for a few minutes, allowing
DNA uptake to occur.
 2.3. Micro-injection
 This technique was originally designed to transform animal cells &
later adapted for plant cells.

 However, in addition to the rigid cell wall the presence of large

vacuoles in plant cell may lead to cell death after vacuole breakage
presents a severe restriction to micro-injection.

 This method is labour-intensive and requires special micro-

equipment for the manipulation of host protoplasts and DNA.

 However, some success in transforming both monocotyledonous and

dicotyledonous species has been achieved employing this technique.
Any question???
Selection and Regeneration of transgenic plants
 Selectable marker Genes
 molecular Analysis of Transgenic Plants
Greenhouse field growth of transgenic
plants
 Selectable Marker Genes (SMg)
 selectable portions on most transformation vectors are prokaryotic
antibiotic resistance enzymes, which will also confer resistancy
when they are expressed in plant cells.
 some commonly used marker genes are:
A. neomycin phosphotransferase (npt-II) gene and hygromycin
phosphotransferase (hpt) gene
B. chloramphenicol acetyltransferase (cat) gene
C. phosphinothricin acetyltransferase genes (bar and pat genes)
D. β-glucuronidase gene (gus)
E. luciferase gene
F. green fluorescent protein (gfp)
 A. neomycin phosphotransferase (npt-II) gene and
hygromycin phosphotransferase (hpt) gene
 Neomycin phosphotransferase-II (npt-II) is a small bacterial enzyme
which catalyses the phosphorylation of a number of
aminoglycoside antibiotics including neomycin and kanamycin.

 The reaction involves transfer of the γ-phosphate group of

adenosine triphosphate (ATP) to the antibiotic molecule, which
detoxifies the antibiotic by preventing its interaction with its target
molecule - the ribosome.

 The hygromycin phosphotransferase (hpt) gene, conferring

resistance to the antibiotic hygromycin, is also commonly used as
selection marker.
 B. chloramphenicol acetyltransferase (cat) gene
 The chloramphenicol resistance (cat) gene encodes the enzyme
chloramphenicol acetyltransferase (CAT) and was the first bacterial
gene to be expressed in plants.

 The enzyme specifically acetylates chloramphenicol antibiotics,

resulting in the formation of the 1-, 3-, and 1,3-acetylated
derivatives, which are inactive.

 Although not used as a selection system in plants, the gene is used

frequently as a reporter gene in plant promoter studies.
C. phosphinothricin acetyltransferase genes (bar and pat
genes)
 A commonly used herbicide is phosphinothricin (PPT, also known as
Glufosinate).

 This compound binds to and inhibits glutamine synthethase, which is

an important enzyme in the nitrogen metabolism and ammonium
fixation pathways.

 PPT-induced glutamine synthetase inhibition results in elevated

cellular ammonium levels and cell death.

 The enzyme phosphinothricin acetyltransferase (PAT), first identified

in Streptomyces hygroscopicus, acetylates and thus detoxifies PPT.

 This allows transformed cells, or complete transgenic plants, to

survive and grow in the presence of PPT.
 D. β-glucuronidase gene (gus)
 The E. coli β-glucoronidase gene adapted as a reporter gene
 β-glucuronidase, catalyses the cleavage of a wide variety of β-
glucuronides,
 β-glucuronides are available commercially as spectrophotometric,
fluorometric and histochemical substrates.
 GUS gene feature that make it useful reporter gene for plant studies.
to date lack detectable inherent glucuronidase activity in plants.
 it is easily, sensitively & cheaply assayed both in vitro & in situ
 sufficient for fixation & enabling histochemical localization in
cells / tissue.
preferred substrate for tissue localization of GUS is 5-
bromo-4chloro-3-indolyl-β-d-glucuronide(x-gluc).
advantage of these substrates; the indoxyl group produced
upon enzymatic cleavage dimerizes to indigo which is
virtually insoluble in an aqueous environment.
 β-glucuronidase expression measured by PNPG based
spectrophometric assay of powdery mildew infected & mock
treated plant
 Gus gene expression under different promoter deletion variants
confirmed by Gus staining and microscopic picture
 E. luciferase gene
 The luciferase (luc) gene isolated from Photinus pyralis (firefly)
encodes the enzyme catalysing the ATP/oxygen-dependent
oxidation of the substrate luciferin, resulting in the emission of
light (bioluminescence).

 As a reporter, the gene is the basis of highly sensitive assays for

promoter activity and for protein targeting sequences, involving
the measurement of light emission using liquid scintillation counter
photomultipliers, luminometers, x-ray film exposure or sensitive
camera film.
 F. green fluorescent protein (gfp)
 GFP is a widely used marker protein in modern biological research.

 protein shows green fluorescence upon exposure to blue light.

originally, the protein was isolated from the jellyfish Aequorea
victoria.

 GFP is widely applied for studies addressing gene expression or

promoter efficiency as well as protein localization, stability
and degradation.
 Molecular analysis of transgenic plants
 analysis of transgenic plants at the molecular level is mainly
performed by PCR and Southern blot analysis.

 PCR indicates the presence of the desired transgene within the

plant, Southern blot analysis.

 molecular analysis of the protein expression levels can be assayed by

enzyme-linked immunosorbent assay elISA or immuno staining of
plant tissue.

 expression of a gene of interest can also be assayed by determining

the presence and quantity of the corresponding RNA transcript, e.g.
by applying a modified PCR protocol (reverse transcriptase PCR)
[RT-PCR]).
 Greenhouse and field growth of transgenic plants
 Safety assessments begin with concept of product GM crop should
evaluate in green house as well as confined area to avoid gene flow
in wild type in the environment
 No variety is released without substantial safety evidence

Research on safety
◦ Nutrient and chemistry same as non-GMO
◦ –no allergens
◦ Transfer and/or breakdown of trait
◦ Environmental safety
Independent researchers
◦ Animal studies
◦ Environmental studies
General steps of genetic transformation of plants
1. Gene isolation
2. Gene cloning
3. Introducing the gene into an expression vector
4. Gene transfer
5. Selection of transformant cells
6. Regeneration of transformant cells
7. Proving integration of transgene into the genome
8. Examination and breeding of transgenic plants
9. Registration of transgenic plant,
10. Commercialization of transgenic foods