Plant Tissue Culture 3

Dr. Siddhartha Dutta

1
 Plant Transformation Techniques
moving a specific piece of DNA (usually a foreign gene ligated to a plasmid)
into cells

Transient Stable
(Transfer of DNA to express for (Transfer of DNA to stably integrate
only a short period of time) into host genome)

Monitoring and detection of plant transformation

• Marker genes: Reporter or scoreable or screenable genes
Selectable marker genes

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Transient • Promoter activity study
• Comparison of the promoter activity

• Identification of minimal promoter

Use of reporter genes
(through promoter deletion study)

• Cellular/subcellular localization of
Stable proteins

• Tissue specific expression of genes

3
 Reporter Genes

A test gene whose expression results in quantifiable phenotype

Ideal characters:
i. Detection with high sensitivity.
ii. Low endogenous background.
iii. There should be a quantitative assay.
iv. Assay should be nondestructive.
v. Assay should require a minimal amount of
effort and expense.

4
 Selectable Markers
• Selection of transformed cells
Enable the transformed cells to survive on media containing toxic levels
of selection agent while nontransformed cells get killed
(antibiotic, antimetabolite and herbicide resistance genes, hormone biosynthetic genes and genes
conferring resistance to toxic levels of amino acids or amino acid analogs.)

5
Chimaeric or transgene constructs
The nuclear plant gene consists of different regions, each involved in different
functions of transcription and translation of mRNA.

Required to be present in the vector for efficient expression in the plant

Chimeric gene construct

Sequence components of a chimeric gene construct.

Constitutive expression,
tissue specific expression
environmentally inducible expression

Commonly used: Cauliflower mosaic virus (CaMV) 35S RNA promoter

Other e.g. maize ubiquitin I promoter, rbcS ribulose bisphosphate carboxylase
small subunit; Adh1 alcohol dehydrogenase; nos nopaline synthase; and the
rice actin promoter 6
 GENE TRANSFER METHODS

Vectorless or direct DNA transfer Vector mediated gene transfer.

• Physical gene transfer methods • Agrobacterium mediate
• Chemical gene transfer methods.. • Virus mediate

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Vector mediated gene transfer
Agrobacterium mediated transformation
• Agrobacterium are gram-negative and rod shaped
• Found near soil level at the junction of plant stem and root.
A. tumefaciens: It induces crown gall disease.
A. rhizogenes: It induces hairy root disease.
A. radiobacter: It is an avirulent strain

A. tumefaciens
• Induces tumors called crown galls (tumor at root-shoot zone)
• Contain large plasmids called tumor-inducing plasmid (Ti plasmid)

Bacteria do not penetrate into the plant cells that are converted into tumor cells.
Rather, bacteria penetrate into the intercellular spaces and into injured cells
and attach themselves only to the wall of healthy plant cells. Only a small part
of the Ti plasmid, the T-DNA or transferred DNA, is transferred to and
integrated into the host plant nuclear genome

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Tumor induction by Agrobacterium tumefaciens.
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 Organization of Ti plasmid (Tumor inducing plasmid)

Octopine / nopaline
Component 1:
T-region

Plant cells in the tumor acquire two new properties:

1. These show phytohormone independent growth.
2. Contain unusual amino acid derivatives known as opines. 10
Left border/ Right border:
• 25-bp highly conserved directly repeated DNA sequence
• Any DNA sequence that is located between these borders is transferred to the
plant

The naturally occurring T-DNA encoded “oncogenes” are necessary for

tumorigenesis and opine production but not necessary for T-DNA transfer

These oncogenes can be deleted, thereby disarming the T-DNA.

Such disarmed Ti plasmids are capable of transferring T-DNA to plants but will
not cause tumors.

In these disarmed plasmids novel foreign genes can be inserted between the T-
DNA borders.

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Component 2:
Virulence genes (vir)

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Component 2:
Virulence genes (vir)

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 Component 3: Chromosomal genes (chvA, chvB)

• Genes chvA and chvB code for exopolysaccharide production, which are
important for attachment of the bacterium to the plant cell.

T-DNA transfer
Wounding

Synthesis of acetosyringone by the plants

Activate and induce the expression of the vir genes

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A. Aactivation of the vir genes via VirA and VirG

B. Key events in the induction of vir genes and T-DNA transfer

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 C. Key events in the transfer of T-DNA to plant cell

Use of Agrobacterium in plant transformation

Agrobacterium vectors:
T-DNA is disarmed by making it nononcogenic, simply by deleting all its oncogenes
and substituting them with an insert (desirable gene) between the regions of the left
and right border.

Restriction enzyme sites are also added into the plasmids (to aid in cloning the gene
Of interest).

Drawback : Large size of plasmid

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 Binary vectors system of delivering the foreign gene

Unit 1: A shuttle (more commonly referred to as a binary) vector that contains gene
of interest between the T-DNA borders

Unit 2: A helper Ti plasmid that provides the vir gene products to facilitate transfer
into plant cells

Shuttle
Should contain:
(i) multiple cloning site,
(ii) a broad host range origin of replication functional in both E. coli and
A. tumefaciens
(iii) selectable markers for both bacteria and plants,
(iv) TDNA border sequences

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Example of a typical binary Ti vector system.

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Agrobacterium tumefaciens mediated
transformation of explants.

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 Agroinfection: Agrobacterium mediated virus infection

Agroinfection/ Agroinoculation. : The delivery of viral or viroidal sequences to plants

using bacterium as a route.

Plant virus genomes cloned into Ti plasmids can be introduced into plants from
Agrobacterium

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 Floral dip method for Arabidopsis transformation

Tissue culture method for transgenics:

1. Involves a tissue culture and plant regeneration step, this approach is painstaking
and time consuming.
2. It also requires skilled labor and relatively expensive laboratory facilities for its
execution.
3. This method can result in undesired DNA modification and somaclonal variation
during the processes of plant dedifferentiation and redifferentiation, which is mostly
due to the stress imposed by the in vitro cell culture protocol

Simple dipping of developing floral tissues into a solution containing

Agrobacterium tumefaciens, 5% sucrose and surfactant Silwet L-77 (0.05%).
« in planta » transformation

• The optimal growth stage for transformation by floral dip was when
plants contained numerous unopened floral buds
The three main requirements for successful transformation were:
(1) correct plant developmental stage (maximum number of unopened floral
bud clusters)
(2) sugar
(3) surfactant and/or vacuum to aid infiltration. 21
 Dry and harvest seeds with a
A good stage for floral dipping is
sample bag
when a pot of healthy plants
contain approximately 20–30
inflorescences
1 5

Select primary transformants.

Invert plants and dip their aerial Transgenic plantlets are readily
parts in an Agrobacterium cell distinguished from non-transgenic
suspension for few minutes plants by their green true leaves
and roots that penetrate into the
6 selection medium.
2

Wrap the dipped plants with

plastic films to maintain high Stages during the floral dip transformation
humidity for 16–24 h.
method.
3

Remove the plastic covers and

grow plants in a growth chamber
for 1 month.
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4
Viruses mediated gene transfer

“Genetic material incorporated in the genome of a plant virus might be replicated

and expressed in the plant cell along with the other viral genes.”

Biological characteristics of a virus can be suitably selected and manipulated, and

new genes must be incorporated into the virus in such a way that they are
expressed in the plant.

• The replicating genomes of plant viruses are nonintegrative vectors

• Accumulate to high copy numbers in their respective target cells

Caulimoviruses (CaMV) • genome made of double stranded DNA

Gemini Viruses: single stranded (ss) DNA
RNA Viruses: single stranded RNA

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 2. Physical gene transfer methods
(DNA mediated gene transfer (DMGT)
• no natural vector is involved
• direct delivery of naked DNA to
the plant cells

Schemes for the production of transgenic

plants using direct gene delivery methods.

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 1. Electroporation
• Electrical impulses of high field strength are used to reversibly permeabilize cell
membranes to facilitate uptake of large molecules, including DNA.
ELECTROPORATION INTO MAMMALIAN CELLS

1. Used for both transient and stable transfection of mammalian cells.

2. Cells are placed in suspension in an appropriate electroporation buffer and put into an
electroporation cuvette. DNA is added, the cuvette is connected to a power supply,
and the cells are subjected to a high-voltage electrical pulse of defined magnitude and
length

Culture and harvest the transfected cells

For stable transformation: Grow cells 48

hr (about two generations) in
nonselective medium, then transfer to For transient expression: Incubate cells 50 to
antibiotic-containing medium. 60 hr, then harvest cells for transient
expression assays.
 ELECTROPORATION INTO MUSCLE OR SKIN

1. Plasmid DNA in the appropriate diluent is injected into the tissue.

2. Electrodes are then placed around the injection site and the cells within the tissue are
subjected to a high-voltage electrical pulse of defined magnitude and length.
3. The animals are then allowed to recover and the tissue is evaluated at specified time
points following delivery.

• Factors that can be varied to optimize electroporation effectiveness are pulse width,
number, amplitude and electrode configuration.

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 ELECTROPORATION INTO PLANT PROTOPLASTS
Plant cells are stripped of their cell walls and DNA is introduced into the resulting protoplasts.

• Protoplasts is pulsed with high/low voltage pulses in the chamber (cylindrical in form
with a distance of 1cm between parallel steel electrodes) of
an electroporator.

• Polyethylene glycol (PEG) can increase protoplast transformation frequency.

(assist the association of the DNA with the membrane)

• Difficulty in regenerating plants from protoplasts.

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 2. Particle bombardment/microprojectile/biolistics
• High velocity microprojectiles were utilized to deliver nucleic acids into living cells

• The DNA bearing tungsten or gold particles (1–3 µm in diameter), referred to

as microprojectiles, is carried by a macroprojectile or microcarrier and is
accelerated into living cells.

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• PDS 1000 (gun powder driven device)
or the PDS-1000/He (helium driven particle gun).

• Disc ruptures, compressed helium gas is

suddenly released, which accelerates a
thin plastic sheet carrying
microprojectiles into a metal screen

• Stopping plate or screen stops the

acroprojectile movement but
permits the passage of microprojectiles through
the mesh screen

• Microprojectiles then travel through a

partial vacuum until they reach the target
tissue.

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30
Applications of Microprojectile Bombardment
•Microprojectile bombardment is widely used for introducing foreign genes into plant cells and
producing genetically modified plants with improved traits such as disease resistance and
higher yield.

•Microprojectile bombardment has also been used to generate transgenic animals with specific
desired traits.

•This method of gene delivery allows the study of gene function and expression patterns in
different tissues.

•This method also has applications in gene therapy for delivering therapeutic genes directly
into target tissues to treat genetic disorders, cancer, and other diseases.

•Microprojectile bombardment can also be used to develop DNA vaccines by delivering DNA-
encoding antigens directly into cells.

•It can also be used to deliver fluorescent dyes into cells and tissues to study cellular signaling
processes.

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Advantages of Microprojectile Bombardment
•It is a fast and relatively simple method for delivering desired genetic materials into
cells.
•It can be used to deliver large nucleic acid fragments.
•This method is not dependent on host specificity or species limitations.
•It is a safer method for genetic transformation as it doesn’t require the use of harmful
viruses or toxic chemicals as gene delivery vehicles.
•Unlike other methods that need the cell wall to be removed, gene gun delivery can
penetrate the intact cell wall. This simplifies the process and can be used to transform a
wider range of cells.

Disadvantages:
1. leads to nonhomologous integration into the chromosome, and is characterized
by multiple copies
2. Lack of control over the velocity of bombardment, which often leads to
substantial damage to the target cells.

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 3. Macroinjection
• Injection of DNA solution (5–10µl) by micropipettes into the developing floral
side shoots (tillers) of plants.
• The reported protocol could not be repeated.

4. Microinjection
Direct mechanical introduction of DNA under microscopical control into a specific
target (cell within a multicellular structure such as embryo, ovule, and meristem or
protoplasts, cells or a defined compartment of a single cell.

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 Recipient cells can be immobilized
using methods such as agarose
embedding, agar embedding, poly-
lysine treated glass surfaces and
suction holding pipettes

Glass micropipettes of 0.5–10.0µm diameter

tips are used for transfer of micromolecules
into the cytoplasm or the nucleus of a
recipient cell or protoplast.

Injected cell is cultured properly to ensure its continued growth and development.

Disadvantage:
.
• The microinjection process is slow, expensive and requires highly skilled and
experienced personnel.

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 5. Liposome-mediated transformation
Liposomes: artificial lipid vesicles surrounded by a synthetic membrane of
phospholipids
• DNA enters the protoplasts due to endocytosis of liposomes

i) Adhesion of liposomes to the protoplast

surface.
ii) Fusion of liposomes at the site of adhesion.
iii) Release of plasmids inside the cell.

5. Ultrasound-mediated DNA transformation

• Ultrasound is used for stimulating uptake of foreign DNA by plant protoplasts and
leaf segments of tobacco
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• Little success has been achieved by this technique
Chemical gene transfer methods
• Involves plasma membrane destabilizing and/or precipitating agents
• Sample is protoplast
PEG mediated gene transfer

Direct uptake of DNA by protoplasts is stimulated by polyethylene glycol (PEG) and PEG is
the most widely used chemical for this purpose. PEG mediated transformation involves
mixing of freshly isolated protoplasts with DNA and immediately adding PEG dissolved in a
buffer containing divalent cations. This mixture is incubated for 30 minutes; protoplasts are
washed and then plated in Petri plates for culture and growth.

Particular concentration of protoplast suspension is taken in a tube followed

by addition of plasmid DNA (donor and carrier).

40% PEG 4000 (w/v) dissolved in mannitol and calcium nitrate solution is added
and incubated for few minutes

Regeneration of the protoplast

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 Protoplast Isolation

• Plant cell excluding the cell wall

• Term protoplast was introduced by Hanstein in 1880
• The use of cell wall degrading enzymes was soon recognized as the preferred
method to release large numbers of uniform protoplasts.

The ability of isolated protoplasts to undergo fusion and take up

macromolecules offers many possibilities in genetic engineering and crop
improvement.

The experiments involving protoplasts consist of three stages –

i. protoplast isolation
ii. protoplast fusion / gene uptake
iii. development of regenerated fertile plants from the fusion
product(Hybrid).

Mechanical Method
(large and highly vacuolated cells of storage tissues such as onion bulb scales,
radish root and beet root tissue could be used for isolation)
1. The cells are plasmolysed resulting in the withdrawal of contents in the
center of cell. 37
2. The tissue is dissected and deplasmolyzed to release the preformed protoplasts.

Disadvantages:
(i) It is restricted to certain tissues which have large vacuolated cells.
(ii) Yield of protoplasts is generally very low.
(iii) The method is tedious and laborious.
(iv) Viability of protoplasts is low because of the presence of substances released
by damaged cells.

Enzymatic Method
• Developed by Cocking in 1960
• Most suitable source of protoplasts is mesophyll tissue from fully expanded
leaves of young plants or new shoots.
• The mesophyll cells are loosely arranged and enzymes have an easy access to
the cell wall.

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Schematic illustration of
protoplast isolation and
culture procedure
Cellulose and hemicellulose are the
components of primary and secondary
structure of cell wall, while pectin
is a component of middle lamella that
joins the cells.

Pectinase mainly degrades the middle

lamella, while cellulase and hemicellulase
are required to digest the cellulosic and
hemicellulosic components of the cell
wall respectively.

Most commonly used:

• Cellulase (Onozuka) R10 purified from the
molds of Trichoderma reesei
and T. viride.
• pectinase is macerozyme (macerase) which
has been derived from the Rhizopus fungus.

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General conditions :
• pH between 4.7 and 6.0.
• Temp is 25–30°C
• Duration 30min-20hrs
• Balance of osmoticum : The wall pressure that is mechanically supported by cell
wall must be replaced with an appropriate osmotic pressure in the protoplast
isolation mixture and also later in the culture medium.
Mannitol (Mannitol is considered to be relatively inert
metabolically and infuses slowly into the protoplasts.)
• Protoplast purification: get rid of sub-cellular debris, undigested cells, broken
protoplasts

Filtration :stainless steel or nylon mesh (50–100 μm) to remove larger portions

centrifugation :suspension medium to give a final sucrose concentration of about 20% and then
centrifugation is done at about 100x g for 7–10 min
(gradient centrifugation :Ficoll and Percoll are osmotically inert, their use may be
preferable over sucrose flotation)

Washing :in culture medium)

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 Two step or sequential method One step or simultaneous method

Tissue is first treated with a macerozyme or Tissue is subjected to a mixture of

pectinase enzyme which separates the cells enzymes in a one step reaction which
by degrading the middle lamella. includes both macerozyme and cellulase.

Free cells are then treated with cellulase which

releases the protoplasts.

Frequently used staining methods for assessing protoplast viability:

1. Fluorescein diacetate (FDA) staining method: FDA, a dye that accumulates inside
the plasma lemma of viable protoplasts can be detected by fluorescence microscopy
(yellow green).
2. Phenosafranine staining: Phenosafranine is specific for dead protoplasts that
turn red. Viable cells remain unstained by phenosafranine.
3. Calcofluor White (CFW) staining: Calcofluor White can ascertain the viability
of protoplasts by detecting the onset of cell wall formation.

The concentration of protoplasts in a given preparation can be determined by the use

of hemocytometer. (ideal density is 5 × 103 to 106 cells/ml with an optimum of about
5 × 104 protoplasts/ ml. 41
 Methods of protoplast fusion

Spontaneous fusion
protoplast from adjoining cells fuse
through their plasmodesmata to form
multinucleate protoplasts

• Normally isolated protoplast do not fuse with each other because the
surface of isolated protoplast carries negative charges (-10mV to-
30mV ) around the outside of the plasma membrane.

strong tendency in the protoplast to repel each other

Induced fusion
Fusion needs a fusion inducing chemicals which actually reduce the electronegativity of the isolated
protoplast

Chemofusion Electrofusion
isolated protoplasts from the two selected high strength of electric field
parents are mixed in appropriate (100Kv/m) for some micro seconds
proportions and treated with 15–45 % PEG are applied this lead to fusion.
(1,500–6,000 MW) solution.
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Culture techniques for protoplast:
1. Agar culture: protoplast cultured with agar culture medium
2. Liquid culture
3. Liquid droplet method: suspending protoplasts in culture media and
pipetting 100–200 μl droplets into plastic petri dishes.
4. Hanging droplet method: Small drops (40–100 μl) of protoplast suspension
are placed on the inner side of the lid of a petridish.
5. Feeder layer (nurse cells): Protoplasts are plated on this feeder layer at low
density in a thin layer of agar medium.
6. Co-culturing: It is the culturing of two types of protoplasts viz. slow growing
and fast growing. The fast growing protoplasts presumably provide the other
species with growth factors and undefined diffusible chemicals, which aid the
regeneration of a cell wall and cell division.
Culture medium for protoplast:
• Similar to those of cultured plant cells with certain standardization
-devoid of ammonium as it is detrimental to its survival
-Increased calcium concentration may be important for membrane
stability.
-Mannitol and sorbitol for maintaining the osmolarity
-one or more auxins plus one or two cytokinins to stimulate protoplast
division and growth.
Environmental factors for culture: similar to the general condition for protoplast
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isolation described earlier PLUS 200-5000 lux of illumination.
 Light units: Photosynthetic photon flux density (PPFD)
µmol photons/ m2/s .
lux meter. PPFD (µmol m-2 s-1) Lux
Sunlight 54
Cool White Fluorescent Lamps 74

PROTOPLAST DEVELOPMENT
Cell Wall Formation
• Protoplasts in culture generally start to regenerate a cell wall within a few hours
after isolation and may take two to several days to complete the process under
suitable conditions.
• Can be tracked by staining it with 0.1% calcofluor white fluorescent stain

Growth, Division and Plant Regeneration

• cell wall formation is required before cytokinesis occurs
• The first cell division generally occurs within 2–7 days. The second division
occurs within a week, and by the end of the second week in culture, small
aggregates of cells are present.
• After 3 weeks, small cell colonies are visible
• Macroscopic colonies are transferred to an osmotic free medium to develop
callus.
• The callus may then be induced to undergo organogenic or embryogenic
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differentiation leading to the formation of plants.
 FUSION PRODUCTS-THE HYBRIDS AND CYBRIDS

• The nuclei of two protoplasts may or may not fuse together even after fusion of
cytoplasms.

• The binucleate cells are known as heterokaryon or heterocyte .

• When nuclei and cytoplasm both are fused, the cells are known as somatic hybrid or
synkaryocyte .

• When only cytoplasms fuse and genetic information from one of the two nuclei is lost is
known as cybrid i.e. cytoplasmic hybrid or heteroplast .

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 SOMATIC HYBRIDIZATION

“sexual hybridization is limited in most cases to cultivars within a species or at

best to a few wild species closely related to a cultivated crop.”
The technique of hybrid production through the fusion of isolated somatic (body)
protoplasts under in vitro conditions and subsequent development of their product
(heterokaryon) to a hybrid plant is known as somatic hybridization.

*nucleus and cytoplasm of both parents are fused in the hybrid cell
*Sometimes, nuclear genome of only one parent but cytoplasmic genes
(plastome) from both the parents are present in the fused hybrid, which
is known as cybrid or cytoplasmic hybrid.

Advantage over sexual hybridization

1. used to overcome the barriers of incompatibility and acts as a method for the genetic
manipulation of plant cells.
2. It provides us with an opportunity to construct hybrids between taxonomically distant
plant species beyond the limits of sexual crossability.
3. Creates cells with new genetic, nuclear as well as cytoplasmic constitutions that otherwise
cannot be obtained.
Steps of Somatic hybridization
Three main phases of protoplast fusion:
i. Agglutination or adhesion
ii. Plasma membrane fusion at localized sites
results in the formation of cytoplasmic bridges
iii. Formation of heterokaryon
Rounding off of the fused protoplasts due to the expansion
of cytoplasmic bridges forming spherical heterokaryon or homokaryon.
2. IDENTIFICATION AND SELECTION OF HYBRID CELLS
About20-25% of the protoplasts are actually involved in the fusion.
a) Chlorophyll deficiency complementation
most frequently used method to isolate somatic hybrids

• two distinct homozygous recessive albino mutants of

Nicotiana tabacum.

A selection scheme involving

complementation of chlorophyll deficient
mutations.
 b) Auxotroph complementation

A summary of somatic hybrid selection

based on auxotroph complementation.

c) Complementation of resistance markers

S2-aminoethyl cysteine (AEC)

5-methyl tryptophan (5MT) resistant parental
lines
(d) Use of visual characteristics:

• Fluorescent labeling (green and red fluorescence in parent lines)

• Based on morphology of callus
 Verification and Characterization of Somatic Hybrids

1. Morphology
2. Isoenzyme analysis
3. Chromosomal constitution
4. Molecular techniques:
Specific restriction patterns of chloroplast and mitochondrial DNA
have been used to great advantage to characterize the nature of
plastoms and chondrioms of somatic hybrids and cybrids.

CHROMOSOME NUMBER IN SOMATIC HYBRIDS

Variation in chromosome number (aneuploidy) is generally observed in hybrids
but frequently chromosome number is more than the total number of both the
parental protoplasts.
The variability in chromosome number of hybrids could be due to following reasons:
i) Multiple fusions give a higher chromosome number.
ii) Fusion of more than two protoplasts with subsequent mitotic irregularities.
iii) In PEG and electro-induced fusions about one-third of fusion products result from
fusions among more than two protoplasts.
iv) Asymmetric hybrids result from fusion of protoplasts isolated from actively dividing
tissue of one parent and quiescent tissue of the other parent. These hybrids are usually
formatted with full somatic complement of one parental species while all or nearly all of the
chromosomes of other parental species are lost during mitotic divisions.
Applications of Somatic Hybridization

•Crop Improvement: Somatic hybridization is used to develop crop varieties

with desirable traits such as disease resistance, stress tolerance, and
improved yield.
•Genetic Diversity Enhancement: By combining genetic material from
different plant species, somatic hybridization contributes to the enrichment of
genetic diversity in breeding programs.
•Creation of Novel Varieties: This technique enables the creation of novel
plant varieties with unique combinations of traits not found in traditional
breeding programs.
•Biotechnological Research: Somatic hybridization is instrumental in studying
gene expression, functional genomics, and understanding the mechanisms of
plant development and stress responses.

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Somatic Hybridization Examples

•Pomato: Created by merging tomato and potato protoplasts, resulting in a

single plant that produces both tomatoes and potatoes.

•Wheat-Agropyron hybrids: Used to transfer genes for disease resistance and

environmental adaptation from wild grasses to cultivated wheat.

•Citrus intergeneric hybrids: Generated by fusing protoplasts from different

citrus species, producing hybrids with improved fruit quality and disease
resistance.

•Brassica napus (rapeseed) hybrids: Utilized to introduce novel traits like

herbicide resistance and increased oil content from related Brassica species.

•Banana breeding: Employed to combine traits for disease resistance and

improved fruit quality from different banana varieties.

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 CYBRIDS

cytoplasmic hybrids / cybrids

“nucleus is derived from one

parent and cytoplasm is derived from both”

Schematic illustration of somatic hybridization

which can produce complete hybrids (left) or
cytoplasmic hybrids (right).
• Offers a unique opportunity to study the interaction of the cytoplasmic organelles.

Interparental recombination of mitochondrial genomes and independent

assortment of chloroplasts and mitochondria following cell fusion results in plants
with novel combinations of nuclear/plastid/mitochondria genomes

Methods to produce cybrids

(a) fusion of a normal protoplast with an enucleate protoplast
(b) fusion between a normal protoplast and a protoplast containing non-viable nucleus
(c) elimination of one of the nuclei after heterokaryon formation
(d) selective elimination of chromosomes at a later stage
 RNA-guided CRISPR-Cas nuclease system
edit parts of the genome by removing, adding or altering sections of the DNA sequence.

Mechanism of genome editing: Double-strand break (DSB) induced by nucleases can be repaired by non-homologous
end joining (NHEJ) or homology-directed repair (HDR) pathways. NHEJ can introduce random insertions or deletions
(indels) of varying length at the site of the DSB. Alternatively, HDR can introduce precise genomic modifications at the
target site by using a homologous DNA donor template.
 RNA-guided CRISPR-Cas nuclease system
edit parts of the genome by removing, adding or altering sections of the DNA sequence.

• An enzyme called Cas9: This acts as a pair of ‘molecular

scissors’ that can cut the two strands of DNA at a specific
location in the genome so that bits of DNA can then be
added or removed.

• Piece of RNA called guide RNA (gRNA): This consists of

a small piece of pre-designed RNA sequence (about 20
bases long) located within a longer RNA scaffold. The
scaffold part binds to DNA and the pre-designed
sequence ‘guides’ Cas9 to the right part of the genome.
This makes sure that the Cas9 enzyme cuts at the right
point in the genome.
The guide RNA is designed to find and bind to a specific sequence in the DNA.
(The guide RNA has RNA bases that are complementary to those of the target DNA
sequence in the genome.

The guide RNA will only bind to the target sequence and no other regions of the
genome.

The Cas9 follows the guide RNA to the same location in the DNA sequence and
makes a cut across both strands of the DNA.

At this stage the cell recognizes that the DNA is damaged and tries to repair it.

Scientists can use the DNA repair machinery to introduce changes to one or more genes in
the genome of a cell of interest.
 Clustered
Regularly
First discovered in the sequences of DNA from
Interspaced Escherichia coli bacteria
Short
Palindromic
Repeats
and CRISPR-associated protein 9

Short palindromic Repeats

CRISPER

Non-identical Spacer DNA

• Each spacer DNA is unique
• Matches with certain bacteriophages DNA

associated with CAS genes

helicase
CAS nuclease
Immune system of bacteria that could fight the bacteriophages
 Case 1: when the bacteria has the spacer DNA specific to the bacteriophage

The crRNA-CAS breaks the viral DNA

Case 2: when the bacteria do not have a spacer DNA specific to the bacteriophage

Class 1 CAS Class 1 CAS Class 1 CAS

protein protein protein
Tracer RNA: holds the crRNA in place
 (guide RNA/ gRNA)

• gRNA contain the information where it is going to cut

• Cas9 is going to cut
How we use it:

Create a gRNA which has a

part complementary to the
target DNA sequence

Target DNA sequence in genome

Silence a gene

Double Strand Break

Insert a desired DNA sequence:

Foreign DNA sequence that we want to put in

(Donor DNA)
 What is the PAM sequence for CRISPR ?
In order for Cas9 to function, it also requires a
specific protospacer adjacent motif (PAM) that
varies depending on the bacterial species of the
Cas9 gene. The most commonly used Cas9
nuclease, derived from S. pyogenes, recognizes a
PAM sequence of NGG that is found directly
downstream of the target sequence in the
genomic DNA, on the non-target strand.

Not all sequences can serve as a CRISPR/Cas target site; this is because all target sites
require a PAM sequence immediately adjacent to its target. PAM is required for Cas
enzyme function and PAM sequence serves as a binding signal for Cas nuclease; without a
PAM sequence, Cas enzyme does not know where to bind and where to cut the sequence.
Thus, PAM sequence is required for all current CRISPR/Cas systems used for genome
editing.
Based on the DNA double strand break
(DSB) repair mechanism, CRISPR can
directly cause gene knockout (silencing)
by insertion or deletion of a couple of
nucleotides and repaired by non-
homologous end join (EHEJ); however, if
the homologue-directed repair (HDR)
happed, with a DNA donor, CRISPR/Cas
genome editing can be used to replace an
undesirable gene or over express
(knockin) and an individual gene.
Limitations
•difficult to deliver the CRISPR/Cas material to mature cells in large numbers, which remains
a problem for many clinical applications.
•not 100% efficient, so even the cells that take in CRISPR/Cas may not have genome editing
activity.
•not 100% accurate, and “off-target” edits, while rare, may have severe consequences,
particularly in clinical applications.
 Advantage: Eliminating Transgenic Sequences Through Genetic Segregation

Schematics showing the main strategies for isolating transgene-free and genome-edited
plants Eliminating transgenic sequence through genetic segregation. CRISPR/Cas DNA
(represented by red double helix) is delivered into plant cells using Agrobacterium
tumefaciens or particle bombardment. The transgenic plants are isolated, and then genome
edited plants are selected through target site genotyping. The transgene-free and genome
edited plants are isolated from progenies of transgenic genome edited plants which is
facilitated by counter-selection
Gu X, Liu L and Zhang H (2021 Transgene-free Genome Edit in in Plants [Link] Ed. 3:805317.
doi: 10.3389/fgeed.2021.805317 72
Delivering editors in a DNA independent manner. CRISPR/Cas9 RNA or Ribonucleoproteins
(RNPs) are delivered into plant cells by polyethylene glycol (PEG)-, virus- or particle
bombardment mediated transformation, and then transgene-free and genome edited plants
are isolated from all the regenerated seedlings by target site genotyping. Mutation on target
site is represented by yellow star and transgene integration by red dot.

73
Comparison of traditional genetic engineering and CRISPR-Cas editing technique
Ahmad A, Jamil A and Munawar N (2023) GMOs or non-GMOs? The CRISPR Conundrum. Front. Plant Sci. 14:1232938. 74
doi: 10.3389/fpls.2023.1232938
 • the use of antibiotic resistant marker genes for the selection of transformants was also a
concern in their public acceptance.

• Majority of the transgenic crops contain genes from unrelated species, transferred
through Agrobacterium, to improve crops against insects or to withstand herbicides.
These crops could induce pest resistance by releasing toxins in soil and destroying crop
biodiversity, thus could have an adverse environmental impact.

• GM crops having bacterial or insect genes have raised health concerns such as allergic
reactions which have been reported in humans in different countries

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Depending on the repair outcomes of DSBs, CRISPR mediated modifications are classified
into three main categories:
Site-Directed Nuclease types 1, 2, and 3 (SDN1, SDN2, and SDN3),
➢ In SDN1, DSBs are repaired through NHEJ repair system which introduces indels (adds
or deletes nucleotides) without using any repair template.

➢ In SDN2, a microhomologies-mediated repair template is used to add, delete, or

replace very few (2-10) specific nucleotides at the target site.

• The resultant plants in both SDN1 and SDN2 are indistinguishable from conventionally
bred plants, and thus could be considered non-GM.
• Most countries like the US, Japan, India, Australia, and Ecuador consider SDN1 and
SDN2 modified plants safe and do not regulate them under conventional GM
regulations.

In SDN3, a repair template through homologous recombination is used to insert a gene

segment or whole gene at the targeted site, resulting in transgenic or cis-genic plants,
consequently, triggering regulatory oversight depending on the nature and origin of the
introduced DNA segment

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 There are TWO main regulatory triggers for the regulation of GM crops in the world

•Product-based regulation: This approach focuses on the final characteristics

or traits of the GM product, regardless of the methods used to create it. It
assesses the safety and impact of the product itself. The USA follows this
model, meaning that regulators evaluate GM crops based on their final form
(traits like pest resistance or herbicide tolerance) rather than how they were
produced.
•Process-based regulation: In this system, the emphasis is on how the GM
crop was developed, specifically the techniques and processes of genetic
modification. The EU follows this regulatory framework, which means that crops
produced using genetic engineering are subject to strict regulations, regardless
of their end-product characteristics.

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Timeline of articles describing the application of the CRISPR/Cas9 method in cereals within the first decade after the CRISPR discovery (2013-
June, 2023).
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“In late 2023, we saw the first-ever approval of CRISPR-based medicine: Casgevy, a cure
for sickle cell disease (SCD) and transfusion-dependent beta thalassemia (TDT).”
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contains five functional genes designated:
All the hemoglobins formed from the different -like globins carry oxygen in the
β blood, but they exhibit somewhat different properties that are suited to specific
δ roles in human physiology. For example, hemoglobins containing either the A γ or
Aγ G γ polypeptides are expressed only during fetal life. Because these fetal
hemoglobins have a higher affinity for oxygen than adult hemoglobins, they can
Gγ
effectively extract oxygen from the maternal circulation in the placenta. The
ε lower oxygen affinity of adult hemoglobins, which are expressed after birth,
permits better release of oxygen to the tissues, espcially muscles, which have a
high demand for oxygen during
exercise

Sickle cell disease (SCD) and another genetic disorder affecting the hemoglobin in red blood
cells, transfusion-dependent beta thalassemia (TDT), were among the first targets of CRISPR-
based treatments. CRISPR Therapeutics and Vertex, the makers of Casgevy, as well as a
number of other groups, take the approach of inducing expression of fetal hemoglobin (HbF),
a kind of hemoglobin usually only found in fetuses and very young infants. If the gene for fetal
hemoglobin is turned “on” in adults, it can do the job of the healthy adult hemoglobin that
individuals with SCD and TDT are missing.
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