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1. • The plastid is a major double-membrane organelle found
in the cells of plants, algae, and some other eukaryotic
organisms.
• Plastids are the site of manufacture and storage of
important chemical compounds used by the cell
• the types of pigments in a plastid determine the cell's
color
• They have a common evolutionary origin and possess a
double-stranded DNA molecule that is circular, like that of
prokaryotic cells
Plastid

2. Proplastids: undifferentiated
plastids
Etioplasts: the predecessors of
chloroplasts
Chromoplasts: coloured
plastids, for pigment synthesis
and storage
Leucoplasts: colourless plastids
1. Amyloplasts: for starch
storage and detecting
gravity (for geotropism)
2. Elaioplasts: for storing fat
3. Proteinoplasts: for storing
and modifying protein

3. •Chloroplast Plastids are plant cellular organelles with a
~120–150kb genome size
•The chloroplast genome most commonly includes around
100 genes
•Genome present in 1,000–10,000 copies per cell
•Maternally inherited in most angiosperm plant species
•Plastid genomes resemble bacterial genomes in many
aspects and also contain some features of multicellular
organisms, such as RNA editing and split genes (Exon-
Intron)
Chloroplast

5. Chloroplasts originated from endosymbiosis around 1.5
billion years ago, when a cyanobacterial cell was engulfed
by heterotrophic eukaryote .
The chloroplast genome is heavily reduced compared to
that of free- living cyanobacteria. Over time, many parts of
the chloroplast genome were transferred to the nuclear
genome of the host.
Chloroplasts may contain 60–100 genes whereas
cyanobacteria often have more than 1500 genes in their
genome.
Chloroplast genome reduction and
gene transfer

6. • Duplicate genes encoding each of the four subunits (23S, 16S,
4.5S, and 5S) of the ribosomal RNA (rRNA) used by the
chloroplast
• 37 genes encoding all the transfer RNA (tRNA) molecules used
for translation within the chloroplast.
• 4 genes encoding some of the subunits of the RNA polymerase
used for transcription within the chloroplast
• A gene encoding the large subunit of the enzyme RUBISCO
(ribulose bisphosphate carboxylase oxygenase)
• 9 genes for components of photosystems I and II
• 6 genes encoding parts of the chloroplast ATP synthase
The genome of the chloroplasts
found in Marchantia polymorpha
( liverwort)

8. • A transplastomic plant is a genetically modified plant in
which genes are inactivated, modified or new foreign genes are
inserted into the DNA of plastids like the chloroplast instead of
nuclear DNA.
• The major advantage of this technology is that in many plant
species plastid DNA is not transmitted through pollen, which
prevents gene flow from the genetically modified plant to other
plants.
Transplastomic plant
Genetically modified plants must be safe for the environment and
suitable for coexistence with conventional and organic crops
• Towards such safety, a major hurdle is posed by the potential
outcrossing of the transgene via pollen movement
• Plastid transformation, which yields transplastomic plants in which
the pollen does not contain the transgene, not only increases
biosafety, but also facilitates the coexistence of genetically modified,
conventional and organic agriculture

9. Transformation History of chloroplast
•Plastid transformation was first achieved in unicellular algae.
•Tobacco was the first higher plant in which chloroplast
transformation was successfully performed
•A protocol for plastid transformation of an elite rapeseed cultivar
(Brassica napus L.) has been developed.
Generally, three key conditions have to be full- filled to achieve
plastid transformation
1- A method of DNA delivery into the chloroplast
2- The presence of active homologous recombination machinery in
the plastid
3- The availability of highly efficient selection and regeneration
protocols for transplastomic cells

10. Biolistic approach
Polyethylene glycol (PEG) treatment of protoplasts
Biolistic Method :
Plastid vector DNA is coated onto high-density tungsten or gold
microprojectiles (0.6–1 μM diameter), which are then delivered at
high velocity first through the cell wall and membrane, and then
through the double-plastid membrane
Methods of Chloroplast Transformation
Two methods are available to stably transform plant plastids

11. PEG Method:
Protoplasts are plant cells with their wall removed by enzyme
treatment.
Treatment of freshly isolated protoplasts with PEG allows
permeabilization of the plasma membrane and facilitates uptake of
DNA. Subsequently, with a mechanism largely uncharacterized, the
plasmid DNA passes the plastid membranes and reaches the stroma
where it integrates into the plastome (genome of plastid) as during
biolistic transformation.
A relatively small number of species have been transformed using this
approach , mainly because it requires efficient isolation, culture and
regeneration of protoplasts, a tedious and technically demanding in
vitro technology. On the positive side, no special equipment is needed.

12. Chloroplast transformation in Chlamydomonas:
Chlamydomonas comprises a single large chloroplast with about
hundreds of copies of its genome. Initial integration occurs in only
one copy of the polyploid plastome resulting in heteroplasmic.
Repeated sub-cloning and selection result in recovery of
homoplasmic clone .

14. Regulatory sequences
• The gene expression level in plastids is predominately
determined by promoter and 5′-UTR and 3′-UTR elements
• suitable 5′-untranslated regions (5′-UTRs) including a
ribosomal binding site (RBS) are important elements of plastid
expression vectors
• In order to obtain high-level protein accumulation from
expression of the transgene, strong promoter is necessary
• Most laboratories used the strong plastid rRNA operon (rrn)
promoter (Prrn)
• Stability of the transgenic mRNA is ensured by the 5′-UTR and
3′-UTR sequences flanking the transgenes.
• The most commonly used 5′-UTR and 3′-UTR is psbA/TpsbA
Plastid expression vectors

15. Insertion sites
• Plastid expression vectors possessed left and right flanking
sequences each with 1–2 kb in size from the host plastid
genome, which are used for foreign gene insertion into plastid
DNA via homologous recombination
• The site of insertion in the plastid genome is determined by
the choice of ptDNA segment flanking the marker gene and
the gene of interest

16. Selection marker genes
• Since ptDNA (plastid DNA) is present in many copies, selectable
marker genes are critically important to achieve uniform
transformation of all genome copies during an enrichment
process that involves gradual sorting out non-transformed plastids
on a selective medium .
Selection marker gene used in chloroplast transformation :
 A gene confers resistance to spectinomycin and streptomycin
(aadA)
 The bacterial bar gene (encoding phosphinothricin
acetyltransferase
(PAT)
 Betaine aldehyde dehydrogenase (BADH) gene which confers
resistance to betaine aldehyde.

17. Chloroplast transformation Nuclear transformation
Reduced gene dispersal in the
environment due to maternal
inheritance
There is gene dispersal in the
environment due to its parental
nature
Multiple copy (high ploidy) of plastids
results higher expression and
accumulation of foreign proteins
Nuclear is not in high ploidy results
lower expression and accumulation of
foreign proteins
Single promoter for expression of
multi- subunit complex protein from
polycistronic mRNAs
Several promoters for each genes to
drive expression of respective
subunits
Simultaneous expression of several
genes as it contains prokaryotic gene
expression system
Do not have prokaryotic expression
system can’t undergo simultaneous
expression of several genes
Homologous recombination avoids
position effects and gene silencing
Random integration presents position
effects and gene silencing

18. Advantages of chloroplast engineering:
1. No position effect:
Absence of position effect due to lack of a compact chromatin
structure and efficient transgene integration by homologous
recombination.
Avoids inactivation of host gene by transgene integration.
2. Disulfide bond formation:
Ability to form disulfide bonds and folding human proteins results in
high-level production of biopharmaceuticals in plants.
3.Risk of transgene escape:
Chloroplast genome is maternally inherited and there is rare
occurrence of pollen transmission. It provides a strong level of
biological containment and thus reduces the escape of transgene
from one cell to other.

19. 4. Expression level:
It exhibits higher level of transgene expression and thus higher level
of protein production due to the presence of multiple copies of
chloroplast transgenes per cell
5. Expression of toxic proteins:
Foreign proteins observed to be toxic in the cytosol are non-toxic
when accumulated within transgenic chloroplasts as they are
compartmentalized inside chloroplast.
6. No Gene Silencing:
Gene silencing or RNA interference does not occur in genetically
engineered chloroplasts.
A lack of epigenetic interference allowing stable transgene
expression.

20. 7. Multiple gene expression:
Multiple transgene expression is possible due to polycistronic mRNA
transcription.
8. Homologous recombination :
Chloroplast transformation involves homologous recombination and is
therefore precise and predictable.
This minimizes the insertion of unnecessary DNA that accompanies in
nuclear genome transformation.
This also avoids the deletions and rearrangements of transgene DNA,
and host genome DNA at the site of insertion.

21. Chloroplast transformation has been achieved in these plants
• tobacco • lettuce • Arabidopsis • tomato • carrot • oilseed rape •
potato, • Cabbage • cotton • Petunia • soybean • Sugarcane • sugar
beet • Rice • Eggplant • cauliflower • poplar

22. Integration of transgenes into the cotton cultures was confirmed by
PCR using internal primers, first primer anneals to the flanking
sequence and second primer anneals to the transgene region. An
expected size of PCR product was amplified and this confirmed
integration of the transgenes in different cell cultures of plant
Integration of the transgenes into plastid genome were investigated by
Southern blot analysis. genomic DNA from transformed and
untransformed cultures was digested with appropriate restriction
enzymes, transferred to nitrocellulose membrane and probed with P32-
radiolabel .
Confirmation of transgene integration into chloroplast
genome

23. 1. Transformation frequencies are much lower than those for
nuclear genes.
2. Prolonged selection procedures under high selection pressure
are required for the recovery of transformants.
3. The methods of transgene transfer into chloroplasts are limited,
and they are either expensive or require regeneration from
protoplasts.
4. Products of transgenes ordinarily accumulate in green parts
only.
Limitations of Chloroplast Transformation

24. Aims of chloroplast genetic engineering applications include
• Crops resistance to insects, bacterial, fungal and viral diseases
• Plants with different types of herbicides, drought, salt and cold
tolerance
• Production of many vaccine antigens
• Production of biopharmaceuticals and industrial enzymes
• Production of biofuels