The document discusses chloroplast transformation, detailing the unique characteristics and advantages of targeting chloroplasts for genetic engineering in plants, including high levels of protein production and gene containment. It outlines key milestones in chloroplast engineering, methods of DNA delivery, and the design of vectors for effective transformation. Additionally, it highlights the applications of chloroplast transformation in improving agronomic traits and producing pharmaceuticals.

1. Chloroplast Transformation and its
Applications to Plant Improvement
MD JAKIR HOSSAIN
Doctoral Student
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
Presented By

2. What is Chloroplast ?
 Chloroplast is a plastid containing chlorophyll and other pigments
occurring in plants and eukaryotic algae and which possess their
own genome or plastome, besides nuclear genome that carry out
photosynthesis.
Fig- Diagrammatic Presentation of Chloroplast

3.  Genetic materials in plants is distributed into nucleus, plastids
and mitochondria.
 There are up to 300 plastids in one plant cell.
 In most angiosperm plant species (80%) plastids are strictly
maternally inherited.
What is Chloroplast ?

4. Organization of Chloroplast Genome
- Size ~120–150 (30-201) kb that encode ~120 genes, responsible for
gene expression, photosynthesis and metabolism
 Example- 107 kb in Cathaya argyrophylla , 218 kb in Pelargonium
- 1,000-10,000 copies of Genomes in a single cell
- In case of Arabidopsis thaliana nucleus encode about 2100
chloroplast proteins and the whole chloroplast genome encodes for
117 proteins.

5. Organization of Chloroplast Genome
 Circular double-stranded DNA through construction of -
 complete genome maps,
 a large single copy (LSC),
 a small single copy (SSC), and
duplication of a large (~25 kb) Inverted region (IRs)
 The number of copies of plastomes per leaf cell-
-1000 to 1700 in Arabidopsis thaliana and
- up to 50,000 in Triticum sp.

6. Why Chloroplast is a Unique Transformation tool ?
 Sources of Enormous Advantages
-sequestration of carbon, production of starch, and evolution of oxygen, synthesis of amino
acids, fatty acids, and pigments, and key aspects of sulfur and nitrogen metabolism
 Have diverse functions
• Chromoplasts– for pigment synthesis and storage
• Gerontoplasts – control dismantling of photosynthetic apparatus during senescence
• Leucoplasts –– monoterpene synthesis
 An attractive alternative to nuclear gene transformation
-High protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs,
and gene containment through the lack of pollen transmission.

7.  Precursor for Photosynthesis
Getaway to high-throughput genome sequencing
- more than 230 photosynthetic organisms including 130 higher plants.
 An excellent tool for phylogenetic and evolutionary studies
-Endosymbiosis -cyanobacterial cell - engulfed by heterotrophic eukaryote
-Chloroplast organelle -evolved from photosynthetic bacteria
 Storage Compartment for Biosynthetic Pathways.
Why Chloroplast is a Unique Transformation tools ?

8. Why Chloroplast is a Unique Transformation tools ?
Protein accumulator - soluble proteins and intrinsic membrane proteins.
 Cellular location for valuable recombinant products
 Own genetic systems and genomes, high copy number,
transcription translation machinery.
 Plastid posses prokaryotic gene expression machinery.

9. Why Plastid transformation is Preferred?
• High level of transgene expression and protein accumulation
• The possibility of co-expressing several transgenes in operons
• The precise transgene integration by homologous recombination .
• The feasibility of expressing multiple proteins from polycistronic
mRNAs
• Regeneration of crop plants with higher resistance to biotic and
abiotic stresses and molecular pharming.

10. • Absence of epigenetic effects
• Uni-parental inheritance is commercially favored
• Easy transgene stacking in operons
• Foreign protein accumulation of up to > 40% of TSP,
70% in Tobacco
• Absence of position effects due to lack of a compact
chromatin structure
• Efficient transgene integration by homologous
Why Plastid transformation is Preferred?

11. Plastid Transformation- Superior than Nuclear Transformation
Chloroplast transformation Nuclear transformation
Reduced gene dispersal - maternal
inheritance
Extensive Gene dispersal -parental
nature
Higher expression and accumulation
of foreign proteins
Lower expression and accumulation
of foreign proteins
Efficient multiple gene expression multiple gene expression is very
poor
Single promoter for expression of
multi-subunit complex protein
Several promoters for xpression of
respective subunits
Simultaneous expression of several
genes - 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
Transgenes is much higher; up to
18%.
Transgenes expression of total
proteins-0.5 - 3%

12. Traits Nuclear genome Plastid genome
Chromosomes Two copies of each of many chromosomes
the number of chromosomes per diploid
cell is species -specific
~60 copies of a single circular
chromosome per plastid
Genes per
chromosome
Could be thousands ~120–150
Arrangement
and operons
transcription
of genes
Each gene is separate (individually
transcribed )
Many genes are in
( transcribed together)
Comparison of the nuclear and plastid genome

13. Milestones of Chloroplast engineering
Year Milestones
DNA
Delivery
Approach Selection
1988 1st stable plastid transformation in
Chlamydomonas reinhardtii
Biolistic Homologous
Targeting
Photosynthetic
Competence
1990 1st stable plastid transformation in
Nicotiano tabacum
Biolistic Homologous
Targeting
Spectinomycin
1993 1st high level foreign protein expression
in Nicotiano tabacum
PEG Homologous
Targeting
Spectinomycin
kanamycin
1995 New agronomic traits: Bacillus
thuringiensis
Marker gene elimination: Co-
transformation
Biolistic Homologous
Targeting
Spectinomycin
1998 1st stable plastid transformation in
Arabidopsis thaliana
Biolistic Homologous
Targeting
Spectinomycin
1999 1st stable plastid transformation in
Solanum tuberosum and 1st stable
plastid transformation in Oryza sativa
Biolistic Homologous
Targeting
Spectinomycin

14. Milestones of Chloroplast engineering
Year Milestones DNA
Delivery
Approach Selection
2000 1st human protein expression in Nicotiano
tabacum
Biolistic Homologous
Targeting
Photosynthetic
Competence
2001 1st stforeign protein in Lycopersicon
esculentum (tomato), Marker gene elimination:
CRE-lox, New agronomic traits: glyphosate
tolerance and PPT resistance
Biolistic Homologous
Targeting
Spectinomycin
2002 1st stable plastid transformation in
Porphyridium sp.
PEG Homologous
Targeting
Spectinomycin
2003 Chlamydomonas reinhardtii: Foot-and-mouth
disease virus VP1 protein expression, 1st
stable plastid transformation in Brassicacea
(oil seeds) and Phytoremediation: Murkery
Biolistic Homologous
Targeting
Spectinomycin
2004 1st stable plastid transformation in Gossypium
hirsutum (cotton), 1st stable plastid
transformation in Glycin max (soybean), PHB
polymer expression in Linum
usitatissimum L. (flax)
Biolistic Homologous
Targeting
Spectinomycin
Aph-A6
npt 2

15. Chloroplast transformation requires
1. A chloroplast specific expression vector
2. A method for DNA delivery
3. An efficient selection for the transplastome

16. key conditions to achieve plastid transformation
Generally, three key conditions have to be full-filled to achieve plastid
transformation:
(1) A robust method of DNA delivery into the chloroplast
(2) The presence of active homologous recombination machinery in
the plastid, and
(3) The availability of highly efficient selection and regeneration
protocols for transplastomic cells

17. Steps in chloroplast genetic engineering
• Aseptic growth of plant on MS medium
• Biolistic particle treated with vector and other chemicals
• Injection of recombinant DNA Plasmid into chloroplast using
Gene gun or other methods.
• After 2 days, leaves cut into section and transferred to medium
containing an antibiotic ,for recombinant selection.

18. Steps in chloroplast genetic engineering
 Green calli formed on the bleached leaf are sub-cultured on the
same medium Calli formed shoots
 These shoots were rooted on MS medium to obtain plants,
express the desired protein.

19. Presently, both Biolistic and PEG (polyethylene Glycol) treatment of
protoplasts have been used to DNA delivery
 The first one consists in bombarding of tissue or cells with DNA
coated particles.
 The second method treats isolated protoplasts with PEG.
 Micro injection is also used (femto syringe)
METHODS USED IN GENE DELIVERY INTO PLASTIDS

20. METHODS USED IN GENE DELIVERY INTO PLASTIDS
Table 1- Chloroplast transformation methods and selection conditions for different plant species
Plant spieces Methods Selection Expressed genes
Chlamydomonas reinhardtii Bombardment photosynthetic
competence
atpB
Nicotiana tabacum (tobacco) Bombardment Spectinomycin rrn16
Nicotiana tabacum (tobacco) PEG Spectinomycin rrn16
Nicotiana tabacum (tobacco) Bombardment Kanamycin nptII
Nicotiana tabacum (tobacco) Bombardment Spectinomycin uidA
Nicotiana tabacum (tobacco) Bombardment Spectinomycin Bar & aadA, Bt
Arabidopsis thaliana Bombardment Spectinomycin aadA
Solanum tuberosum (potato) Bombardment Spectinomycin aadA & gfp
Rice Bombardment Spectinomycin aadA & gfp
Lycopersicon esculentum (tomato) Bombardment Spectinomycin aadA
Brassica napus (oilseed rape) Bombardment Spectinomycin aadA & cry1Aa10
Gossypium hirsutum (Cotton) Bombardment Kanamycin aphA-6
Glycine max (Soybean) Bombardment Spectinomycin aadA

21. Fig. Steps in volves in Plant plastid engineering by Gene gun

22. BIOLISTIC METHODS OF GENE DELIVERY
Advantages
Simple operation and high efficiency makes it a favorable
No need to obtain protoplast as the intact cell wall can be
penetrated.
This device offers to place DNA or RNA exactly where it is
needed into any organism.
Disadvantages
The transformation efficiency may be lower than
Agrobacterium- mediated transformation.
Associated cell damage can occur.
 The target tissue should have regeneration capacity.

23. PEG METHODS OF GENE DELIVERY
 PEG-mediated transformation of plastids requires enzymatically
removing the cell wall to obtain protoplasts, then exposing the
protoplasts to purified DNA in the presence of PEG.
 The protoplasts first shrink in the presence of PEG, then lyse due
to disintegration of the cell membrane. Removing PEG before the
membrane is irreversibly damaged reverses the process.
 Treatment of freshly isolated protoplasts with PEG allows
permeabilization of the plasma membrane and facilitates uptake of
DNA.

24. PEG METHODS OF GENE DELIVERY
Plasmid DNA passes the plastid membranes and reaches the
stroma where it integrates into the plastome 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.

25. PEG METHODS OF GENE DELIVERY
Advantages
 A large number of protoplasts can be simultaneously transformed.
 This can be successfully used for a wide range of plant species
with adequate modifications.
Disadvantages
 The DNA is susceptible for degradation and rearrangement.
 Random integration of foreign DNA into genome may result in
undesirable traits.
 Regeneration of plants from transformed protoplasts is a difficult
task.

26. Plant species Gene introduced
Nicotiana tabacum rrn16, nptII, uidA, hST, cry, cry9Aa2, Bar & aadA, rbcL, DXR, gfp,
Cor 15a-FAD7, Delta(9) desaturase , AsA2, PhaG & PhaC
Solanum tuberosum aadA & gfp
Oryza sativa aadA & gfp
Solanum lycopersicon aadA, Lyc
Brassica napus aadA & cry1Aa10, aadA
Daucus carota dehydrogenase (badh)
Gossypium hirsutum aphA-6
Glycine max aadA
Lactuca sativa gfp
Brassica oleracea gus & aadA
Lettuce gfp
Brassica oleracea aadA & uidA
Beta vulgaris aadA & uidA
Solanum melongena aadA
Arabidopsis thaliana pre-Tic40-His
Zea mays ManA
A List of Some Transplastomic Plants that has Engineered for Various Agronomic Traits

27. Vector Design for Chloroplast Transformation
Fig. Basic design of a typical vector for transforming the plastid genome
P- Promoter and direction of transcription, T- Terminators, White circles- UTRs, The
thin dotted lines with arrows indicate homologous recombination.

28. Vector Design for Chloroplast Transformation
Selectable Marker genes-
 Spectinomycin resistance- The most efficient and routinely used
 16S rRNA (rrn16) gene- Initially used and selected by spectinomycin
resistance with low efficiency.
 aadA (aminoglycoside 3′ adenylyltransferase) gene- Dominant
marker gene that confers resistance to streptomycin and spectinomycin by
inactivation of antibiotics.
 Plastid expressed GFP (green fluorescent protein)- a visual marker
for identification of plastid transformants at the early stage of selection and shoot
regeneration.
 The npt II- Transformation efficiency was low, i.e. about one transplastomic
line per 25 bombarded samples

29. Vector Design for Chloroplast Transformation
Selectable Marker genes-
 neo gene-yielded 34 kanamycin resistant clones out of Bombardment of 25
leaves
 The bacterial bar gene, encoding phosphinothricin acetyltransferase
(PAT)- it was not good enough
 Betaine aldehyde dehydrogenase (BADH) gene- efficiency was 25-
fold higher with betaine aldehyde (BA) selection than with spectinomycin in
tobacco

30. Insertion sites-
 Insertion of foreign DNA in intergenic regions of the plastid
genome had been accomplished at 16 sites, most commonly used
insertion sites are - trnV-3'rps12 ,trnI-trnA and trnfM-trnG
 The trnV-3'rps12 and trnI-trnA sites- located in the 25 kb inverted
repeat (IR) region of ptDNA and a gene inserted into these sites would be
rapidly copied into two copies in the IR region.
Vector Design for Chloroplast Transformation

31. Insertion sites-
 The trnfM-trnG site- Located in the large single copy region of the
ptDNA, and the gene inserted between trnfM and trnG should have only
one copy per ptDNA.
 The pPRV series vectors- Targeting insertions at the trnV-3'rps12
intergenic region, the most commonly used vectors in tobacco and yield
high levels of protein expression
 The trnI and trnA genes- These two tRNAs are located between the
small (rrn16) and large (rrn23) rRNA subunit genes and the operon is
transcribed from promoters upstream of rrn16.
Vector Design for Chloroplast Transformation

32. Vector Design for Chloroplast Transformation
Regulatory sequences
 The level of gene expression in plastids is predominately
determined by regulatory sequences such as promoter as well as 5′
UTR elements .
 Strong promoter is required to ensure high mRNA level for high-
level of protein accumulation e.g. rRNA operon (rrn) promoter (Prrn).
 Most commonly used promoter is CaMV 35S promoter of
cauliflower mosaic virus which drives high level of transgene
expression in dicots.

33. Vector Design for Chloroplast Transformation
Reporter genes used in plastids
 GUS (β-glucuronidase), chloramphenicol acetyl transferase (CAT),
and GFP(Green Fluorescent Protien)
 The enzymatic activity of GUS can be visualized by histochemical
staining
 GFP is a visual marker that allows direct imaging of the
fluorescent gene product in living cells.
GFP has been used to detect transient gene expression.
GFP has also been fused with AadA and used as a bi-functional
visual and selectable marker

34. How Chloroplasts are Transformed ?
Fig- Sorting ptDNA at the organelle and cellular

35. How Chloroplasts are Transformed ?

36. Selection of Transplastomic
 Common selection marker used for plastid transformation is the
bacterial spectinomycin resistance gene aadA (3´aminoglycoside-
adenyltransferase).
 Transplastomic clones are identified as green shoots on
spectinomycin medium.
 Spectinomycin inhibits greening and shoots regeneration of wild
type.
 After integration, Homoplastomic cells obtained by several
rounds of cell division and organelle segregation.

37. Confirmation of transgene integration into chloroplast genome
 Integration of transgenes into the chloroplast genome can be
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 can be
investigated by Southern blot analysis.

38.  Genomic DNA from transformed and untransformed cultures
Can be digested with appropriate restriction enzymes,
transferred to nitrocellulose membrane and probed with P32-
radio-label .
 Transformed chloroplast genomic DNA digested with
restriction enzymes yielded an expected 3.3 kb size hybridizing
fragment.
Confirmation of transgene integration into chloroplast genome

39. Applications of chloroplast Transformation to Plant Improvement
 The expression of foreign genes in chloroplasts offers several advantages
over their expression in the nucleus:
1. Improvement of Agronomic traits
- Biotic stresses or Insect and Diseases resistance
- Abiotic stresses or Drought and Salinity tolerance
2. Production of biopharmaceuticals and vaccines in plants
3. Metabolic pathway engineering
4. Research on RNA editing
5. Phytoremediation
6. Production of Industrial enzymes and Biofuels

40. Agronomic trait development through Chloroplast Transformation
Enhanced traits Site of
integration
Regulatory
sequences
Transgenes Efficiency of
expression
Resistance to Phthorimaea
operculella
trnI/trnA Prrn/ggagg/rbcL Bt cry9Aa2 ~10% of TSP
Tolerance to high
temperature stress
rbcL/accD Prrn/rbcL 3′ panD > 4-fold β-alanine
Drought tolerance trnI/trnA Prrn/ggagg/psbA tps1 >169-fold
transcript
Resistance to herbicide rbcL/rbcL psbA/psbA/3′rbL Hppd 5% TSP
Salt tolerance trnI/trnA Prrn/T7 10/rps16 Badh 93–101 μM g−1
FW
Cold-stress tolerance prs14/trnG Prrn/T7
g10/TrbcL
HTP, TCY,
TMT
NR
Resistance against rice blast
fungus
trnI/trnA Prrn/Trps16 MSI-99 89.75 μg g−1 FW
Broad-spectrum resistance
against viral/bacterial/
phloem-feeding insects
trnI/trnA 5′psbA/3′psbA Pta 7.1–9.2% TSP
Resistance against whitefly
and aphid
trnI/trnA 5′psbA/3′psbA Bgl-1 >160-fold enzyme

41. Production of biopharmaceuticals and vaccines in plants
 Protein drugs made by plant chloroplasts overcome most of these
challenges like expensive fermentation systems, prohibitively expensive
purification from host proteins, the need for refrigerated storage and transport.
 E7 HPV type 16 protein is an attractive candidate for anticancer vaccine
development in Tobacco.
 Plastid transformation systems became successful in the oral delivery of
vaccine antigens against cholera, tetanus, anthrax, plague, and canine
parvovirus.
 Above 7.6% Protein accumulation . Example- OspA

42. Phytoremadiation
 Phytoremediation is a safe and cost-effective system for cleaning up
contaminated environments using plants.
 Two bacterial genes encoding two enzymes, mercuric ion reductase
(merA) and organomercurial lyase (merB), were expressed as an
operon in transgenic tobacco chloroplasts.
 Phytoremediation of toxic mercury was achieved by engineering of
tobacco chloroplast with metallothionein enzyme.

43. Production of industrial enzymes and biomaterials
 To produced the highest level of the poly (p-hydroxybenzoic acid
(pHBA) polymer (25% dry weight) in normal healthy plants poly
hydroxy butyrate (PHB) was designed using an operon extension
strategy
 To date, the highest levels of PHB have been achieved in plastids
due to the high flux of the PHB pathway substrate acetyl-CoA
through this organelle during fatty acid biosynthesis

44. Metabolic Pathway Engineering
 Plastid- ‘biosynthetic centre of the plant cell’
 The most complex metabolic pathway- synthesis of the bioplastic
polyhydroxybutyrate (PHB) , cause male sterility (b-ketothiolase expression )
and severe growth retardation
 1st in Tobacco, Recent- in tomato to alter carotenoid biosynthesis
towards producing fruits with elevated contents of β-carotene.
 Successful example of engineering a nutritionally important biochemical
pathway in non-green plastids by transforming the chloroplast genome.