Chloroplast transformation allows for the integration of foreign genes into the chloroplast genome. This is beneficial as it provides high levels of transgene expression without epigenetic effects or position effects. Chloroplast transformation requires a chloroplast specific expression vector, a method for DNA delivery such as biolistics, and an efficient selection marker such as spectinomycin resistance. Successful transformation is confirmed by PCR and Southern blot analysis showing integration of the transgene into the chloroplast genome. Applications of chloroplast transformation include production of biopharmaceuticals, vaccines, industrial enzymes, and biomaterials as well as phytoremediation.

1. Chloroplast Transformation and its
Applications to Plant Improvement
Presented by
Jajati Keshari Nayak
Dept of MBGE
ID-55493 jajatikesharinayak00@gmail.com

2. What is organell transformation.??
• Chloroplast and mitochondria are semi autonomous
organ.They have their own DNA and produces protein. So
transfer of gene of interest to these targated organelle is
called organell transformation.

3. WHY CHLOROPLAST..??
Nuclear genome is large and contains several copies of the
same gene.
Presence of introns,
Cis-elements
Purification of protein is quite diificult I nuclear genome.
Absence of epigenetic effects
 Uni-parental inheritance is commercially favored
Easy transgene stacking in operons
Absence of position effects due to lack of a compact
chromatin structure

4. Contd…
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.

6. 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

7. 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.

10. Chloroplast transformation
requires :
• A chloroplast specific expression vector
• A method for DNA delivery
• An efficient selection for the
transplastome

11. 1. A chloroplast specific expression
vector

12. FLOW CHART OF TRANSFORMATION

14. 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

16. GENE GUN

17. 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.
• 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

19. Vector Design for Chloroplast
Transformation
• 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

20. 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

23. 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 highlevel
of protein accumulation e.g. rRNA operon (rrn) promoter (Prrn).
• Most commonly used promoter is CaMV 35S promoter cauliflower
mosaic virus which drives high level of transgene expression in dicots.

25. 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.

26. APPLICATION
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

27. 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

28. 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