Chloroplast Transformation and its

Applications to Plant Improvement

Presented by
Jajati Keshari Nayak
Dept of MBGE
ID-55493 jajatikesharinayak00@gmail.com
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.
 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
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.
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
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.

Chloroplast transformation requires
:

• A chloroplast specific expression vector

• A method for DNA delivery

• An efficient selection for the

transplastome
1. A chloroplast specific expression
vector
FLOW CHART OF TRANSFORMATION
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
GENE GUN
 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
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
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
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.
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.
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
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
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