This presentation is about chloroplast transformation, the importance of chloroplast transformation on nucleus transformation and strategies for making marker-free transplastomic plant

1. Chloroplast transformation
By: Shahnam Azizi
Department of Biotechnology, Faculty of
Agriculture, Azarbaijan Shahid madani
University, Tabriz, Iran
Spring 2018
1

2. Plastids classification
Chloroplast (Importance, Genomic feature, Endosymbiosis and so
on)
Chloroplast transformation (History, Methods, Requirement)
Chloroplast transformation vs. Nucleus transformation
Advantage of chloroplast transformation
Limitation of chloroplast transformation
Aims of chloroplast genetic engineering
Constriction of marker free transplastomic plants
Conclusion
content
2

3. Plastid
• 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
3

4.  Proplastids:
undifferentiated plastids
 Etioplasts: the
predecessors of
chloroplasts
 Chromoplasts: coloured
plastids: for pigment
synthesis and storage
 Leucoplasts: colourless
plastids: for monoterpene
synthesis
 Amyloplasts: for starch
storage and detecting
gravity (for geotropism)
 Elaioplasts: for storing fat
 Proteinoplasts: for storing
and modifying protein4

5. Chloroplast
Plastids are plant cellular organelles with a ~120–150kb
The chloroplast genome most commonly includes around 100 genes
Genome present in 1,000–10,000 copies per cell (Bendich,1987)
Maternally inherited in most angiosperm plant species (Hagemann,
2004)
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)
5

6.  cpDNA regions includes Large Single-Copy (LSC) & Small Single-Copy
(SSC) regions, and Inverted Repeats (IRA & IRB).
 Variation in length mainly due to presence of inverted repeat (IR)
 Conifers and a group of legumes lack Inverted Repeats.
Chloroplast genome
6

7. Chloroplast genome reduction and gene transfer
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: A process called endosymbiotic gene
transfer
Chloroplasts may contain 60–100 genes whereas cyanobacteria often
have more than 1500 genes in their genome
7

8. The genome of the chloroplasts found in Marchantia
(polymorpha (a liverwort
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
Genes for 19 of the ~60 proteins used to construct the chloroplast ribosom
8

9. Chloroplast genome in a Liverworth
9

10. Transplastomic plant
• A transplastomic plant is a genetically modified plant in which
the new genes have not been inserted in the nuclear DNA but
in the DNA of the chloroplasts.
• 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.
10

11. Biological containment and agricultural coexistence of GM plants
• 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
11

12. transformationHistory of chloroplast
Plastid transformation was first achieved in
unicellular algae called Chlamydomonas reindhartii
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.
12

13. 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
3- The availability of highly efficient selection and
regeneration protocols for transplastomic cells
13

14. Biolistic approach
Polyethylene glycol (PEG) treatment of protoplasts
Methods of Chloroplast Transformation:
Two methods are currently available to stably transform plant plastids:
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
14

15. 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 as
during biolistic transformation.
PEG Method :
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.
15

16. processes for chloroplast genome transformation
untranslated regions
16

17. 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
(Adapted from Day A and Clermont MG. 2011. The chloroplast transformation toolbox:
selectable markers and marker removal. Plant Biotechnology Journal 9, 540–553)17

18. Regulation sequences
• The gene expression level in plastids is predominately
determined by promoter and 5′-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 nessesary
• 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
18

19. 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
• Insertion of foreign DNA in intergenic regions of the plastid
genome had been accomplished at 16 sites (Maliga, 2004)
• Three of the insertion sites, trnV-3'rps12，trnI-trnA and
trnfM-trnG, were most commonly used (Maliga, 2004).
• The trnV-3'rps12 and trnI-trnA sites are located in the 25 kb
inverted repeat (IR) region of ptDNA and thus a gene
inserted into these sites would be rapidly copied into two
copies in the IR region.
• The trnfM-trnG site is 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.19

20. 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 (Kittiwongwattana et al., 2007).
• The first selection marker gene used in chloroplast transformation
was plastid 16S rRNA (rrn16) gene (Svab et al., 1990).
• Other selection marker gene used in chloroplast transformation :
 aadA gene confers resistance to spectinomycin and streptomycin
 npt II
 neo gene
 The bacterial bar gene (encoding phosphinothricin acetyltransferase
(PAT))
 Betaine aldehyde dehydrogenase (BADH) gene which confers
resistance to betaine aldehyde.
20

21. Commonly used promoters, un-translated regions and insertion
sites for chloroplast transformation
Popular insertion
sites
3′-UTRs5′-UTRsPromoter
rbcL-accD
Trnl-trnA
rp132-trnL
petA-psbJ
3’rps12/7-trnV
Trn16/V-3’rps12/7
23srrnA-16srrnA
trnfM-trnG
atpB-rbcL
trN-trnR
Ycf3-trnS
rps7-ndhB
rbcL
rps16
petD
psbA
Ggagg
T7G10
rbcL
atpB
psbA
cry2a
PpsbA
Prna
PrbcL
psaA
atpI
21

22. Chloroplast transformation Nuclear transformation
Reduced of 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
Efficient multiple gene expression in
single transformation event
Efficiency of single transformation for
multiple gene expression is very poor
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 silencing22

23.  High level expression of foreign proteins may have deleterious
phenotypic effects (Magee et al., 2004) and impose a significant
metabolic burden on the plant.
 Deleterious effects of constitutive transgene expression can
occur if gene products are harmful to the transformed plant.
 If plastid transformation carried out in food crops, inadvertent
contamination of the food chain with the plant-produced
chemical or pharmaceutical must be prevented.
 This could be accomplished by making transgene expression
dependent on an inducer.
It is highly desirable to limit transgene expression to certain
tissues, organs and/or developmental stages for following reasons
23

24. 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 inadvertent inactivation of host gene by transgene
integration.
2- Disulfide bound formation
Ability to form disulfide bonds and folding human proteins results
in high-level production of biopharmaceuticals in plants.24

25. 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.
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
 Remains unaffected by phenomenon such as pre or post-
transcriptional silencing.
25

26. 5- Expression of edible vaccine
High level of expression and engineering foreign genes without
the use of antibiotic resistant genes makes this compartment
ideal for the development of edible vaccines.
6- Codon usage
Chloroplast is originated from cyanobacteria through
endosymbiosis. It shows significant similarities with the bacterial
genome. Thus, any bacterial genome can be inserted in chloroplast
genome.
26

27. 7- 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.
8- No Gene Silencing
Gene silencing or RNA interference does not occur in genetically
engineered chloroplasts
A lack of epigenetic interference allowing stable transgene expression
27

28. 9- Multiple gene expression
Multiple transgene expression is possible due to polycistronic
mRNA transcription.
Multigene expression
(Adapted from Daniell H, Khan MS, Allison L. 2002. Milestones in chloroplast genetic
engineering: an environmentally friendly era in biotechnology. Trends in Plant Science 7
(2): 84-91.)
28

29. 10- 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
29

30. A vector containing a selectable marker (yellow) under the control of
plastid expression signals (promoter, 5´ UTR and 3´ UTR , shown in
green) flanked by chloroplast sequences (shown in purple). Homologous
recombination takes place between the flanking targeting arms and
recipient plastid genome (plastome).
10- Homologous recombination
30

31. 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
31

32.  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
32

33. Critical challenges in chloroplast transformation
Recalcitrant nature of cereal species to existing regeneration protocols
is daunting so developing efficient shoot regeneration system is very
critical
Optimizing the level of expression as massive expression of foreign
proteins is resulting in phenotypic alterations of transplastomic plants
Lack of appropriate tissue specific regulatory sequences
Problem of gene expression in non-green plastids
Unintended homologus recombination that hinder efficient recovery of
transplastomic transformants containing the desired transgene
Degradation of foreign proteins is a limiting factor for accumulation of
foreign proteins in transgenic chloroplasts
Low frequency transgene dispersion might occur due to occasionally
parental/biparental transmission of plastids and via transgene transfer
to nuclear genome33

34. 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
34

35. Aims of chloroplast genetic engineering applications include
Resistance to insects, bacterial, fungal and viral diseases
Different types of herbicides, drought, salt and cold tolerance
Cytoplasmic male sterility
Metabolic engineering
Phytoremediation of toxic metals
Production of many vaccine antigens
Biopharmaceuticals
Industrial enzymes
Biofuels35

36. Why and how?
36

37. Reasons to produce marker-free transplastomic plants
1- Potential metabolic burden imposed by high levels of marker gene
expression. Marker genes are designed for high levels of expression so
that the initial few integrated copies can protect the cells. As a
consequence, when the homoplastomic state is reached, the marker
gene product can make up 5% to 18% of the total cellular soluble
protein
2- The second reason to remove the marker gene is the shortage of
primary plastid selective markers, which at this point include only
genes that confer resistance to spectinomycin and streptomycin (aadA)
or kanamycin (neo or kan and aphA-6 ). If multiple engineering steps
are required, the marker genes might have to be removed to enable
repeated selection for the same marker.
3- The third issue is opposition to having any unnecessary DNA in
transgenic crops, especially anti-biotic resistance genes
37

38. Strategies developed for plastid marker gene excision
I. Homology-based excision via directly repeated
Sequences
II. Excision by phage site-specific recombinanses
III. Transient cointegration of the marker gene
IV. Cotransformation-segregation
38

39. Homology-based marker gene excision via directly repeated
sequences
39

40. Excision by phage site-specific recombinanses
1-Transplastomics carry marker gene flanked by two directly
oriented recombinase target sites
2-Removal of marker gene by introduction of a gene encoding a
plastid-targeted recombinase in the plant nucleus: recombinases
(Cre and Int)
40

41. Two recombinases (Cre and Int) were tested for plastid marker
gene excision
The Cre recombinase derives from the P1 bacteriophage and
excises target sequences flanked by directly oriented 34 bp loxP
sites
fC31 phage integrase, Int, marker gene flanked with directly
oriented non-identical phage attP (215 bp) and bacterial attB (54
bp) attachment sites, which are recognised by Int recombinase.
Absence of homology between the attB and attP sites and the
absence of pseudo-att sites in ptDNA=> Int better than Cre
41

42. Marker gene excision by phage site-specific recombinanses
Marker gene excision by the phage Cre or Int site-specific recombinases. Site-
specific recombinases (Cre/Int) expressed from nuclear genes ( cre/int ) excise
marker genes ( mg) from TP1-ptDNA after import into plastids. Excision of the
marker gene by phage recombinases via the target sites (triangles) yields marker-
free TP2-ptDNA carrying only the gene of interest (goi) and one recombinant copy
of the recombinase recognition sequence
42

43. The cre gene has been introduced into the plant nucleus by three
methods
Stable transformation of the nucleus was obtained using an Agro-
bacterium binary vector that instantly yielded marker-free
transplastomic plants. Although the plants regenerated from tissue
culture were plastid marker free, they now carried a nuclear cre
transgene that had to be segregated away in the seed progeny
the cre gene was introduced into the transplastomic plants by
pollination. Although introduction of cre by pollination took longer, it
appears that non-specific Cre-induced re-arrangements between
homologous ptDNA sequences were absent or occurred significantly
less often than in directly transformed plants
Cre was expressed transiently from T-DNA introduced by
Agroinfiltration, exploiting the observation that not every T-DNA
delivery results instable integration Of particular significance for
transplastomic engineering
43

44. Transient cointegration of the marker gene to obtain marker-free plants
44

45. Cotransformation-segregation to obtain marker-free plants
45

46. Note
Although the technology to
obtain marker-free transplas-
tomic plants is available, no
transplastomic crops are yet
grown commercially
46

47. Conclusion
 Chloroplast has a pivotal role in life-sustaining.
 Gene gun and PEG treatment is most used techniques for
chloroplast transformation
 High copy number, Polycistronic gene expression,
uniparental transition, homologous recombination is the
important characteristic of chloroplast transformation.
 Int-att and Cre/loxp is most and efficient methods for
making the marker-free transplastome plant
47

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