chloroplast genomics and biotechnology

1. Chloroplast Genomics and
Biotechnology
Submitted by:
Nidhi Singh

2. Outline
• Introduction
• History
• Genome organization
• Advantages
• Different Transformation system
• Molecular biology of Chloroplast Transformation
• Chloroplast functional genomics by reverse genetics
• Transgenic Chloroplast in Biotechnology
• Challenges
• Case study
• Perspectives
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3. INTRODUCTION
• Chloroplast word derived from Greek word : chloros – green and plast –
organelle or cell.
• One of the form of a plastid such as Amyloplast for storing starch,
Elaioplast for fat storage, Chromoplast for pigment synthesis and storage.
• Derived from proplastid.
• Present in plant cell and capture light energy from sun to produce free
energy via photosynthesis.
10/12/2010 3(R .Bock, 2001)

4. • Transplastomic technologies allow precise targeted integration of trait
genes into chloroplasts without marker genes.
• Industrial and therapeutic proteins expressed in chloroplasts accumulate to
extraordinarily high levels providing an attractive production platform for
manufacture of high-value products for industry and health ,which is both
sustainable and carbon-neutral.
• Maternal inheritance of chloroplast genes prevents the pollen-mediated
spread of transgenes providing a natural form of gene containment for the
next generation of Biotech crops .
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5. 10/12/2010
Chloroplast evolution
Source:cps.ci.cambridge.ma.us
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6. 10/12/2010
Plastid differentiation
Source: biofortified.org 6

7. History
• Theory of chloroplast inheritance, 1909, by Erwin baur, in Pelargonium
and Mirabilis.
• Presence of unique DNA in chloroplast, 1963, by Ruth sagar and
Masahiro R. Ishida , in Chlamydomonas.
• Circular DNA molecule in chloroplast,1971, by Manning et al., in
Euglena.
• Physical map of chloroplast,1976, by Jhon R. Bedbrook & Lawrence
Bogorad, in Maize.
• Physical map of Tobacco chloroplast DNA,1980, by Jurgenson &
Bourque.
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8. • First chloroplast transformation, 1988, by Boynton and Gillham , in
Chlamydomonas.
• First chloroplast transformation in higher plants, 1990, by Pal Maliga &
coworkers, in Tobacco.
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(Pal Maliga, 2004)
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9. Genome organization
• Gene content highly conserved.
• Semiautonomous organelles.
• Highly polyploid genome.
• Circular double stranded DNA.
• Prokarotically organized.
• Most plant genome have two identical copies of 20 to 30 kb inverted
repeats separating a large single copy and small copy region.
10/12/2010 (Adrian C. Barbrook et al., 2010) 9

10. • Chloroplast DNA constitute 10 to 20 percent of total cellular DNA
content.
• Genome size ranges from Acetabularia spp. with 1.5Mbp size and
Ostreoccocus tauri having 86 genes only.
• Nucleomorph, feature of some complex chloroplast having retention of
eukaryotic nucleus and located between the inner and outer chloroplast
membrane.
(Adrian C. Barbrook et al., 2010)
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11. 10/12/2010
Rice chloroplast DNA
Source: shigen.nig.ac.jp
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12. (Adapted from: TRENDS in Biotechnology, May 2005 Vol.23 No.5)10/12/2010 12

13. Different Transformation
system
• Direct DNA delivery methods such as particle gun or Biolistic method.
• Microinjection techniques.
• Embryogenesis.
• Organogenesis via protoplast or leaves.
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(Dheeraj Verma et al., 2007)

14. 10/12/2010 14(Adapted from: Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)

15. 10/12/2010 15(Adapted from: Plant Physiology, December 2007, Vol. 145, pp. 1129–1143,)

16. e
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(Adapted from: Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)

17. Molecular biology of Chloroplast
Transformation
• Stable transformation system depends on integration of the transforming
DNA into the plastid genome by homologous recombination.
• Sequence to be introduced into the plastid genome must flanked on both
side by region of homology with the chloroplast genome.
• Primary transplastomic event results hetroplasmic cells.
• Hetroplasmy is unstable so it will resolve into homoplasmy .
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(Ralph Bock, 2001)

18. 10/12/2010 18
(Adapted from : Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)

19. Analysis of DNA isolated from putative transplastomic shoots
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PCR analysis :
Lane 1: untransformed plant
Lanes 2 to 4: transformed lines
lane 1kb+: DNA marker
Lanes 5 to 7: transformed lines
PCR products of
3P/3M primers
PCR product with 5P/2M
primers
chloroplast genome is probed with a
radiolabeled flank fragment
Sothern blot analysis:
Lane 1: Untransformed plant
lanes 2 and 4: homoplasmic transplastomic plant
lane 3: heteroplasmic transplastomic plan.
(Adapted from : Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)

20. Chloroplast functional genomics
by reverse genetics
• Reverse genetics: study from genotype to phenotype.
• Used for functional characterization of chloroplast genome.
• Approaches used:
1. Knock out analysis
2. Site directed mutagenesis
• Model system
1. Chlamydomonas reinhardtii
2. Tobacco
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(Ralph Bock, 2001)
(Adapted from: PLASTID TRANSFORMATION IN HIGHER
PLANTS, 2004)

21. Strategy
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(Ralph Bock, 2001)

22. Arabidopsis Chloroplast 2010 Project
• Mutation of gene At1g10310
• A: At1g10310 gene model and two insertion
alleles, Rectangles: exons , black rectangle:
5’ & 3’ UTR, Solid lines: introns &
intergenic regions, Gray triangle : T-DNA
insertion.
• B: At1g10310 steady-state mRNA levels in
wild-type Columbia (WT Col), a line derived
from a wild-type segregant plant (WT
Segregant), SALK_125505C (T-DNA), and
RIKEN mutant line 15-1699-1 (Ds).
• C: Seed fatty acid composition of a line
derived from a wild-type segregant plant
(WT Segregant), the Columbia ecotype (WT
Col), and the two mutant lines, T-DNA and
Ds.
• Differences between the mutant and the wild
type of greater than 15% .
10/12/2010 22(Adapted from: Plant Physiology, February 2010, Vol. 152, pp. 529–540)

23. Transgenic Chloroplast in
Biotechnology
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24. 10/12/2010 24
(Adapted from: Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)

25. 10/12/2010 25
(Adapted from : Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)

26. Challenges
• Chloroplast transformation has been accomplished in relatively few
species.
• Unavailability of the genome sequence.
• The chloroplast transformation vectors utilize homologous flanking
regions for recombination and insertion of foreign genes.
• species-specific chloroplast vectors for transformation of grasses
• Its transformation is a tissue culture-dependent process, a better
understanding of DNA delivery, selection, regeneration, and progression
toward homoplasmy is essential to achieve it in different taxonomic
groups
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27. Case study
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29. Introduction
• Rapeseed (Brassica napus L.) is one of the most important oil-producing
crops worldwide.
• Foreign genes escape through pollen from transgenic rapeseed plants to
other closely related species under complicated ecological environment.
• Natural crosses occur among B. napus, B. rapa and B. Juncea.
• Chloroplast genes inheriting in a strictly maternal fashion, minimizes the
possibility of out crossing of transgenes.
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30. Materials and methods
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32. • Construct rapeseed chloroplast transformation vector pCL308 targets the
expression cassette into the trnI-trnA region of the rapeseed chloroplast
genome.
• Transform the chloroplast by bombardment of cotyledons, two days after
bombardment cut it into 0.3x 0.3 cm explants & 80 explants were placed per
Petri dish on B5 medium for callus induction.
• After 6 to 8 week of culture, spectinomycin resistant calli started to grow.
• Shoot producing spectinomycin resistant calli were transferred to a shoot
regenerating medium.
• Regenerated roots were transferred in to rooting medium.
• Transplastomic rapeseed resistant to spectinomycin were selected for
transferring onto B5 medium.
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33. Optimization of the culture system for efficient
regeneration of rapeseed
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Construction of
rapeseed chloroplast
transformation
vector
PCR analysis of transgene integration into the
rapeseed
chloroplast genome

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Analysis of the chloroplast vector
cassette in transformed
rapeseed by Southern blot hybridization
Northern blot analysis of
aadA mRNA in the
transplastomic
plants

36. Inheritance of the transplastomic aadA gene
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37. Upshot
• A callus induction and regeneration system derived from cotyledonary
tissues of elite rapeseed cultivars was developed and successfully used for
rapeseed chloroplast transformation.
• This chloroplast transformation method may serve as the basis of a
method to investigate further integration of genes into the chloroplast
genome of rapeseed to breed cultivars with improved agronomic
characters.
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44. Perspectives
• Important task for the coming years will be implementing plastid
transformation in the major crops.
• Identifying bottlenecks in the recalcitrant species and combining suitable tissue
culture systems with efficient molecular tools.
• Exploitation of gene maintenance mechanisms in chloroplasts is expected to
lead to improvements in transplastomic technologies and the design of
transplastomic crops.
• Now reached a more mature phase when it is expected to make a broader
impact through agricultural and industrial applications.
• Biotechnological applications of this new and exciting area of science are
underpinned by fundamental research on the genes present in chloroplasts.
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45. DISCUSSION
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