Lecture on chloroplast transformation through genetic engineering. Merits of introducing foriegn gene in chloroplast of plant cell.

1. CHALOROPLAST
TRANSFORMATION

2. INTRODUCTION
It is possible to use of transgenic plants to study nuclear gene function and
regulation and improve agronomically important crop plants. There are several
alternative methods to produce of transgenic plants developed specifically for
transformation of nuclear genome of higher plants. Chloroplast transformation is
an important tool for biotechnological applications and has gained much interest
in the regulation of gene expression and plant physiology.

3. CHLOROPLAST
– Chloroplasts are sub cellular organelles (plastids) of plant cells .
– present in shoots and leaves of green plants.
– contain pigment called chlorophyll.
– also present in several forms as colorless plastids (amyloplasts).

4. CHLOROPLAST

5. Historical perspective
– Ist achieved in the alga Chlamydomonas reinhardtii.
– In higher plants like Tobacco due to its ease of culture and regeneration,
gained significant attention for chloroplast transformation.
– In the following figure it is described that 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 clones.

6. Figure

7. CHLOROPLAST
TRANSFORMATION
– Multistep processes are involved to achieve chloroplast transformation.
– When the foreign DNA is delivered into plasmids, initially only a few
copies of the plastome are transformed resulting in-to heteroplasmic state .
– Then all copies of the plastome contains the transgene leading to the state
of homoplsamy, where all the plastomes of the chloroplasts present in the
cell are transformed.

8. Conditions for Chloroplast
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.

9. METHODS FOR CHLOROPLAST
TRANSFORMATION
– by several ways
– But to date only biolistic method and polyethylene glycol (PEG)
treatment have yielded stable chloroplast transformation .

10. Biolistic Method:
– Commonly used for genetic transformation of plants.
– plant cell wall is usually impermeable to foreign DNA.
– gene gun method has shown better results against the various barriers .
– Gene transfer through a gene gun is part of the biolistic method.
– During this method DNA or RNA construct adheres to biological inert
particles to form a DNA complex .
– DNA/particle complex is bombarded on a targeted tissue in a vacuum.
– bombardment is achieved by accelerating a powerful shot to the targeted
tissue

11. Continued…
– high density of the DNA/particle complex increases the bombardment
speed which may result in effective transformation .
– Such a complex once formed is directly transferred to the plant cell.

12. Biolistic Method

13. PEG Treatment
– PEG and Agrobacterium- mediated transformation method was also
employed in the early days.
– Polyethylene glycol is widely used in transformation work. Despite its
efficiency, PEG mediated transformation is far behind than the biolystic
approach. Foreign DNA is taken by protoplast in presence of PEG and
transported by unknown process from cytoplasm into the chloroplast and
finally integrated into the genome.

14. –ADVANTAGES OF
CHLOROPLAST
TRANSFORMATION

15. 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 and remains unaffected by phenomenon such as pre or
post-transcriptional silencing

16. Risk of transgene escape
– It provides a strong level of biological containment and thus reduces the
escape of transgene from one cell to other.

17. 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.

18. Gene silencing/ RNA interference
– Gene silencing or RNA interference does not occur in genetically
engineered chloroplasts.

19. Di-Sulphide bond formation
– It has ability to form disulfide bonds and folding human proteins results
in high-level production of biopharmaceuticals in plants

20. Multiple gene expression
– It is helpful in multiple transgene expression due to polycistronic mRNA
transcription.

21. 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.

22. USE OF THE CHLOROPLAST ENGINEERING
– Chloroplast transformation techniques has been used to address plastid
genetic and molecular biology such as characterization of promoter
strength, trans - splicing, mRNA stability, photosynthetic function, to
achieve targeted disruption of plastid genes, to examine the requirements
for expression of foreign gene. On the other hand, methods for chloroplast
transformation have made it possible to identify sequence elements that
regulate chloroplast gene expression in vivo at the level of transcription,
transcript stability, translation and photosynthetic complex assembly.
Chloroplast can also be used for transferring of foreign genes.

23. CONCLUSION AND PROSPECTS
– Chloroplast genome has become the target of many plant genetic
transformation efforts due to its enormous advantages over nuclear genome of
the plant. The nuclear transgenic approach is incapable to develop products
when higher-level transgene expression and multigene engineering is a
requirement. Chloroplast transformation is expected to offer unique
advantages in the advancement of different biotechnological applications;
including, phytoremediation, production of industrial enzymes, biofuels,
biomaterials, molecular farming for the production of antibiotics, vaccines,
biopharmaceuticals and conferring agronomic traits. Chloroplast
transformation has been achieved only to tobacco, lettuce, Arabidopsis,
tomato, carrot, oilseed rape, potato, cabbage, cotton, petunia, soybean,
sugarcane, sugar beet, rice, eggplant, cauliflower and poplar.

24. REFERENCES
– Igloi, G. L. and H. Kossel. 1992. Transcriptional apparatus of chloroplast. Crit.
Rev. Plant Sci. 10: 525-558.
– Svab, Z., P. Handukieviczç, and P. Maliga. 1990. Stable transformation of plastids
in higher plants. Proc. Natl. Acad. Sci. USA. 87: 8526-8530.
– Mullet, J. E. 1988. Chloroplast development and gene expression. Ann. Rev.
Plant Physiol. Mol. Biol. 39: 475-502.
– Gruissem, W., and J. L. Tonkyn. 1993. Control mechanisms of plastid gene
expression. Crit. Rev Plant Sci. 12: 19-55.

25. Continued…
– Jana Ř. 2010. Potential of chloroplast genome in plant breeding. Czech J
Genet Plant Breed. ;46(3):103–13.
– Grevich JJ, Daniell H.2015. Chloroplast genetic engineering: recent
advances and future perspectives. Crit Rev Plant Sci. 24:83–107.