Chloroplasts can be genetically engineered through transformation because they have their own genetic systems and DNA located in structures called chloroplast nucleoids. Transformation involves delivering a chloroplast-specific expression vector containing the gene of interest, flanked by chloroplast DNA sequences to facilitate homologous recombination-mediated integration into the chloroplast genome. Successful methods for DNA delivery include biolistics and polyethylene glycol-mediated transformation, with biolistics being preferred. Transformed chloroplasts are called transplastomes and confer advantages like high-level expression of foreign genes without risks of transgene escape or gene silencing seen in nuclear transformation.

1. Chloroplast Transformation

2. Chloroplast:
Site of photosynthesis.
Endosymbiotic event.
Have their own genetic systems.
1 – 900 chloroplasts per plant cell.
cpDNA is packed into discrete structures called
chloroplasts nucleoids.
Approx. 10,000 cpDNA per cell.

3. The chloroplast is genetically engineered
because of the following factors:
The risk of transgene escape.
Low expression level.
Gene silencing.
Difficulty in gene stacking.
Codon usage.

4. Transformation of chloroplast:
History:
1988 : Putting the foreign genes into chloroplast
genome.
Late 1990 : Several biotech companies have
initiated major programmes on chloroplast
transformation and from then it’s continuing.

5. Chloroplast transformation requires:
A chloroplast specific expression vector.
A method for DNA delivery through a double
membrane of the chloroplast.
An efficient selection for the transplastome.
Note:
Transplastome:
genetically modified plant in which the new
genes have not been inserted in the nuclear
DNA but in the DNA of the chloroplasts.

6. Gene Gun
DNA of interest chloroplast DNA
(homologuous recombination occurs).
Advantages:
Much better chance of localizing transgene
compared to when transgenes are in nuclear
genome.
Disadvantages:
Not suitable for all crops.
Into chloroplast

7. DNA delivery into plastids
Two successful methods include biolistics and
polyethylene glycol mediated transfer.
Biolistics is preffered as it is less time
consuming and demanding.
Integration of foreign DNA into plastid genome
occurs via homologous recombination.
Homologous recombination operates in plastids
at a high efficiency.

8. A Chloroplast Specific Expression
Vector
Depends on the integration of the foreign DNA
into the chloroplast genome by homologous
recombination.
>400bp of homologous sequence on each side
of the construct is generally used to obtain
chloroplast transformants at a reasonable
frequency.
Chloroplast specific promoters and termination
signals.

9. Cont…
Transcribed as operons, which allows more
than two ORFs to transcribe under the same
promoter.
The selectable marker and the gene of interest
are placed between the promoter and the
terminator which are flanked by the 5’ and 3’
untranslated regions.

10. 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 sides by region
of homology with the chloroplast genome.
Primary transplastomic event results
heteroplasmic cells.
Heteroplasmy is unstable so it will resolve into
homoplasmy.

12. Marker Removal:
Recombination between directly repeated
sequences excises the intervening DNA
sequence and one copy of the direct repeat.
The breakage and joining of DNA strands
involved in recombination can be mediated by
the native homologous recombination
machinery present in plastids.

13. Advantages:
Hyper expression.
Multigene engineering.
Maternal inheritance.
Gene containment.
No vector sequences.
No position effect.
No gene splicing.
No pleiotropic effects.