Chloroplast transformation

What is?

Chloroplasts perform indispensable functions of photosynthesis and respiration. Modification of

chloroplast genes, and even introduction into them of some selected transgenes could improve
photosynthesis or even enhance crop yields. in addition, transgenes resistance to herbicides,
insects, etc. and those encoding pharmaceutical proteins, etc. could be transferred and expressed
in chloroplasts as this offers unique advantages.

Why genetically engineer

The risk of transgene escape
chloroplasts?
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.

Low 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 posttranscriptional silencing.
gene silencing
absence of position effects due to
lack of a compact chromatin
structure and efficient transgene
integration by homologous
recombination

Chloroplast transformation requires:

1.

A chloroplast specific expression vector.

2.

A method for DNA delivery through a double membrane of the

chloroplast.

3. An efficient selection for the transplastome.

Methods

Many unique methods used for chloroplasts transformation. These are

Particle gun/ Biolistics - gene gun
PEG (polyethylene glycol)
microinjection
Particle gun is the most widely used and more effective way of
chloroplasts transformation. This method yields a high efficiency rate, and
can be used to transform a variety of explants. The first successful
transformation of chloroplast in higher plants was gained in 1989, when
Spectinomycin resistance was transferred into tobacco.
Protoplasts take up DNA in presence of PEG (polyethylene glycol), this
DNA also transported by some yet unknown means into the chloroplasts
where it may become integrated into the chloroplast genome.

The currently used ways have only been optimized for the transformation
of tobacco, and only a limited succeed has been gained with tomato and
few other species.

Biolistics DNA delivery

biological + ballistic
Using high-pressure He gas as propellant
Tungsten or gold particles
Leaves, cotyledons, or cultured cells

PEG-mediated
transformation
expose
the protoplasts to purified DNA in the presence of PEG
regeneration step required

Agrobacterium-mediated
transformation
unsuccessful as yet
Galistan expansion
femtosyringe
This is a novel approach involves the
microinjection of DNA into chloroplast
(Knoblauch et al., 1999) and is not widely
used.
The heat-induced expansion of a liquid
metal, galistan, within a glass syringe forces
the transformation plasmid DNA through a
capillary tip with a diameter of approximately
0.1 mm.

Advantages Chloroplasts are batter able to express bacterial genes than are nuclei.
No risk of the transgenes being transmitted through pollen to non transgenic
plants of the same species, or to those of related species.
High level of transgene expression ensures a high level of mortality of the
pest organism.

Disadvantages
Chloroplast transformation frequencies are much lower than those for
nuclear transformation.
The methods of transgene transfer into chloroplasts are limited, and they are
either expensive, or require regeneration from protoplasts.
Products of tansgenes ordinarily would accumulate in green plant parts only.

 Applications of chloroplast
engineering
Chloroplast transformation can be
used in the production of transgenic
plants with herbicide resistance,
insect resistance, viral resistance,
fungal resistance, abiotic and biotic
stress tolerance, production of
biopharmaceuticals etc.

THANK YOU