Chloroplasts are sub cellular organelles (plastids) of plant cells generally considered to be derived from the symbiotic cyanobacteria. They are present in shoots and leaves of green plants and contain pigment called chlorophyll. They are also present in several forms as colorless plastids (amyloplasts) in roots and as colored plastids (chromoplasts) in fruits. A cell comprises variable number of plastids, each plastid containing many copies of genome (50 to 100). Plastid genomes resemble bacterial genomes in many aspects and also contain some features of multicellular organisms, such as RNA editing and split genes. Most of the proteins are encoded in the plant nucleus, synthesized and then imported into the chloroplast.

Chloroplast transformation: historical perspective

First stable chloroplast transformation was achieved in the alga Chlamydomonas reinhardtii. In addition, the aadA marker and methods for removal of marker were first demonstrated. In higher plants, Tobacco due to its ease of culture and regeneration, gained significant attention for chloroplast transformation. Tobacco protoplasts were co-cultivated with Agrobacterium but the resulted transgenic lines showed the unstable integration of foreign DNA into the chloroplast genome.

The candidate genes were introduced in isolated intact chloroplasts and then into protoplasts resulting in transgenic plants. Gene gun, a transformation device, was developed by John Sanford to enable the transformation of plant chloroplasts without using isolated plastids.

Advantages of chloroplast transformation

Chloroplast transformation offer several advantages compared with nuclear transformation which are as follows-

1. 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.

2. 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.

3. 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.

4. Gene silencing/ RNA interference

- Gene silencing or RNA interference does not occur in genetically engineered chloroplasts.

5. 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.

6. Disulphide bond formation

- Ability to form disulfide bonds and folding human proteins results in high-level production of biopharmaceuticals in plants.

7. Multiple gene expression

- Multiple transgene expression is possible due to polycistronic mRNA transcription.

8. 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.

9. 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.

10. 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.

Transformation methods for chloroplast transformation

• Biolistic/Particle bombardment method involves the introduction of Escherichia coli plasmids containing a gene of interest and marker gene into chloroplasts or plastids. The insertion of foreign genes into plasmid DNA occurs by homologous recombination via the sequences flanking at the insertion site. First successful chloroplast transformation was performed in Chlamydomonas reinhardtii by particle bombardment method. Simple operation and high transformation efficiency makes it a favorable way for plastid or chloroplast transformation.

• PEG-mediated and Agrobacterium- mediated transformation method was also employed in the early days. After the first chloroplast transformation in Chlamydomonas reinhardttii, the stable plastid transformation has also been established in higher plants, Nicotiana tobacum, Arabidopsis, rape, Lesquerella, rice, potato, lettuce, soybean, cotton, carrot and tomato.

However, plastid transformation is routinely performed only in tobacco because of higher efficiency of transformation in tobacco than in other plants.

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.

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