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The following points highlight the top five methods of chloroplast transformation in higher plants. The methods are: 1. Vectors for Plastid Transformation 2. Engineering for High-Level Protein Production 3. Biolystic Transfer 4. EG Mediated Transformation 5. Galistan Expansion Femtosyringe Method.
Method # 1. Vectors for Plastid Transformation:
Vectors used for plastid transformation utilize left (LTR) and right (RTR) targeting regions to direct inserting of transgene into the plastid region. Some of the commonly used plasmid transformation vectors are plasmid repeat vector (pRV) and vector pRB94 and pRB95. Expression vector for chloroplast transformation contains two-open reading frame under the control of chloroplast-specific promoter and termination signal (Fig. 19.1).
Presence of homologous sequence facilitate two recombination events which consequently responsible for the insertion of marker gene and genome of interest into the LRT and RTR regions of the plastid. Some of the well-known plastid transformation vectors are plasmid repeat vector, and other is pRB95. Some of the expression system facilitated the expression of ribosome binding site region inserted at intergenic regions allowing production of target protein from polycistronic mRNA transcript (Fig. 19.2).
Method # 2. Engineering for High-Level Protein Production:
Strategies for production of high amount of recombinant proteins have been adopted like utilization of strong promoter, and stable mRNA transcript determined by 5′ untranslated (UTR) and 3′ untranslated (UTR) of the transgene. Considering these facts several plastid expression vectors are designed to contain 5′ regulatory region PL casette and 3′ regulatory region (T casette), strong sigma 70-type PEP promoter of the rRNA operon promoter (prnn).
The 3′ UTR regulatory sequence of mRNA include RNA stem loop structure, which acts as a inefficient transcription terminator. Most 3′ UTR T casettes are derived from PbSA, rbcL and rps 16 genes. The 5′ UTR (PL casette) region play important role in translation efficiency. In addition, careful optimization of transgene codons, despite its prokaryotic nature of expression system, resulted in high protein production.
Method # 3. Biolystic Transfer:
Biolystic is one of the efficient approaches for plastid transformation. Effective penetration and high transfer frequency are some of the plus points of biolystic method. There are number of bacteria and viruses are known to infect chloroplast.
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It’s envelop is made up of double membrane and actually considered that chloroplast transformation was considered to be virtually impossible. However, invention of biolystic genegun technology paved the pathway of direct delivery of target gene into the living cell.
Tungsten or gold particles coated with plasmid DNA is shot through chloroplast envelop. Efficient plastid transformation system for liverport (Marchantia polymorpha L) by bombarding plasmid coated gold projectiles was developed recently by Chiyode (2007).
It is fortunate that DNA is deposited in a chloroplast and successfully integrated into the chloroplast genome. For details on biolystic method refer chapter on plant transformation technique.
Method # 4. PEG Mediated Transformation:
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.
Method # 5. Galistan Expansion Femtosyringe Method:
The existing microinjection method in which recipent cells damaged by the release of cellular contents into the needle after injection have raised fresh look into the designing of novel approach for chloroplast transformation of wide range of species. A novel galistan femtosyringe method designed for chloroplast transformation involves microinjection of foreign DNA into chloroplast (Fig. 19.3).
The heat induced expansion of a liquid metal within a glass syringe forces the foreign DNA through a minute capillary top with a diameter of approximately 0.1 µm. The liquid metals employed in the specialized femtosyringe are generally galistan, an alloy of gallium, indium and tin.
