Chloroplast transformation

What is chloroplast transformation, why it is better than nuclear transformation. Advantages and disadvantages of chloroplast transformation.

1. Chloroplast Transformation and its Applications to Plant Improvement Presented by Jajati Keshari Nayak Dept of MBGE ID-55493 [email protected]

2. What is organell transformation.?? Chloroplast and mitochondria are semi autonomous organ.They have their own DNA and produces protein. So transfer of gene of interest to these targated organelle is called organell transformation.

3. WHY CHLOROPLAST..?? Nuclear genome is large and contains several copies of the same gene. Presence of introns, Cis-elements Purification of protein is quite diificult I nuclear genome. Absence of epigenetic effects Uni-parental inheritance is commercially favored Easy transgene stacking in operons Absence of position effects due to lack of a compact chromatin structure Efficient transgene integration by homologous recombination.

4. Contd … High level of transgene expression and protein accumulation The possibility of co-expressing several transgenes in operons The precise transgene integration by homologous recombination . The feasibility of expressing multiple proteins from polycistronic mRNAs Regeneration of crop plants with higher resistance to biotic and abiotic stresses and molecular pharming .

5. WHAT IS CHLOROPLAST ??? Chloroplast is a plastid containing chlorophyll and other pigments occurring in plants and eukaryotic algae and which possess their own genome or plastome , besides nuclear genome that carry out photosynthesis

6. Why Chloroplast is a Unique Transformation tools..??? Protein accumulator - soluble proteins and intrinsic membrane proteins. Cellular location for valuable recombinant products Own genetic systems and genomes, high copy number, transcription translation machinery. Plastid posses prokaryotic gene expression machinery.

7. Chloroplast transformation requires : A chloroplast specific expression vector A method for DNA delivery An efficient selection for the transplastome

8. 1. A chloroplast specific expression vector

9. FLOW CHART OF TRANSFORMATION

10. BIOLISTIC METHODS OF GENE DELIVERY Advantages Simple operation and high efficiency makes it a favorable No need to obtain protoplast as the intact cell wall can be penetrated. This device offers to place DNA or RNA exactly where it is needed into any organism. Disadvantages The transformation efficiency may be lower than Agrobacterium- mediated transformation. Associated cell damage can occur. The target tissue should have regeneration capacity

11. GENE GUN

12. PEG METHODS OF GENE DELIVERY PEG-mediated transformation of plastids requires enzymatically removing the cell wall to obtain protoplasts, then exposing the protoplasts to purified DNA in the presence of PEG. The protoplasts first shrink in the presence of PEG, then lyse due to disintegration of the cell membrane. Removing PEG before the membrane is irreversibly damaged reverses the process. Treatment of freshly isolated protoplasts with PEG allows permeabilization of the plasma membrane and facilitates uptake of DNA. A relatively small number of species have been transformed using this approach, mainly because it requires efficient isolation, culture and regeneration of protoplasts, a tedious and technically demanding in vitro technology

13. Vector Design for Chloroplast Transformation Spectinomycin resistance- The most efficient and routinely used 16S rRNA (rrn16) gene- Initially used and selected by spectinomycin resistance with low efficiency. aadA (aminoglycoside 3′ adenylyltransferase ) gene- Dominant marker gene that confers resistance to streptomycin and spectinomycin by inactivation of antibiotics. Plastid expressed GFP (green fluorescent protein)- a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The npt II- Transformation efficiency was low, i.e. about one transplastomic line per 25 bombarded samples

14. Insertion sites Insertion of foreign DNA in intergenic regions of the plastid genome had been accomplished at 16 sites, most commonly used insertion sites are - trnV-3'rps12 , trnI-trnA and trnfM-trnG The trnV-3'rps12 and trnI-trnA sites- located in the 25 kb inverted repeat (IR) region of ptDNA and a gene inserted into these sites would be rapidly copied into two copies in the IR region

15. Regulatory sequences The level of gene expression in plastids is predominately determined by regulatory sequences such as promoter as well as 5′ UTR elements . Strong promoter is required to ensure high mRNA level for highlevel of protein accumulation e.g. rRNA operon ( rrn ) promoter ( Prrn ). Most commonly used promoter is CaMV 35S promoter cauliflower mosaic virus which drives high level of transgene expression in dicots.

16. Confirmation of transgene integration into chloroplast genome Integration of transgenes into the chloroplast genome can be confirmed by PCR using internal primers, first primer anneals to the flanking sequence and second primer anneals to the transgene region. An expected size of PCR product was amplified and this confirmed integration of the transgenes in different cell cultures of plant Integration of the transgenes into plastid genome can be investigated by Southern blot analysis .

17. APPLICATION Production of biopharmaceuticals and vaccines in plants Protein drugs made by plant chloroplasts overcome most of these challenges like expensive fermentation systems, prohibitively expensive purification from host proteins, the need for refrigerated storage and transport. E7 HPV type 16 protein is an attractive candidate for anticancer vaccine development in Tobacco. Plastid transformation systems became successful in the oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus. Above 7.6% Protein accumulation . Example- OspA

18. PHYTOREMADIATION Phytoremediation is a safe and cost-effective system for cleaning up contaminated environments using plants. Two bacterial genes encoding two enzymes, mercuric ion reductase ( merA ) and organomercurial lyase ( merB ), were expressed as an operon in transgenic tobacco chloroplasts. Phytoremediation of toxic mercury was achieved by engineering of tobacco chloroplast with metallothionein enzyme

19. Production of industrial enzymes and biomaterials To produced the highest level of the poly ( p -hydroxybenzoic acid ( pHBA ) polymer (25% dry weight) in normal healthy plants poly hydroxy butyrate (PHB) was designed using an operon extension strategy To date, the highest levels of PHB have been achieved in plastids due to the high flux of the PHB pathway substrate acetyl-CoA through this organelle during fatty acid biosynthesis