1. Biotechnologies Influencing Agriculture: Molecular Pharming Betty Ikawati, Rahmi Yuniarti, Nita

2. Plant made Pharmaceutical - Wave of Agricultural Biotechnology First Generation : Agronomic traits Second Generation : Nutritional Enhancement traits Third Generation : Plants/animals as Factories

3. Molecular Pharming by Chloroplast Transformation Chloroplast transformation for transgene containment Chloroplasts : - class of plastids - organelle - from cyanobacteria (blue-green bacteria ) - contain chlorophyll

4. transformation of the chloroplast  1988 : - putting the foreign genes into chloroplast genome  Late 1990 : several biotech companies have initiated major programmes on chloroplast transformation  1998 : Chloroplast transformation has been touted at least as far back as 1998 as a means of “containing” transgenes; that is, preventing them from transferring to non-GM crops or wild relatives through pollen, and hence preventing the creation of transgenic herbicide tolerant weeds. The theory is that chloroplasts are inherited exclusively through the female line.

5. Other benefits of chloroplast transformation Chloroplast transformation involves homologous recombination In practice, the inserted transgene has short DNA sequence tails added at each end, the tails are homologous to sequences on the chloroplast target gene, which thus initiate homologous recombination. Once the transgene is inserted into the chloroplast chromosome, the target gene is disrupted. The disruption of the target gene is expected to alter the growth and metabolism of the plant

6. Leaf discs are bombarded with plasmid constructs containing a selectable antibiotic resistance marker physically linked to the gene of interest, flanked by DNA for inserting into the correct site of the chloroplast genome. The antibiotic resistance marker most frequently used is the aadA gene encoding resistance for spectinomycin and streptomycin, driven by the promoter of the chloroplast encoded 16S rRNA gene. this transformation procedure applied to tobacco, Arabidopsis or oil seed rape, generates plants in which all the chloroplast genomes are uniformly transformed (a condition referred to as homoplasmic), despite the fact that tobacco leaf cells may contain 100 chloroplasts, each containing 100 copies of the chloroplast genome.

7. Transformation of the chloroplast genome by bombarding tobacco leaves with microprojectiles coated with DNA. Following bombardment, leaf discs are placed onto antibiotic-containing medium (panel A). Transgenic plants are regenerated from the transformed tissue that is able to develop green chloroplasts (panel B)

8. Another advantage of chloroplast transformation is that foreign genes can be over-expressed, due to the high gene copy number, up to 100 000 compared with single-copy nuclear genes. And there does not seem to be gene-silencing and other instability that plague nuclear transformation. The gene product is retained inside the chloroplasts or can in principle be targeted to a specific compartment in the chloroplast.

9. Plant Made pharmaceuticals The manufacturing of bulk active pharmaceutical ingredients derived from plants A transgenic plant/seed is developed to perform as a high production system The plant/seed containing the active pharmaceutical ingredient is harvested The active pharmaceutical ingredient is isolated and processed to be used in pharmaceuticals