**2. Chloroplast biotechnology—an overview**

Transgenic technology is the technology of the day to develop crop plants with desired traits but crucial traits need to be engineered through plastid genome instead of nuclear genome [4]. It is an amazing organelle where more than 120 genes from various sources have been integrated and expressed. This organellar genome has well been explored for a wide variety of applications including crops with elevated level of resistance against biotic (insects, bacterial, viral, and fungal diseases) and abiotic stresses (salinity, drought, and cold); phytoremediation of toxic metals, cytoplasmic male sterility [5]; and production of biopharmaceuticals, vaccine antigens, industrial enzymes, biomaterials, and biofuel [6]. Hyperexpression of recombinant protein in plant expression system is only possible through plastid transformation. The high ploidy number of the plastid genome results in higher level of protein expression, and up to 70% total soluble protein is reported to be produced in tobacco [7]. Moreover, hyperexpression of therapeutic proteins and vaccine antigens in chloroplasts (leaves), leucoplasts (roots), or chromoplasts (fruits) makes it ideal organelle for the oral delivery of vaccine antigens against tetanus, cholera, anthrax, canine parvovirus, and plague [8]. Other salient advantages include possibility of multigene engineering, absence of gene silencing, position effect, epigenetic, complete absence of pleiotropic effects due to subcellular compartmentalization, and transgene containment due to maternal inheritance of plastids in most of the crops [9].

Plastid transformation was first established in unicellular green algae (*Chlamydomonas reinhardtii*) followed by model tobacco plant. It has now been well established in economically valuable crops (rice, brassica, cotton, carrot, spinach, lettuce, etc.) and even in woody plant like popular. Small circular plastid genome (plastome) facilitates targeted engineering, which has been exploited not only for basic research but also for the applied research [10]. Most of the genes present in plastome have been characterized through functional genomics. Organellar transcription and translation have been thoroughly elucidated to understand transcriptional and translational machinery of the plastids. Even the proteins involved in cross-talk between chloroplast and nucleus have been worked out. Further, plastid transformation is the most ideal technology to develop marker-free transgenic plants where antibiotic resistance genes, used for the selection of putative transformants, are not acceptable by the ultimate consumer. Different techniques have been developed to produce marker-free plants in order to facilitate the acceptance of transplastomic crops. In spite of so many advantages of plastid transformation technology, there are still difficulties impeding expansion of this technology to economically valuable crops particularly monocots. These include lack of species-specific regulatory sequences, inefficient selection system, metabolic burden in case of hyperexpression and unavailability of green plastids in monocots.
