Plastid Genome Engineering and its Potential Applications: A Review

Plastid genome engineering is a credible tool for the basic biotechnological research and various innovative techniques have led to the better understanding of the complex processes involved in the plastid transformation. Plastids in higher plants are the major biosynthetic centers for photosynthesis which is the main source of energy requirement. Plastids have their own genome i.e. plastome which is maternally inherited in most angiospermic plant species. Although production of transgenic plants has traditionally been through expression of transgene in the nucleus, but plastid transformation is considered more attractive and efficient target for genetic engineering due to several advantages over nuclear transformation including high level of foreign protein, eliminating the risk of cross pollination with weeds, absence of silencing mechanism and ability to engineer multiple genes rather than a single gene. The potential utility of plastid genome engineering has been explored in development of crops with various agronomic traits, development of vaccine, biopharmaceuticals, therapeutic proteins, biomaterials and industrial enzymes, which will definitely prove beneficial in near future. Plastid transformation is still to be fully utilized for product commercialization, because of the problems associated with protein purification and expression level control. This review article highlights the various possibilities and potential applications of plastid genome engineering for generation of marker free transplastomic plants, improvement in agronomic traits and role of plastids in the production of cost effective biopharmaceuticals and biomaterials.

The plant cell's genetic information is localized in the nucleus along with DNA in chloroplast and mitochondria for DNA replication, transcription and protein synthesis. Plastids are the major biosynthetic centers for photosynthesis in plant cells and eukaryotic algae, which is the primary source of food production (Wang et al. 2009). The plastids genome called as plastome is a circular double-stranded DNA molecule of size 120 to 160 kb, present in 1,000-10,000 copies per cell in different plant species and contains 100-120 highly conserved unique genes which are maternally inherited in most angiosperm plant species. The expression of transgenes in the nucleus led to the production of transgenic plants for basic and applied purposes worldwide, but the possibility that transgenes may escape via pollen, contaminating non-transformed plants has given scope for a new field of genetic engineering i.e. plastid transformation (Ruf et al. 2007;Daniell 2007). These plants with transformed plastid genomes are termed as transplastomic (Maliga 1993). Plastid transformation is a tissue culture dependent Print ISSN : 1974ISSN : -1712 Online ISSN : 2230-732X process which involves integration of transgene that encodes a selectable marker by two homologous recombination events, followed by exposure of plastids to the selective agent and finally elimination of untransformed plastid genome copies in the tissue culture medium containing antibiotics (Bock 2001 Plastid transformation is routinely done in tobacco and the efficiency of transformation is much lower in other plants than in tobacco (Maliga 2004). The possibilities and obstacles to extend this technology to higher crops which regenerate through somatic embryogenesis has been discussed by many authors (Daniell et al. 2002;Lee et al. 2006;Clarke and Daniell 2011). The chloroplast genome of closely related plant species was not found conserved. Due to the lack of conservation of intergenic spacer regions of the chloroplast and the species specificity of this regulatory sequences have put forward the process of development of highly efficient species specific transformation vectors for integration and expression of transgenes in chloroplast (Daniell et al. 2016). First commercial development of an oral drug produced in commercial lettuce cultivar using species specific chloroplast transformation vector was published by Su et al. in 2015. This may open up new era in plastid genome engineering to introduce and express novel genes in the engineered plants for oral delivery of pharmaceuticals and vaccines, which will reduce expensive purification, cold storage, transportation and short shelf life of current protein drugs. In this review, we will discuss advances made so far for generation of marker free transplastomic plants, which is the need of hour for public acceptability of the genetically modified crops, transplastomic plants for expression of agronomically important traits and role of plastids in the production of cost effective biopharmaceuticals and biomaterials in plants.

Development of marker free transplastomic plants
The marker genes are required for the selection of transplastomic plants. After selection of transplastomic plants, the marker genes are eliminated for the biosafety concern to release antibiotic resistant gene in the field crops and the high level expression of marker gene will increase metabolic burden on the plant . The marker free plants can be obtained by direct repeats or Cre-lox recombination approaches.
In the Cre-loxP site specific recombination, marker gene (flanked by two directly oriented lox sites, 34 bp) and gene of interest are introduced into the plastid genome without Cre activity. When marker elimination is required, a gene encoding nuclear plastid targeting Cre activity is introduced into the nucleus and subsequent import in plastids excises sequences between two lox sites (Corneille et al. 2001). Another site specific recombinase (phiC31 phage integrase) have been used for the excision of aadA marker gene, flanked by directly oriented non identical phage attP (215 bp) and bacterial attB (54 bp) attachment sites. The marker gene thus removed after nuclear transformation of transplastomic plants with integrase gene encoding a plastid targeted integrase enzyme (Kittiwongwattana et al. 2007).
Both the systems (Cre-lox and Int-att) are equally efficient for obtaining marker free plants, but Int-att appears to be better choice as plastid DNA contains pseudo lox sites recognized by Cre (Corneille et al. 2003;Lutz et al. 2004;Kittiwongwattana et al. 2007). Alternatively, the removal of marker gene via directly repeated sequences (Iamtham and Day 2000), transient co-integrative (Klaus et al. 2003;2004) and cotransformation-segregation (Kindle et al. 1991;Ye et al. 2003) approaches may be used to obtain marker free plants, but due to some limitations, these approaches are not commonly used to obtain marker free plants. Recently, removal of aadA marker gene was achieved by using mycobacteriophage Bxb1 recombinase and attP/attBII recognition sites (Shao et al. 2014). Several antibiotic-free selectable markers such as D-amino acid oxidase (Gisby et al. 2012), isopentenyl transferase (IPT) (Dunne et al. 2014) and anthranilate synthase α-subunit (ASA2)

Engineering of plastid genome for agronomic traits
The engineering of plastid genome for agronomic traits is important to feed worldwide increasing population (Clarke and Daniell 2011). Hence several agronomic traits for crop improvement, including herbicide resistance, insect resistance, draught tolerance, salt, water and temperature tolerance have already been engineered via plastid transformation (Verma and Daniell 2007;Repkova 2010). The major advances have already been made by expressing heterologous cry genes for delta-endotoxin from Bacillus thuriengiensis via engineering chloroplast genome. Plastid expression of Bt gene in important major crops has not yet reached commercial development, as market is saturated with Bt crops that avoid the use of expensive chemical pesticides . Different cry genes have been expressed in different crops against a range of pests by plastid transformation (Kota et al. 1999;De Cosa et al. 2001;Gatehouse 2008;Chakrabarti et al. 2006;Liu et al. 2008;Kim et al. 2009;Dufouramantel et al. 2005).
The authors suggested that targeting of cry genes to chloroplast confers a high level plant resistance to different insects, thus providing an efficient strategy for crop insect management. The RNA interference (RNAi) concept was also used for engineering chloroplast genome for insect resistance Zhang et al. 2015). The study conducted by  used lepidoptern chitin synthase (Chi), cytochrome P450 monooxigenase and V-ATPase as RNAi targets, which are essential proteins required for insect survival. The transcripts level of targeted genes were reduced to almost undetectable levels in the insect midgut, which resulted in significant reduction in net weight of larvae and population rate. In another study, Zhang et al. (2015) introduced dsRNA via chloroplast genome to target insect β-actin gene to provide resistance against Colorado potato beetle. The expression of dsRNAs via chloroplast genome explore the possibility of use of RNAi approaches to confer desired agronomic traits or to downregulate dysfunctional genes following oral delivery of dsRNA bio-encapsulated within the plant cell .
Plastid transformation has also been used for the development of plant varieties which are resistant to bacterial and fungal diseases. Disease resistant tobacco was developed by expressing MSI-99, an antimicrobial peptide which conferred resistance to fungal pathogen Colletotrichum destructive in tobacco (DeGray et al. 2001). Transplastomic plants inhibited the growth of pregerminated spores of Aspergillus flavus, Fusarium moniliforme and Verticillium dahlia and Pseudomonas syringae pv tabaci bacteria by more than 95% compared with non-transformed control plant, which suggested that MSI-99 expressed in tobacco chloroplasts can provide significant protection from both bacterial and fungal pathogens. The research conducted by Wang et al. (2015) showed that MSI-99 expressed in tobacco chloroplast is capable of providing protection against rice blast, one of the most dangerous fungal rice disease.
The possibility of plastid genome engineering for weed control has been explored in several studies. Plastid expression of bar gene which encode herbicide inactivating phosphinothricin acetyltransferase (PAT) enzyme led to high level enzyme accumulation and conferred field tolerance to glufosinate (Daniell et al. 1998;Lutz et al. 2001). Plastid expression of bacterial 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme in transgenic chloroplast of tobacco and soybean resulted in strong herbicide tolerance (Dufouramantel et al. 2007). In another study, plastid expression of a variant form of the 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene conferred higher resistance to the broad-spectrum herbicide, glyphosate (Roudsari et al. 2009).
Every plant is exposed to various biotic and abiotic stress factors such as drought, salinity and freezing which affect plant's growth and ultimately crop production. Plastid genetic engineering has successfully been used for the development of abiotic stress tolerance in plants. Trehalose, an osmoprotectant accumulated under stress conditions can play a significant role in protecting plant cells against damage caused by these stresses. The expression of trehalose phosphate synthase 1 (TPS1) gene in chloroplast has no phenotypic variation (Lee et al. 2003) as compared to nuclear transgenic plants. The study conducted by Kumar et al. (2004) clearly showed that transgenic carrot plants expressing badh (betaine aldehyde dehydrogenase) gene accumulated glycine betaine which showed tolerance to high conc. of NaCl up to 400 mmol/l, the highest level of salt tolerance reported among genetically engineered crops. A gene for choline monooxygenase (BvCMO) from beet (Beta vulgaris) was expressed via plastid genetic engineering in tobacco (Zhang et al. 2008). Transplastomic plants accumulated glycine betaine, an osmoprotectant in leaves, roots and seeds and showed tolerance to toxic level of choline and salt/drought stress when compared to wild type plants. Khan et al. (2015) also highlighted that expression of ArDH gene in tobacco chloroplast increases tolerance to high conc. of NaCl up to 350 mM due to expressed ArDH gene encoding enzyme arabitol dehyrogenase, which is responsible for reduction of D-ribulose to D-arabitol.
The chloroplast targeted codA gene from Arthrobacter globiformis for transgenic rice has been developed for water stress tolerance which showed higher photosystemII activity and better physiological performance under water stress conditions (Kathuria et al. 2009). Temperature stress resistance, an important agronomic trait can be successfully achieved by expressing E. coli panD gene which catalyses the decarboxylation of L-aspartate to generate β-alanine and CO 2 . Transplastomic plants expressing panD was able to endure high temperature stress than that of wild type plants (Fouad and Altpeter 2009;Wani et al. 2015). Further, Chen et al. (2014) showed that by using protease inhibitors and chitinase in transgenic tobacco confer resistance against insects, pathogens and abiotic stress.

Engineering of plastid genome for production of biopharmaceuticals
One of the most fascinating applications of plastid genetic engineering is being used for the production of biopharmaceuticals. Plant cell expressing therapeutic proteins can be lyophilized and stored indefinitely at room temperature without losing their efficacy (Kwon et al. 2013). So, the use of high level expression of particular protein in edible leaves permits oral delivery and hence reduces production cost by eliminating the purification step. Plastid transformation has made enormous advances in the field of molecular farming for the production of high end biopharmaceuticals. More than 40 biopharmaceuticals and vaccine antigens have been expressed in the chloroplast genome by different researchers .
The first therapeutic protein, human somatotropin (hST) was expressed in a soluble, biologically active and disulfide bonded form (Staub et al. 2000), since then many researchers have expressed both bacterial and viral vaccine antigen genes in plastid genome. The vaccines developed have only been experimented on mice and developing effective vaccines for human use is still in the progress (Daniell et al. 2009). Most therapeutic proteins were expressed in tobacco chloroplast for initial evaluation and for the oral delivery of drugs, its usefulness was limited due to high alkaloid content. After extensive optimization of plastid transformation protocols in lettuce, therapeutic proteins were subsequently expressed in lettuce (Ruhlman et al. 2010) which is the only reproducible transplastomic system for oral delivery of biopharmaceuticals and vaccines. Some algae have also been explored for production of vaccine antigens.

Engineering of plastid genome for production of biomaterials
Plastid transformation has been used for the production of many industrially valuable biomaterials such as enzymes, amino acids and polyester. The plastid transformation has been used to produce p-hydroxybenzoic acid (pHBA), which is major monomer in liquid crystal polymers. The transplastomic tobacco plants expressing ubiC gene produced highest level of pHBA polymer in normal healthy plant (Vitanen et al. 2004). The chloroplast produced enzymes offer several advantages over traditionally produced enzymes including significantly reduced cost, improved stability and no need for enzyme purification. Genes for thermostable xylanase enzyme used in pulp and paper industry were successfully expressed in tobacco chloroplast (Leelavathi et al. 2003;Kim et al. 2011 Trichoderma reesei in tobacco chloroplast. Chloroplast produced enzyme showed wider pH optima and thermostability than E. coli produced enzyme. Plastid transformation have also been used for the production of amino acid, tryptophan (Tsai et al. 2005) and polyester, polyhydroxybutyrate (Lossl et al. 2003).

CONCLUSION
The chloroplast genome has become innovative target for plant genetic engineering due to several advantages over nuclear transformation. Although more than 100 transgenes have been stably integrated and expressed in tobacco chloroplast genome till date, however, extension of technology to other crop plants is limited by several factors including non-availability of chloroplast genome sequences and optimization of plastid transformation protocols in different crop species. Plastid transformation is routinely carried out in tobacco, while efficiency of transformation is low in other crop plants. Plastid transformation have been used for engineering of several important agronomic traits such as insect resistance, herbicide resistance, draught, salt, water and temperature tolerance in an eco-friendly manner which will definitely enhance crop productivity. Generation of marker free transplastomic plants, which is the need of hour for public acceptability of the genetically modified crops will facilitate public acceptance in near future. The plastid transformation is being used for the production of valuable vaccines antigens and therapeutic proteins.
This technology has not resulted in product commercialization, because of problem associated with protein purification and in nascent stage as experimented only in animal model. Most of the therapeutic proteins and vaccine antigens are produced in tobacco plastid genome which cannot be used for oral administration because of its toxic alkaloid contents. At present lettuce is the only reproducible system used for production of therapeutic proteins. Therefore, it is necessary to use those plant species that can be used as a system for oral delivery of biopharmaceuticals and vaccines.
Further studies with edible crops will be needed in near future for successful implementation of plastid genetic engineering for oral administration of drugs.