1. INTRODUCTION:

Plant transformation is a remarkable tool that enables the introduction of any desired gene from many organisms across the different kingdoms into plants and supports genetic engineers by serving as an exceptional plant breeding technique to produce plants with unique and functional genotypes^[1]. Plant transformation is essential for scientific research, agriculture and recombinant protein production. It helps in better understanding of the functionality of introduced genes. For instance, in Nicotiana tabacum, the Ycf9 gene was disrupted by the insertion of aadA marker gene through homologous recombination at trnS/trnG site which led to the discovery that the protein encoded by the Ycf9 gene is required for the stable association of the antenna protein CP26 with Photosystem-II^[2].

Plant transformation maybe achieved by direct gene transfer methods such as particle bombardment^[3], electroporation^[4], polyethylene glycol mediated transformation^[5], glass bead agitation^[6] or by Agrobacterium-mediated indirect transformation^[7]. The technique employed for transformation depends on the host plant. For instance, Agrobacterium-mediated transformation is mostly implemented in dicotyledons, for which Agrobacterium serves as a natural pathogen while direct gene transfer techniques are preferred in monocotyledons^[8]. The direct methods are used to transform not only the nucleus but also the cell organelles. One such emerging technique is plastid transformation, where instead of the nuclear genome of the cell, the chloroplast genome is transformed resulting in the uptake and integration of the transgene. Plastid transformation has helped to improve the characteristics of important crops, by enhancing their agronomic traits to improve yield^[9] and increase their nutritive value^[10,11] and induce resistance/tolerance against abiotic and biotic stresses^[12,13], by the introduction of foreign genes or by the overexpression of endogenous genes.

Plastid transformation was first successfully accomplished through microprojectile bombardment by replacing the mutant gene with wild type atpB gene which produces ATP from ADP in the presence of a proton gradient across the membrane, to restore the photosynthetic capacity of the unicellular organism Chlamydomonas reinhardtii^[^14]. Later, the same was executed in a higher plant, Nicotiana tabacum to introduce the 16S rRNA gene through microprojectile bombardment^[15^]. Plastid transformation is an evolving technique which has been implemented predominantly in model organisms viz, Chlamydomonas, Nicotiana species. However, the commercial success of the technology depends on its reach to important crops, which depends on designing efficient vector systems and innovative transformation methods^[16].

2. A GREENER TECHNIQUE:

Horizontal gene transfer (HGT) is the lateral transfer of genetic information between unrelated or distantly-related species and it establishes phylogenetic relationship between genomes and impacts the evolution of species^[17]. For instance, Striga hermonthica, a parasitic plant which affects monocots received a nuclear gene of Sorghum bicolor through its continual parasitic activity over the years, which was revealed by expression tag analysis^[18]. Although HGT is crucial for engendering genetic variation, the transfer of gene from transgenic crops into wildtypes may lead to development of undesired characteristics and can be detrimental to the ecosystem^[17]. In transgenics, the foreign gene inserted is expected to be confined within the system it was integrated into. But in nuclear transformation, the frequency of gene transfer through pollination is tremendously high, which can result in the loss of biodiversity of the ecosphere. For instance, the transfer of herbicide resistance gene from a transgenic plant into weeds led to generation of superweeds, which have become immune to the herbicide^[19].

In chloroplast transformation, the transgene introduced in the plastid is maternally inherited into the subsequent generations which maintains the genes through the generations. For instance, in an experiment performed by Shashi Kumar et al. in 2004, when pollen from a transplastomic line was used to pollinate an emasculated non-transgenic line, it was observed that the F₁ seedlings exhibited only the non-transgenic characters^[20]. This property of gene containment makes it more environment-friendly in comparison to nuclear transformation, thus making it a greener technique.

3. A CLEANER TECHNIQUE:

In the process of transforming plants, antibiotic resistant genes or herbicide resistant genes, which are mostly of prokaryotic origin are used as selectable markers to isolate the transformed units from the non-transformed units. Despite its role in the selection, the gene does not play any role in transcription or expression of the gene of interest. But the presence of these genes in a transgenic system poses a bio-safety hazard because they may get transferred to other organisms in the ecosystem^[21]. Clean-gene technology enables the production of transgenic plants in the absence of a selectable marker or by the excision of the selectable marker gene by a recombinase enzyme, by flanking the marker gene with its recognition sites^[22]. For instance, the expression cassette of VP6 viral protein in plastids, from Rotavirus is a potential source for an oral subunit vaccine for gastroenteritis, involved the use of a Cre/lox system (where Cre recombinase enzyme cleaves loxP recognition site) in Nicotiana tabacum to remove the selectable marker gene, aadA which confers resistance to spectinomycin and streptomycin^[23]. Also, the use of scorable markers such as gfp (green fluorescent proteins)^[24,25] and lux (luminescence genes)^[26] are encouraged to differentiate between transformed and untransformed lines. Plastid transformation not only serves as a ‘green’ technique but can also be used as a ‘clean-gene’ technique.

4. OVEREXPRESSION OF PROTEINS:

The standard number of chloroplasts present in the mesophyll cells of dicots such as Arabidopsis and Triticum aestivum is approximately 100 per cell. Since each chloroplast holds nearly 100 genomes, it allows for multiple copies of the transgene to be integrated into the plastid genomes^[27]. Due to the absence of RNA interference in chloroplast, there is no gene silencing observed in the system, therefore the level of protein expression is marginally higher in plastid transformation. This overproduction of recombinant proteins by chloroplast transformation paves the way for plants to be used as ‘molecular factories’^[28]. The limitations faced in nuclear transformation such as poor protein stability due to lack of appropriate post-translational modifications required to produce a functional protein, highly variable transgene expression due to illegitimate recombination and reduced transgene expression caused by gene silencing, hinders molecular farming in plants. With the advent of plastid transformation, the above limitations may be overcome^[29,30]. For instance, when the PlyGBS gene which codes for a lytic protein from Streptococcus agalactiae was expressed in the chloroplast of Nicotiana tabacum, an expression up to 70% of the total soluble protein was observed. The expressed lytic protein can be used as a proteinaceous antibiotic against Group A and Group B pathogenic Streptococci^[31]. Molecular farming through chloroplast transformation is highly promising with the current focus towards biodegradable plastics^[32,33] and mass production of therapeutics^[34]. Furthermore, optimizing the codon in accordance with the host species genome enhances the production of the recombinant proteins^[35].

5. VECTOR CONSTRUCTION AND TRANSFORMATION:

In chloroplast transformation, constitutive or inducible promoters are used to drive expression of the transgene, which makes the proteins to be expressed at exponential levels. In chloroplast genome many genes can be expressed using a single promoter^[36] and hence in plastid transformation, transgenes can be organized in the vector as polycistronic transcription units attached to a single promoter. With complex post translational modification ability, the polycistrons are converted into functional proteins. The commonly used promoters include, psbA (Photosystem II protein D1 precursor), rbcL (Rubisco bisphosphate carboxylase), psaA (Photosystem I P700 chlorophyll apoprotein A1) and atpI (ATP synthase protein I). While designing vectors, the transgene is flanked on both the sides by UTR (untranslated region) sequences. The 5’UTR sequence is placed upstream of the coding sequence which is recognized by the ribosome to bind and initiate translation. The 3’UTR sequence is placed downstream of the coding sequence which plays a critical role in termination of translation. The commonly used 5’UTRs include 5’UTRs of rbcL, atpB, psbA, Ggagg and T7G10. The commonly used 3’UTRs include 3’UTRs of rbcL, rps16 and psbA^[37]. The transgene gets integrated into the plastome through homologous recombination between the wildtype plastid DNA and the vector carrying the transgene. Some popular insertion sites include rbcL-accD, trnl-trnA, 3’rps12/7-trnV, trnfM-trnG, 23srrnA-16srrnA, atpB-rbcL and rps7-ndhB (Table 1-4). Hence the concern over position effect is eliminated unlike in the case of nuclear transgenic lines. Further the designed vector is transferred into the organelle using direct gene transfer techniques, such as particle bombardment, which is the most preferred method over polyethylene glycol (PEG) mediated transformation, glass bead agitation and electroporation.

6. PLASTID TRANSFORMATION IN LOWER ORGANISMS:

Chlamydomonas reinhardtii was the first unicellular algae in which plastid transformation was achieved^[14]. Later other lower organisms including liverworts such as, Marchantia polymorpha was also transformed with vectors to integrate aadA gene at the trnI/trnA site employing particle bombardment technique^[38,39]. Many therapeutic proteins with potential treatment options to various diseases, have been expressed in Chlamydomonas reinhardtii. Fourteenth human fibronectin type III domain (14FN3), human vascular endothelial growth factor (VEGF), high mobility group protein B1 (HMGB1), have been produced in Chlamydomonas reinhardtii through particle bombardment by replacing the psbA locus. VEGF has been proved to have potential to treat erectile dysfunction, pulmonary emphysema and depression, HMGB1 facilitates important functions in wound healing including activation of endothelial cell and innate immune cells, and maturation of dendritic cell. The recombinant proteins were produced at the rate of 2-3% of total soluble protein (TSP)^[40]. In the course of developing a vaccine for malaria, Pfs25^[41,3], Pfs48/45^[42] antigens from Plasmodium falciparum was engineered in Chlamydomonas reinhardtii through particle bombardment with Kanamycin as the selectable marker. The antibodies generated by the above vaccine intrude in the completion of parasite’s life cycle in the mosquito mid-gut, thus preventing transmission.

Renewable fuel is in high demand all over the world, as the existing fuel source is depleting. Reifschneider-Wegner et al. expressed a nuclear cphydA gene ([FeFe] hydrogenase) from Chlamydomonas reinhardtii in its plastome. As a result, it was observed that the starch and ROS-detoxifying enzyme levels were elevated. Further, the [FeFe] hydrogenase enzyme helps in hydrogen generation from phototrophic organisms which is a promising source of renewable fuel^[43]. The technique has been further extended to other unicellular organisms such as Phaeodactylum tricornutum, in which HBsAg (Hepatitis B surface antigen), which is commonly used as a Hepatitis B vaccine and CL4 monoclonal antibody were expressed up to 0.7% and 8.7% of TSP respectively^[44]. Further, the Cry11Aa gene from Bacillus thuringiensis was expressed in Chlamydomonas reinhardtii up to a level of 0.35% of total cellular protein (TCP) to control Aedes and Culex mosquitoes^[45]. Plastid transformation in lower organisms has been observed to be promising in the production of renewable energy, biopharmaceuticals and towards mosquito control (Table 1).

7. PLASTID TRANSFORMATION IN MONOCOTS:

Many staple foods across the world such as Oryza sativa, Zea mays, Triticum aestivum, Hordeum vulgare, which constitute a dominant part of the diet belong to the class of monocotyledons. Application of plastid transformation in monocotyledons to improve important agronomic traits would be beneficial. Although plastid transformation of monocots is desired, it is not well-established due to lack of appropriate regeneration protocols suitable for cereals^[46]. But some progress has been made in transforming rice plastids with gfp in the intergenic spacer trnI-trnA through particle bombardment^[46,47]. Despite the low regeneration efficiency, homoplastomic state was achieved through repeated sub-culturing once in 20 days^[47]. The transformation and regeneration protocols have to be improved for successful transformation of monocots, to acquire new characteristics or to improve the expression of inherent genes.

Table 2: Biopharmaceuticals produced in Nicotiana tabacum through plastid transformation using aadA marker

S. No.	Transgene	Source	Function	Site of integration	Expression	Reference
1	Fraction 1 outer capsule (F1-V) antigen	Yersinia pestis	As plague vaccine	trnI/trnA	14.8% TSP	70
2	Insulin-like Growth Factor 1 (IGF-I)	Human	Tissue renewal and repair	trnI/trnA	32% TSP	35
3	Cardiotrophin-1 (hCT-1)	Human	Potential therapeutic cytokine	trnV/3′rps12	5% TSP	71
4	Transforming Growth Factor-β3 (TGFβ3)	Human	Therapeutic protein used to reduce scarring	rbcL/accD	12% TLP	72
5	Envelope Protein Domain III (EDIII)	Dengue virus	To elicit serotype-specific neutralizing antibodies	trnfM/trnG	1.6% TP (Total Protein)	73
6	VP6 protein	Rotavirus	Treatment of gastroenteritis	rbcL/accD	>15% TSP	23
7	Retrocyclin-101 (RC101) and Protegrin-1 (PG1)	RC101 – Synthetic Human pseudogene, PG1 - Porcine	Potent broad spectrum antimicrobials	trnI/trnA	RC101 - 32-38% TSP, PG1 - 17-26% TSP	74
8	Alpha1-Antitrypsin (A1AT)	Human	Therapeutic protease inhibitor	rbcL/accD	2% TSP	75
9	Amidase (Pal) and Lysozyme (Cpl-1)	Phages	Treatment of pneumonia	trnfM/trnG	Pal - 30% of TSP, Cpl-1 -10% of TSP	76
10	2L21 (immunogenic peptide)	Canine Parvovirus	As a subunit vaccine	trnI/trnA	6% TSP	77
11	Hemagglutinin Neuraminidase Neutralizing Epitope (HNE)	Paramyxoviruses	As vaccine against Newcastle disease	16S-trnI/trnA	0.5% TSP	78
12	Exotoxin epitopes	Corynebacterium diphtheriae, Bordetella pertussis, and Clostridium tetani	Vaccination during infancy	trnN/trnR	0.8% TSP	79
13	Acid Alpha Glucosidase (GAA)	Human	Treatment of Pompe disease	trnI/trnA	190 μg per g of freeze-dried leaf material	80
14	Epidermal Growth Factor (hEGF)	Human	Maintenance and repairing of epithelial tissues	rrn16/trnI	6% TSP	81
15	β-site APP cleaving enzyme (BACE)	Human	Treatment of Alzheimer disease	trnI/trnA	2% TSP	82

Table 3: Engineering tolerance towards biotic and abiotic stresses using aadA marker in dicots

S. No.	Transgene	Source	Function	Host	Site of integration	Expression	Reference
1	Hydroxy-Phenylpyruvate Dioxygenase (hppd)	Pseudomonas fluorescens	Herbicide tolerance	Nicotiana tabacum	rbcL/accD	-	83
2	Plant chitinase (BjCHI1)	Brassica juncea	Inhibits fungal phytopathogens and gram-negative bacteria	Nicotiana tabacum	rbcL/accD	-	84
3	γ-tocopherol methyltransferase (γ-tmt)	Arabidopsis	Sugar export and starch metabolism under stress	Nicotiana tabacum	trnA/trnI	7.7% TLP	85
4	β-glucosidase (bgl1)	Trichoderma reesei	Protection from aphids/whiteflies	Nicotiana tabacum	trnI/trnA	1.9-fold increase	86
5	Pinelliaternata agglutinin (pta)	Pinellia ternate	Protection against Myzuspersicae, Bemisiatabaci&Helicoverpazea	Nicotiana tabacum	trnI/trnA	9.2% TSP	87
6	Cry1Ab	Bacillus thuringiensis	Insecticidal to Plutella xylostella	Brassica oleracea	C16s/C23s	4.8–11.1% TSP	67
7	Mitochondrial Superoxide Dismutase (MnSOD) and Glutathione Reductase (gor)	MnSOD – Nicotiana tabacum and gor-Escherichia coli	Scavenging ROS	Nicotiana tabacum	rrn16/rsp12/7	MnSOD - 9-fold increase, gor - 8-fold increase	88
8	Chloroperoxidase (cpo)	Pseudomonas pyrrocinia	Confers fungal resistance	Nicotiana tabacum	trnI/trnA	-	89
9	Mutated Acetolactate Synthase (mALS)	Arabidopsis thaliana	Herbicide tolerance	Nicotiana tabacum	rbcL/accD	-	90
10	Choline monooxygenase (BvCMO)	Beta vulgaris	Improves salt and drought tolerance	Nicotiana tabacum	trnfM/trnG	GlyBet - 0.25 μmol/g FW	91

Table 4: Transfer of valuable traits from various sources into dicots using aadA marker

S. No.	Transgene	Source	Function	Host	Site of integration	Expression	Reference
1	β-Mannanase (man1)	Trichoderma reesei	Lignocellulosic hydrolysis for biofuel production	Nicotiana tabacum	16S-trnI/trnA	25 units of mannanase per gm fresh weight	92
2	Glutathione-S-transferase (GST) and His-tagged derivative of the Maltosebinding protein (His6-MBP)	Schistomosoma japonicum	GST - cytoplasmic male sterility; His6-MBP - purification by affinity chromatography	Nicotiana tabacum	rrn16/rps12/7	GST - 7% TSP, His6-MBP - 37% TSP	93
3	Thiolase (phaA), Synthase (phaC) and Reductase (phaB)	phaA, phaC- Acinetobacter sp. and phaB- Bacillus megaterium	Renewable and biodegradable plastic	Nicotiana tabacum	psbA locus	18.8% dry weight PHB in leaf tissue	94
4	Pectin lyase (PelA) and Manganese peroxidase (MnP-2)	PelA - Streptomyces thermocarboxydus and MnP-2 – Phanerochaete chrysosporium	Pectin and lignin degradation for waste degradation	Nicotiana tabacum	rrn16S/3′rps′12	PelA - 66676.25 units per ~470 g of biomass, MnP-2 - 21,715.46 units per ~470 g of biomass	95
5	β-carotene ketolase (CrtW), β-carotene hydroxylase (CrtZ) and Isopentenyl diphosphate isomerase (idi)	CrtWand CrtZ - Brevundimonas sp. and idi- Paracoccus sp.	Production of Astaxanthin (carotenoid)	Lactuca sativa	rbcL/accD	Produced astaxanthin fatty acid esters - 67% of total carotenoids	11
6	β-carotene ketolase (CrtW) and β-carotene hydroxylase (CrtZ)	Brevundimonas sp.	Production of Astaxanthin (carotenoid)	Nicotiana tabacum	rbcL/accD	7.29 mg/ g dry weight	96
7	Fructose-1,6-/sedoheptulose-1,7-bisphosphatase (fbp/sbp)	Synechococcus	Increases the photosynthetic capacity	Lactuca sativa	rbcL/accD	Photosynthetic capacity and productivity were increased 1.3-fold and 1.6-fold, respectively	9
8	Lux operon (luxCDABEG)	Photobacterium leiognathi	Autonomous light emission	Nicotiana tabacum	rps12/trnV, trnI/trnA	The LUX-trnI/trnA plants emitted approx. 25 times more photons than the LUX-rps12/trnV plants	97
9	β-1,4-Endoglucanase (EGPh)	Pyrococcus horikoshii	To produce fermentable sugars for biofuel and ethanol industry	Nicotiana tabacum	trnV/rps7	25% TSP	98
10	Metallothionein (mt1)	Mouse	Phytoremediation of mercury by chelation	Nicotiana tabacum	trnI/trnA	-	99

9. CONCLUSION:

Chloroplast transformation has been shown to offer unique advantages over nuclear transformation. Foreign proteins can be overexpressed and multiple gene expression can be achieved using a single promoter. Chloroplast transformation is projected to offer enormous advantages in the production of therapeutic proteins, industrial enzymes, biofuel and phytoremediation. It can also help in improving plant agronomic traits and can help in introducing cytoplasmic male sterility. Although plastid transformation has been implemented predominantly in model organisms viz, Chlamydomonas and Nicotiana species, the commercial success of the technology depends on its reach to other important food crops such as cereals, fruits and vegetables.

10. ACKNOWLEDGEMENT:

Our sincere thanks to Vellore Institute of Technology for encouraging us to write the review.

11. REFERENCES:

1. Birch, R.G., 1997. Plant transformation: problems and strategies for practical application. Annual review of plant biology, 48(1), pp.297-326.

2. Ruf, S., Biehler, K. and Bock, R., 2000. A small chloroplast-encoded protein as a novel architectural component of the light-harvesting antenna. The Journal of cell biology, 149(2), pp.369-378.

3. Gregory, J.A., Topol, A.B., Doerner, D.Z. and Mayfield, S., 2013. Algae-produced cholera toxin-Pfs25 fusion proteins as oral vaccines. Applied and environmental microbiology, pp.AEM-00714.

4. Xie, W.H., Zhu, C.C., Zhang, N.S., Li, D.W., Yang, W.D., Liu, J.S., Sathishkumar, R. and Li, H.Y., 2014. Construction of novel chloroplast expression vector and development of an efficient transformation system for the diatom Phaeodactylumtricornutum. Marine biotechnology, 16(5), pp.538-546.

5. Nugent, G.D., Coyne, S., Nguyen, T.T., Kavanagh, T.A. and Dix, P.J., 2006. Nuclear and plastid transformation of Brassica oleracea var. botrytis (cauliflower) using PEG-mediated uptake of DNA into protoplasts. Plant science, 170(1), pp.135-142.

6. Economou, C., Wannathong, T., Szaub, J. and Purton, S., 2014. A simple, low-cost method for chloroplast transformation of the green alga Chlamydomonasreinhardtii. In Chloroplast Biotechnology (pp. 401-411). Humana Press, Totowa, NJ.

7. Anami, S., Njuguna, E., Coussens, G., Aesaert, S. and Van Lijsebettens, M., 2013. Higher plant transformation: principles and molecular tools. International Journal of Developmental Biology, 57(6-7-8), pp.483-494.

8. Hofmann, N.R., 2016. A breakthrough in monocot transformation methods.

9. Ichikawa, Y., Tamoi, M., Sakuyama, H., Maruta, T., Ashida, H., Yokota, A. and Shigeoka, S., 2010. Generation of transplastomic lettuce with enhanced growth and high yield. GM crops, 1(5), pp.322-326.

10. Apel, W. and Bock, R., 2009. Enhancement of carotenoid biosynthesis in transplastomic tomatoes by induced lycopene-to-provitaminA conversion. Plant Physiology, 151(1), pp.59-66.

11. Harada, H., Maoka, T., Osawa, A., Hattan, J.I., Kanamoto, H., Shindo, K., Otomatsu, T. and Misawa, N., 2014. Construction of transplastomic lettuce (Lactucasativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids. Transgenic research, 23(2), pp.303-315.

12. De Cosa, B., Moar, W., Lee, S.B., Miller, M. and Daniell, H., 2001. Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nature biotechnology, 19(1), p.71.

13. Hou, B.K., Zhou, Y.H., Wan, L.H., Zhang, Z.L., Shen, G.F., Chen, Z.H. and Hu, Z.M., 2003. Chloroplast transformation in oilseed rape. Transgenic Research, 12(1), pp.111-114.

14. Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M. and Shark, K.B., 1988. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science, 240(4858), pp.1534-1538.

15. Svab, Z., Hajdukiewicz, P. and Maliga, P., 1990. Stable transformation of plastids in higher plants. Proceedings of the National Academy of Sciences, 87(21), pp.8526-8530.

16. Ahmad, N., Michoux, F., Lössl, A.G. and Nixon, P.J., 2016. Challenges and perspectives in commercializing plastid transformation technology. Journal of experimental botany, 67(21), pp.5945-5960.

17. Vogan, A.A. and Higgs, P.G., 2011. The advantages and disadvantages of horizontal gene transfer and the emergence of the first species. Biology direct, 6(1), p.1.

18. Yoshida, S., Maruyama, S., Nozaki, H. and Shirasu, K., 2010. Horizontal gene transfer by the parasitic plant Strigahermonthica. Science, 328(5982), pp.1128-1128.

19. Daniell, H., 2002. Molecular strategies for gene containment in transgenic crops. Nature biotechnology, 20(6), p.581.

20. Kumar, S., Dhingra, A. and Daniell, H., 2004. Stable transformation of the cotton plastid genome and maternal inheritance of transgenes. Plant molecular biology, 56(2), pp.203-216.

21. Yau, Y.Y. and Stewart, C.N., 2013. Less is more: strategies to remove marker genes from transgenic plants. BMC biotechnology, 13(1), p.36.

22. Afolabi, A.S., 2009. Clean Gene Technology and its application to crops. Plant Breeding of Orphan Crops in Africa, p.211.

23. Inka Borchers, A.M., Gonzalez‐Rabade, N. and Gray, J.C., 2012. Increased accumulation and stability of rotavirus VP6 protein in tobacco chloroplasts following changes to the 5′ untranslated region and the 5′ end of the coding region. Plant biotechnology journal, 10(4), pp.422-434.

24. Michoux, F., Ahmad, N., McCarthy, J. and Nixon, P.J., 2011. Contained and high‐level production of recombinant protein in plant chloroplasts using a temporary immersion bioreactor. Plant biotechnology journal, 9(5), pp.575-584.

25. Oey, M., Ross, I.L. and Hankamer, B., 2014. Gateway-assisted vector construction to facilitate expression of foreign proteins in the chloroplast of single celled algae. PloS one, 9(2), p.e86841.

26. Manuell, A.L., Beligni, M.V., Elder, J.H., Siefker, D.T., Tran, M., Weber, A., McDonald, T.L. and Mayfield, S.P., 2007. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast. Plant biotechnology journal, 5(3), pp.402-412.

27. Maliga, P., 2004. Plastid transformation in higher plants. Annu. Rev. Plant Biol., 55, pp.289-313.

28. Sharma, A.K. and Sharma, M.K., 2009. Plants as bioreactors: recent developments and emerging opportunities. Biotechnology advances, 27(6), pp.811-832.

29. Adem, M., Beyene, D. and Feyissa, T., 2017. Recent achievements obtained by chloroplast transformation. Plant Methods, 13(1), p.30.

30. Scotti, N., Rigano, M.M. and Cardi, T., 2012. Production of foreign proteins using plastid transformation. Biotechnology Advances, 30(2), pp.387-397.

31. Oey, M., Lohse, M., Kreikemeyer, B. and Bock, R., 2009. Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. The plant journal, 57(3), pp.436-445.

32. Bohmert-Tatarev, K., McAvoy, S., Daughtry, S., Peoples, O.P. and Snell, K.D., 2011. High levels of bioplastic are produced in fertile transplastomic tobacco plants engineered with a synthetic operon for production of polyhydroxybutyrate. Plant physiology, pp.pp-110.

33. Guda, C., Lee, S.B. and Daniell, H., 2000. Stable expression of a biodegradable protein-based polymer in tobacco chloroplasts. Plant Cell Reports, 19(3), pp.257-262.

34. Lim, S., Ashida, H., Watanabe, R., Inai, K., Kim, Y.S., Mukougawa, K., Fukuda, H., Tomizawa, K.I., Ushiyama, K.I., Asao, H. and Tamoi, M., 2011. Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts. Plant molecular biology, 76(3-5), pp.335-344.

35. Daniell, H., Ruiz, G., Denes, B., Sandberg, L. and Langridge, W., 2009. Optimization of codon composition and regulatory elements for expression of human insulin like growth factor-1 in transgenic chloroplasts and evaluation of structural identity and function. BMC biotechnology, 9(1), p.33.

36. Quesada-Vargas, T., Ruiz, O.N. and Daniell, H., 2005. Characterization of heterologous multigene operons in transgenic chloroplasts. Transcription, processing, and translation. Plant Physiology, 138(3), pp.1746-1762.

37. Jin, S. and Daniell, H., 2015. The engineered chloroplast genome just got smarter. Trends in plant science, 20(10), pp.622-640.

38. Chiyoda, S., Linley, P.J., Yamato, K.T., Fukuzawa, H., Yokota, A. and Kohchi, T., 2007. Simple and efficient plastid transformation system for the liverwort Marchantiapolymorpha L. suspension-culture cells. Transgenic research, 16(1), pp.41-49.

39. Chiyoda, S., Yamato, K.T. and Kohchi, T., 2014. Plastid transformation of sporelings and suspension-cultured cells from the liverwort Marchantiapolymorpha L. In Chloroplast Biotechnology (pp. 439-447). Humana Press, Totowa, NJ.

40. Rasala, B.A., Muto, M., Lee, P.A., Jager, M., Cardoso, R.M., Behnke, C.A., Kirk, P., Hokanson, C.A., Crea, R., Mendez, M. and Mayfield, S.P., 2010. Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonasreinhardtii. Plant biotechnology journal, 8(6), pp.719-733.

41. Gregory, J.A., Li, F., Tomosada, L.M., Cox, C.J., Topol, A.B., Vinetz, J.M. and Mayfield, S., 2012. Algae-produced Pfs25 elicits antibodies that inhibit malaria transmission. PloS one, 7(5), p.e37179.

42. Jones, C.S., Luong, T., Hannon, M., Tran, M., Gregory, J.A., Shen, Z., Briggs, S.P. and Mayfield, S.P., 2013. Heterologous expression of the C-terminal antigenic domain of the malaria vaccine candidate Pfs48/45 in the green algae Chlamydomonasreinhardtii. Applied microbiology and biotechnology, 97(5), pp.1987-1995.

43. Reifschneider-Wegner, K., Kanygin, A. and Redding, K.E., 2014. Expression of the [FeFe] hydrogenase in the chloroplast of Chlamydomonasreinhardtii. international journal of hydrogen energy, 39(8), pp.3657-3665.

44. Hempel, F., Lau, J., Klingl, A. and Maier, U.G., 2011. Algae as protein factories: expression of a human antibody and the respective antigen in the diatom Phaeodactylumtricornutum. PloS one, 6(12), p.e28424.

45. Kang, S., Odom, O.W., Thangamani, S. and Herrin, D.L., 2017. Toward mosquito control with a green alga: expression of Cry toxins of Bacillus thuringiensis subsp. israelensis (Bti) in the chloroplast of Chlamydomonas. Journal of applied phycology, 29(3), pp.1377-1389.

46. Lee, S.M., Kang, K., Chung, H., Yoo, S.H., Xu, X.M., Lee, S.B., Cheong, J.J., Daniell, H. and Kim, M., 2006. Plastid transformation in the monocotyledonous cereal crop, rice (Oryzasativa) and transmission of transgenes to their progeny. Molecules and cells, 21(3), p.401.

47. Wang, Y., Wei, Z. and Xing, S., 2018. Stable plastid transformation of rice, a monocot cereal crop. Biochemical and biophysical research communications, 503(4), pp.2376-2379.

48. Barrera, D.J., Rosenberg, J.N., Chiu, J.G., Chang, Y.N., Debatis, M., Ngoi, S.M., Chang, J.T., Shoemaker, C.B., Oyler, G.A. and Mayfield, S.P., 2015. Algal chloroplast produced camelid VHH antitoxins are capable of neutralizing botulinum neurotoxin. Plant biotechnology journal, 13(1), pp.117-124.

49. Demurtas, O.C., Massa, S., Ferrante, P., Venuti, A., Franconi, R. and Giuliano, G., 2013. A Chlamydomonas-derived Human Papillomavirus 16 E7 vaccine induces specific tumor protection. PLoS One, 8(4), p.e61473.

50. Dreesen, I.A., Charpin-El Hamri, G. and Fussenegger, M., 2010. Heat-stable oral alga-based vaccine protects mice from Staphylococcus aureus infection. Journal of biotechnology, 145(3), pp.273-280.

51. Wang, X., Brandsma, M., Tremblay, R., Maxwell, D., Jevnikar, A.M., Huner, N. and Ma, S., 2008. A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65). Bmc Biotechnology, 8(1), p.87.

52. Yang, Z., Chen, F., Li, D., Zhang, Z., Liu, Y., Zheng, D., Wang, Y. and Shen, G., 2006. Expression of human soluble TRAIL in Chlamydomonasreinhardtii chloroplast. Chinese Science Bulletin, 51(14), pp.1703-1709.

53. Zhou, F., Badillo‐Corona, J.A., Karcher, D., Gonzalez‐Rabade, N., Piepenburg, K., Borchers, A.M.I., Maloney, A.P., Kavanagh, T.A., Gray, J.C. and Bock, R., 2008. High‐level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes. Plant biotechnology journal, 6(9), pp.897-913.

54. Cheng, L., Li, H.P., Qu, B., Huang, T., Tu, J.X., Fu, T.D. and Liao, Y.C., 2010. Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons. Plant cell reports, 29(4), pp.371-381.

55. Scotti, N., Alagna, F., Ferraiolo, E., Formisano, G., Sannino, L., Buonaguro, L., De Stradis, A., Vitale, A., Monti, L., Grillo, S. and Buonaguro, F.M., 2009. High-level expression of the HIV-1 Pr55 gag polyprotein in transgenic tobacco chloroplasts. Planta, 229(5), pp.1109-1122.

56. Fernández‐San Millán, A., Ortigosa, S.M., Hervás‐Stubbs, S., Corral‐Martínez, P., Seguí‐Simarro, J.M., Gaétan, J., Coursaget, P. and Veramendi, J., 2008. Human papillomavirus L1 protein expressed in tobacco chloroplasts self‐assembles into virus‐like particles that are highly immunogenic. Plant biotechnology journal, 6(5), pp.427-441.

57. Maldaner, F.R., Aragão, F.J.L., Dos Santos, F.B., Franco, O.L., Lima, M.D.R.Q., de Oliveira Resende, R., Vasques, R.M. and Nagata, T., 2013. Dengue virus tetra-epitope peptide expressed in lettuce chloroplasts for potential use in dengue diagnosis. Applied microbiology and biotechnology, 97(13), pp.5721-5729.

58. Chan, H.T., Xiao, Y., Weldon, W.C., Oberste, S.M., Chumakov, K. and Daniell, H., 2016. Cold chain and virus‐free chloroplast‐made booster vaccine to confer immunity against different poliovirus serotypes. Plant biotechnology journal, 14(11), pp.2190-2200.

59. Rigano, M.M., Manna, C., Giulini, A., Pedrazzini, E., Capobianchi, M., Castilletti, C., Di Caro, A., Ippolito, G., Beggio, P., De GiuliMorghen, C. and Monti, L., 2009. Transgenic chloroplasts are efficient sites for high‐yield production of the vaccinia virus envelope protein A27L in plant cells. Plant biotechnology journal, 7(6), pp.577-591.

60. Shao, H.B., He, D.M., Qian, K.X., Shen, G.F. and Su, Z.L., 2008. The expression of classical swine fever virus structural protein E2 gene in tobacco chloroplasts for applying chloroplasts as bioreactors. Comptesrendusbiologies, 331(3), pp.179-184.

61. Rosales‐Mendoza, S., Alpuche‐Solís, Á.G., Soria‐Guerra, R.E., Moreno‐Fierros, L., Martínez‐González, L., Herrera‐Díaz, A. and Korban, S.S., 2009. Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants. The Plant Journal, 57(1), pp.45-54.

62. Lakshmi, P.S., Verma, D., Yang, X., Lloyd, B. and Daniell, H., 2013. Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro. PLoS One, 8(1), p.e54708.

63. Albarracín, R.M., Becher, M.L., Farran, I., Sander, V.A., Corigliano, M.G., Yácono, M.L., Pariani, S., López, E.S., Veramendi, J. and Clemente, M., 2015. The fusion of Toxoplasma gondii SAG1 vaccine candidate to Leishmaniainfantum heat shock protein 83‐kDa improves expression levels in tobacco chloroplasts. Biotechnology journal, 10(5), pp.748-759.

64. Gorantala, J., Grover, S., Goel, D., Rahi, A., Magani, S.K.J., Chandra, S. and Bhatnagar, R., 2011. A plant based protective antigen [PA (dIV)] vaccine expressed in chloroplasts demonstrates protective immunity in mice against anthrax. Vaccine, 29(27), pp.4521-4533.

65. Gorantala, J., Grover, S., Rahi, A., Chaudhary, P., Rajwanshi, R., Sarin, N.B. and Bhatnagar, R., 2014. Generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine. Journal of biotechnology, 176, pp.1-10.

66. Kota, M., Daniell, H., Varma, S., Garczynski, S.F., Gould, F. and Moar, W.J., 1999. Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proceedings of the National Academy of Sciences, 96(5), pp.1840-1845.

67. Liu, C.W., Lin, C.C., Yiu, J.C., Chen, J.J. and Tseng, M.J., 2008. Expression of a Bacillus thuringiensis toxin (cry1Ab) gene in cabbage (Brassica oleracea L. var. capitata L.) chloroplasts confers high insecticidal efficacy against Plutellaxylostella. Theoretical and Applied Genetics, 117(1), pp.75-88.

68. Chakrabarti, S.K., Lutz, K.A., Lertwiriyawong, B., Svab, Z. and Maliga, P., 2006. Expression of the cry9Aa2 Bt gene in tobacco chloroplasts confers resistance to potato tuber moth. Transgenic research, 15(4), p.481.

69. Ruiz, O.N. and Daniell, H., 2005. Engineering cytoplasmic male sterility via the chloroplast genome by expression of β-ketothiolase. Plant Physiology, 138(3), pp.1232-1246.

70. Arlen, P.A., Singleton, M., Adamovicz, J.J., Ding, Y., Davoodi-Semiromi, A. and Daniell, H., 2008. Effective plague vaccination via oral delivery of plant cells expressing F1-V antigens in chloroplasts. Infection and immunity, 76(8), pp.3640-3650.

71. Farran, I., Río‐Manterola, F., Íñiguez, M., Gárate, S., Prieto, J. and Mingo‐Castel, A.M., 2008. High‐density seedling expression system for the production of bioactive human cardiotrophin‐1, a potential therapeutic cytokine, in transgenic tobacco chloroplasts. Plant biotechnology journal, 6(5), pp.516-527.

72. Gisby, M.F., Mellors, P., Madesis, P., Ellin, M., Laverty, H., O’Kane, S., Ferguson, M.W. and Day, A., 2011. A synthetic gene increases TGFβ3 accumulation by 75‐fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule. Plant biotechnology journal, 9(5), pp.618-628.

73. Gottschamel, J., Lössl, A., Ruf, S., Wang, Y., Skaugen, M., Bock, R. and Clarke, J.L., 2016. Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems. Plant molecular biology, 91(4-5), pp.497-512.

74. Lee, S.B., Li, B., Jin, S. and Daniell, H., 2011. Expression and characterization of antimicrobial peptides Retrocyclin‐101 and Protegrin‐1 in chloroplasts to control viral and bacterial infections. Plant biotechnology journal, 9(1), pp.100-115.

75. Nadai, M., Bally, J., Vitel, M., Job, C., Tissot, G., Botterman, J. and Dubald, M., 2009. High-level expression of active human alpha1-antitrypsin in transgenic tobacco chloroplasts. Transgenic research, 18(2), pp.173-183.

76. Oey, M., Lohse, M., Scharff, L.B., Kreikemeyer, B. and Bock, R., 2009. Plastid production of protein antibiotics against pneumonia via a new strategy for high-level expression of antimicrobial proteins. Proceedings of the National Academy of Sciences, 106(16), pp.6579-6584.

77. Ortigosa, S.M., Fernández-San Millán, A. and Veramendi, J., 2010. Stable production of peptide antigens in transgenic tobacco chloroplasts by fusion to the p53 tetramerisation domain. Transgenic research, 19(4), pp.703-709.

78. Sim, J.S., Pak, H.K., Kim, D.S., Lee, S.B., Kim, Y.H. and Hahn, B.S., 2009. Expression and characterization of synthetic heat-labile enterotoxin B subunit and hemagglutinin–neuraminidase-neutralizing epitope fusion protein in Escherichia coli and tobacco chloroplasts. Plant molecular biology reporter, 27(3), pp.388-399.

79. Soria-Guerra, R.E., Alpuche-Solís, A.G., Rosales-Mendoza, S., Moreno-Fierros, L., Bendik, E.M., Martínez-González, L. and Korban, S.S., 2009. Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer. Planta, 229(6), pp.1293-1302.

80. Su, J., Sherman, A., Doerfler, P.A., Byrne, B.J., Herzog, R.W. and Daniell, H., 2015. Oral delivery of Acid Alpha Glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of Pompe mice. Plant biotechnology journal, 13(8), pp.1023-1032.

81. Wirth, S., Segretin, M.E., Mentaberry, A. and Bravo-Almonacid, F., 2006. Accumulation of hEGF and hEGF–fusion proteins in chloroplast-transformed tobacco plants is higher in the dark than in the light. Journal of biotechnology, 125(2), pp.159-172.

82. Youm, J.W., Jeon, J.H., Kim, H., Min, S.R., Kim, M.S., Joung, H., Jeong, W.J. and Kim, H.S., 2010. High-level expression of a human β-site APP cleaving enzyme in transgenic tobacco chloroplasts and its immunogenicity in mice. Transgenic research, 19(6), pp.1099-1108.

83. Dufourmantel, N., Dubald, M., Matringe, M., Canard, H., Garcon, F., Job, C., Kay, E., Wisniewski, J.P., Ferullo, J.M., Pelissier, B. and Sailland, A., 2007. Generation and characterization of soybean and marker‐free tobacco plastid transformants over‐expressing a bacterial 4‐hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance. Plant biotechnology journal, 5(1), pp.118-133.

84. Guan, Y., Ramalingam, S., Nagegowda, D., Taylor, P.W. and Chye, M.L., 2008. Brassica junceachitinase BjCHI1 inhibits growth of fungal phytopathogens and agglutinates Gram-negative bacteria. Journal of experimental botany, 59(12), pp.3475-3484.

85. Jin, S. and Daniell, H., 2014. Expression of γ‐tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal‐induced oxidative stresses by reducing reactive oxygen species. Plant biotechnology journal, 12(9), pp.1274-1285.

86. Jin, S., Kanagaraj, A., Verma, D., Lange, T. and Daniell, H., 2011. Release of hormones from conjugates: chloroplast expression of β-glucosidase results in elevated phytohormone levels associated with significant increase in biomass and protection from aphids or whiteflies conferred by sucrose esters. Plant physiology, 155(1), pp.222-235.

87. Jin, S., Zhang, X. and Daniell, H., 2012. Pinelliaternata agglutinin expression in chloroplasts confers broad spectrum resistance against aphid, whitefly, lepidopteran insects, bacterial and viral pathogens. Plant biotechnology journal, 10(3), pp.313-327.

88. Poage, M., Le Martret, B., Jansen, M.A., Nugent, G.D. and Dix, P.J., 2011. Modification of reactive oxygen species scavenging capacity of chloroplasts through plastid transformation. Plant molecular biology, 76(3-5), pp.371-384.

89. Ruhlman, T.A., Rajasekaran, K. and Cary, J.W., 2014. Expression of chloroperoxidase from Pseudomonas pyrrocinia in tobacco plastids for fungal resistance. Plant science, 228, pp.98-106.

90. Shimizu, M., Goto, M., Hanai, M., Shimizu, T., Izawa, N., Kanamoto, H., Tomizawa, K.I., Yokota, A. and Kobayashi, H., 2008. Selectable tolerance to herbicides by mutated acetolactate synthase genes integrated into the chloroplast genome of tobacco. Plant physiology, 147(4), pp.1976-1983.

91. Zhang, J., Tan, W., Yang, X.H. and Zhang, H.X., 2008. Plastid-expressed choline monooxygenase gene improves salt and drought tolerance through accumulation of glycine betaine in tobacco. Plant cell reports, 27(6), p.1113.

92. Agrawal, P., Verma, D. and Daniell, H., 2011. Expression of Trichoderma reesei β-mannanase in tobacco chloroplasts and its utilization in lignocellulosic woody biomass hydrolysis. PloS one, 6(12), p.e29302.

93. Ahmad, N., Michoux, F., McCarthy, J. and Nixon, P.J., 2012. Expression of the affinity tags, glutathione-S-transferase and maltose-binding protein, in tobacco chloroplasts. Planta, 235(4), pp.863-871.

94. Bohmert-Tatarev, K., McAvoy, S., Daughtry, S., Peoples, O.P. and Snell, K.D., 2011. High levels of bioplastic are produced in fertile transplastomic tobacco plants engineered with a synthetic operon for the production of polyhydroxybutyrate. Plant physiology, 155(4), pp.1690-1708.

95. Espinoza-Sánchez, E.A., Álvarez-Hernández, M.H., Torres-Castillo, J.A., Rascón-Cruz, Q., Gutiérrez-Díez, A., Zavala-García, F. and Sinagawa-García, S.R., 2015. Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast. Electronic Journal of Biotechnology, 18(3), pp.161-168.

96. Hasunuma, T., Miyazawa, S.I., Yoshimura, S., Shinzaki, Y., Tomizawa, K.I., Shindo, K., Choi, S.K., Misawa, N. and Miyake, C., 2008. Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering. The Plant Journal, 55(5), pp.857-868.

97. Krichevsky, A., Meyers, B., Vainstein, A., Maliga, P. and Citovsky, V., 2010. Autoluminescent plants. PloS one, 5(11), p.e15461.

98. Nakahira, Y., Ishikawa, K., Tanaka, K., Tozawa, Y. and Shiina, T., 2013. Overproduction of Hyperthermostable β-1, 4-Endoglucanase from the Archaeon Pyrococcushorikoshii by Tobacco Chloroplast Engineering. Bioscience, biotechnology, and biochemistry, 77(10), pp.2140-2143.

99. Ruiz, O.N., Alvarez, D., Torres, C., Roman, L. and Daniell, H., 2011. Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability. Plant biotechnology journal, 9(5), pp.609-617.

Received on 12.04.2019 Modified on 23.05.2019

Research J. Pharm. and Tech. 2019; 12(10):5083-5090.

DOI: 10.5958/0974-360X.2019.00881.3

S. No.	Transgene	Source	Function	Marker	Site of integration	Expression	Reference
1	C2, H7 (VHH monomers) and H7-B5 (heterodimer) antitoxins	Alpacas	Neutralizes botulinum neurotoxin	KanR	psbA	5% TSP	48
2	E7 oncoprotein	Human Papillomavirus	As therapeutic vaccines against HPV-related lesions	aadA	The integration occurs in psaA intron	0.12% TSP	49
3	D2 (fibronectin) fused with CTB	Staphylococcus aureus	As a heat-stable vaccine	aadA	tscA gene	1.6 mg CTB-D2 per 1g of lyophylized algae	50
4	Mammary-Associated Serum Amyloid (M-SAA)	Bovine	To stimulate the production of mucin in the gut	lux	Replace the psbA locus	3% TSP	26
5	Glutamic Acid Decarboxylase (hGAD65)	Human	A potential autoantigenic marker for the diagnosis of type 1 diabetes	aadA	CP3/CP4 primer pair	0.25-0.3% TSP	51
6	Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)	Human	Induces selective apoptosis	aadA	Inserted into the chlL gene	0.43-0.67% TSP	52