Plant genetic modification is one of the important technologies for crop improvement as well as an emerging approach for the production of biopharmaceuticals in plants. The expression of foreign genes in chloroplasts offers several advantages over their nuclear gene expression: polycistronic and enhanced foreign proteins expression, lack of epigenetic interference allowing stable transgene expression. Additionally, transgenic chloroplasts are generally not transmitted through pollen grains because of the cytoplasmic localization and prevent pollen-mediated outcrossing thus providing high level transgene biological containment. Recently, great progress has been made in chloroplast genetic engineering of various crop plants. little more detailsâ€¦ In this article, we review and emphasize on recent studies on plastid genetic engineering of potato for various aspects including current status and the potential for future expansion.
Genetic engineering of potato has been accomplished routinely by Agrobacterium-mediated gene transfer resulting in genotypes with increased tolerance to insects, viruses and nutritionally improved quality traits (Lawson et al., 1990; Perlak et al., 1993; Stark et al., 1992, Sorensen et al. 2000, Kok-Jacon et al 2007, Crowell et al 2008, Hemavathi et al 2010). Since the existing nuclear transformation methods appear to be inadequate in fulfilling these requirements, the targeting of the plastid genome becomes the most attractive alternative method. Chloroplast is a type of specialized plastid which develops from proplastid in meristematic cells. Depending on organ type and environmental conditions, proplastids differentiate into a variety of plastids including amyloplasts, chromoplasts and elaioplasts, with specialized and important functions for the plant. Plastid transcription is a multistep gene regulation system and plays a crucial role in developmental and environmental regulation of plastid gene expression (Shiina et al. 2005). Genes encoded by plastome can be effectively manipulated to attain the desirable quality traits such as novel molecules, increased biotic and abiotic tolerance and photosynthetic efficiency.
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Plastome engineering certainly has myriad applications especially in crop improvement programmes as transformation of the plastid genome (plastome) has several advantages over nuclear transformation. Since plastids are maternally inherited, the introduction of foreign genes into the plastid genome prevents pollen-mediated outcrossing (Daniell et al., 1998; Maliga, 1993; Scott and Wilkinson, 1999). Additionally, plastid transformation offers the possibility of polycistronic operon expression, thus enabling stacking of multiple expressed genes in a single transformation event (Staub and Maliga, 1995). Furthermore, the high ploidy level of the plastome in cells makes feasible high levels of transgene expression (McBride et al., 1995). Since gene integration always occurs by homologous recombination and is neither affected by position effects nor by epigenetic gene-silencing mechanisms (Svab et al., 1990, Bock 2001) which is normally ensued from random insertion of transgenes in nuclear transformation (Kooter et al 1999) and therefore transgene expression is stable in the progeny of transplastomic plants. Daniell and McFadden (1987) provided the first proof of direct uptake and expression of the foreign genes in isolated platids from dark-grown cucumber cotyledons. Latter in 1988 Boynton et al. used high-velocity tungsten microprojectiles for plastome transformation of unicellular alga Chlamydomonas reinhardtii. Since then this concept has been extended to number of crop spesies; tobacco (Svab et al., 1990, Golds et al., 1993; O'Neil et al., 1993), carrot (Kumar et al 2001), cotton (Kumar et al 2001), soybean (Dufourmantel et al. 2004), spinach (To et al 1996), lettuce (Lelivelt et al 2005), sugar beet (De Marchis et al 2009) , potato ( Sidorov et al. 1999, Nguyen et al 2005), tomato (Ruf et al. 2001; Wurbs et al. 2007), egg plant (Singh et al. 2010.), Arabidopsis (Sikdar et al. 1998), Lesquerella (Skarjinskaia et al. 2003), oilseed (Hou et al. 2003), soybean (Dufourmantel et al. 2004), cotton (Kumar et al. 2004b), cauliflower (Nugent et al. 2006), rice (Lee et al. 2006a, b), oil seed rape (Hou et al 2003) and cabbage (Liu et al. 2007).
The expression of foreign genes in plastid genomes not only dramatically enhances the level of gene expression (there are up to 105 copies of the foreign gene in chloroplasts per plant cell) (Bendich 1987), but proteins from plastid transgenes may be expressed at very high levels. Consequently, this technique is currently broadly utilized, and has proven successful for stable delivery of DNA to plastids of number of crops. However, its efficiency and applicability are rather limited, and reports of successful transgene expression are still scanty in potato species. Non-photosynthetic plastids contain an identical genome to that in chloroplasts, but transcription occurs at constitutively low rates (Deng & Gruissem 1988; Piechulla et al. 1985). Gene expression in non green tissue plastids is largely uncharacterized compared with leaf chloroplasts. Recent studies have reported the thorough characterization of gene expression in tuber amyloplasts (Brosch et al. 2007; Valkov et al. 2009), showing that gene expression in such organelles is generally impaired, with multi-step control occurring at transcriptional, post-transcriptional and translational levels. Nevertheless, some mRNAs, such as the transcript of the fatty acid biosynthesis gene accD, displayed relatively high ribosome association in tubers. However, these studies have allowed the tentative identification of candidate regulatory sequences that could potentially improve transgene expression in amyloplasts and other non-green plastids (Valkov et al 2010). In this review we summarise the various aspects of plastid transformation including integration and expression of foreign genes into the plastid genome of potato for various agronomical, industrial and pharmaceutical applications and also discuss the current status and future prospects of plastid transformation in potato.
Plant regeneration system:
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For any successful genetic transformation studies efficient plant regeneration system is a prerequisite. Regeneration system in potato is highly species dependant (reference.)â€¦â€¦â€¦litte more information. Several regeneration systems have been reported in potato using various explants such as leaf (Figueira Filho et al 1994,Wenzler et al. 1989 Hulme et al 1992, Yadav and Sticklen 1995, De Block 1998, Nguyen et al 2005, Banerjee et al 2006), inter nodes (Beaujean et al 1998, Ducreux et al 2005, Chakravarty and Wang-Pruski 2010), petioles (Yee et al 2001, Ducreux et al 2005), Leaf and stem (Visser et al 1989, Gustafson et al 2006) and tubers (Ishida et al 1989, Kumar et al 1995 Dale & Harnpson 1995.) Recently, transformation of potato was extended by the Agrobacterium-mediated biolistic method (Nguyen et al 2010). Using this approach transgenic shoots could be obtained at a similar frequency to that achieved through conventional biolistics. However, efficient and highly reproducible species independent plant regeneration system is yet to be standerdizesd for both Agrobacterium and direct gene transfer methods in potato. Agrobacterium-mediated nuclear transformation of potato using stem and leaf explants is routine, there has been very little effort towards development of biolistic approaches for transformation of this crop. (referenceCraig et al 2005 reportedâ€¦â€¦. Very similar results were shown by Romano et al 2001 â€¦â€¦.(write something like this, in a story form). Although direct gene transfer using the biolistic method is apparently the most widespread technology for plastid transformation, stable introduction of cloned DNA into chloroplast genomes also has been conclusively demonstrated using two alternative protocols. The use of polyethylene glycol (PEG) to introduce foreign DNA into plant chloroplasts has proven possible in isolated protoplasts of lettuce and tobacco (O'Neill 1993, Koop et al. 1996 Eibl et al., 1999; Lelivelt et al., 2005). This method holds out the promise of the capacity to generate more cells with transformed plastids more readily than by the biolistic procedure.(reference). In another approach direct injection of DNA into individual chloroplasts in photosynthetic leaf cells of tobacco has also been possible (Knoblauch et al 1999). Establishment of a reproducible potato plastid transformation protocol requires development of a highly efficient regeneration system, optimization of microprojectile bombardment parameters and a stringent potato specific selection scheme. Sidorov et al (1999) described first stable chloroplast transformation of potato by microprojectile bombardment of leaf explants. Significant differences were observed in transformation frequencies between tobacco and potato. In general, transformation frequency of about one event per bombarded plate has been reported in tobacco (Svab and Maliga, 1993), whereas, in potato on average, one event was recovered from 15 to 30 bombarded plates (Sidorov et al 1999). Potato plastid transformants are generated at 10-30 times lower frequencies than tobacco (Nguyen et al (2005). Valkov et al. 2011 were able to regenerate about one shoot for every bombardment. This efficiency corresponds to 15-18-fold improvement compared to previous reports (Sidorov et al. 1999 and Nguyen et al. 2005).
Despite recent progress, potato plastid transformation is still limited by low transformation frequencies and low gene expression in tubers of transplastomic plants (Sidorov et al. 1999; Gargano et al. 2003 Gargano 2006; Nguyen et al. 2005; Valkov et al. 2011). This low recovery of transplastomic shoots has been ascribed to several factors, such as difference in efficiency of shoot regeneration from potato explants(Valkov et al. 2011), relatively inefficient homologous recombination system, non-optimal homology and length of flanking regions(Nguyen et al. 2005, Sidorov et al (1999), promoter used for the expression of the genes (Valkov et al 2011, Davarpanah et al. 2009), stringent selection for selection of homoplasmic transplastomic events and inefficient gene expression in non-green plastids (Deng & Gruissem, 1988; Piechulla et al. 1985, Brosch et al. 2007; Kahlau and Bock 2008; Valkov et al. 2009).
Plastid transformation system and Trans gene expression:
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It was thought that plastome sequences held little variation from one species to other however, recent plastid genomes sequence analyses are revealing a richness of diversity among plastid genomes that was not expected earlier (Kanamoto et al., 2005; Kimet al., 2005; Daniell et al., 2006, some moreâ€¦â€¦â€¦â€¦). Whilst foreign genes targeting IGS (abbreviate) regions not perturb the endogenous plastid gene function, it is possible to achieve integration without complete homology but recombination and thus transformation efficiency is impaired (DeGray et al., 2001; Zubko et al., 2004). Evaluations of UTRs from different plant species indicates the need of employing species specific regulatory elements such as promoters and translation sequences to elevate the level of foreign protein expression (Kramzar et al., 2006, Volkov et al 2011). It has been shown that transcription from heterologous rrn promoters in transplastomic tobacco would require species-specific activating factors (Sriraman et al., 1998). This was evident in tobacco, when flanking sequences derived from the petunia plastome were used for recombination, transformation efficiency decreased by more than ten fold (DeGray et al. 2001). Earlier, plastid transformation in potato was attempted with vectors designed for homologous tobacco IR flanking sequences. The homologous flanking sequences present in these vectors show very high homology (98-99%) to the corresponding sequences of potato ptDNA (Sidorov et al, 1999, Nguyen et al 2005), therefore the efficient integration of such sequences in potato via homologous recombination was anticipated.(ref?). Similar results were obtained with no apparent reductions in transformation frequencies in tobacco and tomato using homologous or homeologous N. tabacum and S. nigrum ptDNA sequences in transformation vectors (Kavanagh et al. 1999; HorvaÂ´th et al. 2000; Nugent et al. 2005; Nugent et al. 2006), or in transformations of N. benthamiana plastids with tobacco-derived vectors (Davarpanah et al. 2009), indicating that species-specific vectors are not always necessary (Skarjinskaia et al 2003). If a similar level of homology exists among other species, the construction of species-specific vectors may not be required always (Sidorov et al, 1999, Skarjinskaia et al 2003). However, attempts were made to make use of species specific plastid vectors for further improving the transformation efficiency (Dufourmantel et al. 2004; Kumar et al. 2004a, b; Kanamoto et al. 2006). Since the complete potato ptDNA sequence became available recently, vectors with homologous potato flanking sequences were constructed and tested to examine the effect of increasing homology on plastid transformation efficiency (Gargano et al. 2005; Chung et al. 2006, Volkov et al 2011Valkov et al. 2011). Amman & Brosius 1985 reported the first successful expression of gfp gene under bacterial trc promoter in tobacco plastid. However, transplastomic plants expressing gfp gene undere rrn promoter contained approximately 90- fold more gfp than plants using trc or psbA promoters [Newell et al 2003]. Nevertheless, gfp gene driven by rrn promoter was transiently expressed in non-green starch-storing amyloplasts in potato tuber tissue and in chromoplasts in marigold petals, carrot roots and pepper fruits (Hibbard et al 1998) following bombardment of tissue slices suggesting that foreign genes can be expressed in non-green plastids. Nguyen et al. (2005) reported that in transplastomic potato no plastid transformants were recovered when aadA gene is driven by trc promoter. However, this bacterial promoter can give sufficient expression of the aadA gene to select plastid transformants in tobacco. This emphasizes the importance of using a strong plastid promoter for crops with a less optimal regenerative capacity.
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Explain a little about PEP and NEP first. Although both the eubacterial-type (PEP) and phage-type (NEP) RNA polymerases were shown to be active in non-green plastids (Brosch et al. 2007; Kahlau and Bock 2008; Valkov et al. 2009), genes with relatively higher levels of transcripts in tubers (e.g., rrn, clpP, accD, ycf1, ycf2) contain either NEP or multiple PEP and NEP promoters (Hajdukiewicz et al. 1997; Legen et al. 2002; Valkov et al. 2009), suggesting the involvement of NEP in tuber amyloplast gene expression. Indeed, recent data suggest that the two polymerases do not simply mediate gene classs specific transcription in different cells or plastid types, but differential processing, stability, and accumulation of the resulting transcripts and polypeptides (Legen et al. 2002; Cahoon et al. 2004; Zoschke et al. 2007) are involved in regulating gene expression. Alternative 5'- and 3'-UTRs of plastid genes can also play a significant role in transcript stability and translatability (Bock 2001; Maliga 2003, volkov et al 2011).
Attempts were made in order to improve the transformation efficiency by use of novel vectors containing potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy (LSC) region of the plastome (Valkov et al 2011). Variation in accumulation of transcripts, proteins attributed to a differential translatability of the different plastid constructs with different promoters and UTR regions. Although, transcript accumulation in tubers tended to match that in leaves, some differences were apparent between constructs with the same 5' regulatory sequences, but different 3'- UTRs. Plants with the bacterial derived rrnB terminator accumulated 5 to 7 fold more gfp transcripts than plants with psbA and rpoA 3'UTRs, respectively, suggesting a positive effect of the rrnB terminator on mRNA stability.(referenceâ€¦..). A significant positive effect of clpP 5' regulatory sequences on translatability, particularly in non-green plastids was found (Valkov et al 2011) which is in agreement with expression profile analyses that indicated clpP as one of the less downregulated genes in tubers compared to leaves (Valkov et al. 2009). In leaves, accumulation of gfp was about 4% of total soluble protein (TSP) with constructs containing strong promoter of rrn operon with a synthetic rbcL-derived 5â€²-UTR and the bacterial rrnB terminator, where as with clpP promoter and clpP 5â€²-UTR sequence from the clpP gene it was about 0.6%TSP. However in tubers GFP protein expression was equally detectable (up to approximately 0.02% TSP) with plants transformed with both constructs. Transgene expression with the plastid rrn and the bacterial trc promoters in potato tuber amyloplasts ranged from 1 to 20% of that in leaf chloroplasts, respectively, although GFP concentration attainable with the prokaryotic promoter was lower than with rrn one (0.004 vs. 0.05% of total soluble proteins), due to its low efficacy in both tissues (Sidorov et al. 1999; Nguyen et al. 2005, volkov et al 2011). Bae et al. 1998 reported that expression of the rps16 gene was strong in chloroplasts and transcripts were also detectable in amyloplasts, suggesting that the rps16 gene is active in non photosynthetic plastids as well as in photosynthetic plastids that can be used as a new homologous recombination site of plastid transformation for potato cultivars. Since protein accumulation in tubers of plants containing constructs with the rrn promoter is generally accompanied by high expression in leaves, a potential use of the clpP 5' regulatory sequences can be envisaged in cases where recombinant protein accumulation is required in amyloplasts, but not in chloroplasts(Valkov et al 2011). The expression levels achieved in tubers may be sufficient to manipulate the expression of enzymatic proteins for metabolic engineering purposes, but are still too low to exploit tubers of transplastomic plants as a production platform for proteins with pharmaceutical or industrial interest.
Need to modify according to verma et al .
Genetic engineering of higher plant plastids typically involves stable introduction of antibiotic resistance gene as a selection marker along with gene of interest. For any successful plastid transformation efficient marker system is crucial, which is required for organelle sorting out during repeated cell divisions in vitro, in order to achieve regeneration of homoplasmic transplastomic shoots (Bock 2001; Maliga 2004). aadA is the the first used chloroplast specific antibiotic resistance marker, a bacterial aminoglycoside 3''-adenylyltransferase gene (aadA) conferring resistance to a number of antibiotics of the aminoglycoside type, including spectinomycin and streptomycin. (Goldschmidt-Clermont 1991). The relatively low transformation frequency with antibiotic resistance encoded by mutations in 16S ribosomal RNA genes (Svab et al 1990, Staub, & Maliga et al 1992) is most probably due to the recessive mode of action of the rRNA marker during the selection phase and confers antibiotic resistance only to those few chloroplast ribosomes that have received their 16 S rRNA molecule from the very few initially present transformed ptDNA copies. In contrast, antibiotic-inactivating marker genes provide dominant drug resistance to the recipient chloroplast and a single transformed plastid genome copy is sufficient to detoxify the entire organelle (Carrer et al. 1993 and 1995). Transformation efficiency with the chimeric aadA gene is about 100-fold greater than with antibiotic resistance encoded by mutations in 16S ribosomal RNA genes, this increase in the transformation frequency is due to an improved recovery of the newly formed transgenomes by the dominant aadA gene (Svab et al 1990, Staub, & Maliga et al 1992). The most efficient and routinely used selectable markers have been spectinomycin (Svab and Maliga, 1993), and kanamycin selection (Carrer et al., 1993) while the nptII appears to be less efficient, as it produces a significant background of nuclear transformants. In contrary, herbicide resistance bar gene was not found to be suitable for the direct selection of transplastomic lines as glyphosate has deleterious effects on plastid even with the bar gene expressed (approximately 7% of total soluble cellular protein) at a higher level (Ye et al., 2003). In glyphosate-treated cells of cultured tobacco leaf discs, the reticulate network of thylakoid membranes has been lost, indicating the disintegration of the photosynthetic membranes. (Lutz et al., 2001).
Reporter genes include chloramphenicol acetyltransferase (CAT; Daniell et al., 1990), b-glucuronidase (GUS; Ye et al., 1990) and green fluorescent protein (GFP, Hibberd et al., 1998). Green fluorescent protein (GFP) has been widely used as a versatile marker for monitoring of gene expression and protein tagging in plants (Haseloff & Amos 1995Pang et al., 1996; Köhler etÂ al. 1997 Vain et al., 1998, Blackman etÂ al. 1998 ). GFP is an excellent candidate for a reporter which could be used non-destructivly to monitor gene expression in subcellular compartments targeting to chloroplasts (KoÈ hler et al., 1997b) and mitochondria (KoÈ hler et al., 1997a). GFP was transiently expressed in amyloplasts of potato tubers and in chromoplasts of marigold petals, carrot roots and pepper fruits after bombardment (Hibberd et al 1998) suggesting that GFP can be used as a reporter for transient gene expression in chloroplasts and in non-photosynthetic plastids in a range of higher plants. It has been reported that high expression of GFP could affect plant morphology or inhibit plant regeneration (Haseloff and Siemering, 1998). However, a high level of GFP expression (5% tsp) in chloroplasts had no apparent deleterious effects, perhaps due to the compartmentalization (Sidrov et al., 1999). Therefore, GFP expression was valuable for screening of plastid transformants at the early stages of selection and for in vivo monitoring of plastid gene expression in various tissues and organs.
Use of antibiotic selection pressure varies from species to species and low level of spectinomycin generally used for selection of nuclear transformants was not sufficient for inhibition and sorting out of non-transformed plastids (Sidrov et al., 1999). For recovery of plastid transformants in potato, a 10-fold higher selection pressure had to be applied than for production of nuclear transformants (Sidrov et al., 1999).
Incorporation of a selectable marker gene in the plastid genome is essential to uniformly alter the thousands of genome copies in a plant cell. However, once transformation is accomplished, the marker gene becomes undesirable. However, as a consequence of placing a transgene in the chloroplast genome, the antibiotic resistance genes used as selectable markers are highly amplified. Even though chloroplast genes are maternally inherited in most crops1, the possibility of marker transfer to wild relatives2 or microorganisms3 cannot be completely excluded. Therefore, efficient methods for complete marker removal from plastid transformants are necessary. Targeted insertion and the precise deletion of marker gene from transgenic plant genome increase the potential of plant biotechnology for commercial applications and ease public concerns regarding GM crops in addition to trans gene containment. To date numerous stratagies are available for production of marker free nuclear transformants as well as platid transformantas (reviewed in Upadhyaya et al. 2010, Darbani et al 2007, Daniell et al 2001, Klus et al 2004, Day et al 2005, Lutz and Maliga 2007). However, Cre-lox recombination system is one of the best characterized and most widely used systems for these purposes (Lutz et al 2006, Gilbertson 2003, and Hajdukiewicz et al 2001).
Chloroplast is an obvious candidate for increasing photosynthesis efficiency, providing one the attractive avenues to drive increases in crop yields. Crop models predict that substantial increases in canopy photosynthesis could result from incorporating a "better Rubisco" into C3 crop species (Long et al., 2006). In addition improvements in the Rubisco would lead to an increase in the production of food, fiber and renewable energy (Spreitzer and Salvucci, 2002, Genkov et al 2010). Over the past few years, extensive work has been carried out to engineer Rubisco to alter its enzymatic properties (Andrews and Whitney, 2003; Raines, 2006; Tcherkez et al., 2006; Portis and Parry, 2007, Parry et al., 2007). There has been much interest in Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and, in particular, its large chloroplast-encoded catalytic subunit as a target for engineering to increase in net CO2 fixation in photosynthesis. Naturally occurring Rubiscos with superior catalytic turnover rates and better specificity have been found among the red algae and C4 plant species(refâ€¦.) and several approaches have been undertaken to introduce subunit genes from cyanobacteria, algae, sunflower and pea to tobacco or Arabidopsis through nuclear and plastid transformation (reviewed in Parry et al., 2003). Although, the large subunit contains the catalytic active site, Rubisco small subunit can also influence the carboxylation catalytic efficiency and CO2/O2 specificity of the enzyme. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. However, engineering the native (or foreign) S subunit genes (RbcS) in higher plants remains an elusive challenge due to the multiple RbcS copies that are located in the nucleus which essentially precludes RbcS from targeted mutagenic or replacement strategies.(refâ€¦) Multiple attempts to assemble appreciable levels of plastid-synthesized tobacco S subunits showed an apparent necessity for very high levels of plastomic rbcS mRNA expression and reduced levels of the native RbcS message (Whitney and Andrews, 2001b; Zhang et al., 2002; Dhingra et al., 2004). Although the recent development in improving performance of Rubisco seems to be reluctant, this research has provided novel insights into structural and functional relationships and greatly enhanced our understanding of this key enzyme, providing new opportunities to develop more productive crop plnats. Engineering of metabolic and PS activities for increasing sink strength especially in non-leaf sinks, such as tubers will have tremendous potential to improve the potato tuber yield.
Abiotic stress tolerance:
Potato (Solanum tuberosum L.) is relatively vulnerable to abiotic stresses such as drought. Conventional breeding methods to accelerate the abiotic stress tolerance of potato have met with limited success and efforts to improve the abiotic stress tolerance of potatoes are complicated by the genetic complexity of potato, coupled with the need for any newly developed potato varieties to adhere to rigorous yield and quality expectations (Waterer et al 2010). Genetic engineering provides a potentially useful tool for improving abiotic stress tolerance of the potato (Leone et al 1999, Rohde et al 2000, Mora-Herrera and Lopez-Delgado 2007, Hemavathi et al 2009, Hemavathi et al 2010, Shin et al 2010, Waterer et al 2010).
To cope with adverse conditions, many plants express low molecular weight compounds collectively called osmoprotectants and are typically sugars, alcohols, proline, and quaternary ammonium compounds (Glick and Pasternak, 1998). Transplastomic tobacco plants expressing Yeast trehalose phosphate synthase (TPS1) gene in chloroplast showed 169-fold more TPS1 transcript and 15-25 fold higher accumulation of trehalose than the best surviving nuclear transgenic plants and transplastomic chloroplast thylakoid membranes showed high integrity even under osmotic stress with normal growth and without pleiotropic effects(Lee at al. 2003). Another highly effective osmolyte Glycine betaine is known to accumulate in some plants during drought or high salinity. The accumulation of this compound as a result of stress protects the plant by maintaining an osmotic balance within the cell (Robinson and Jones, 1986; Rhodes and Hanson, 1993; Hanson et al., 1991; Hanson and Gage, 1991; Rathinasabapathi, Fouad, and Sigua, 2001). However tomatoes and carrot including potato do not accumulate betaine. Attempts to increase the GB accumulation by over expression of badh gene, engineered via the nuclear genome resulted in only moderate levels of tolerance to salt stress (Flowers, 2004; Rathinasabapathi et al., 2001). However, over expression of badh gene in carrot plastids resulted in high accumulation of glycine betaine and ß-alanine betaine and subsequently conferring a significant level of salt tolerance (Kumar et al., 2004a). Tang et al. 2006 developed oxidative stress-tolerant transgenic potato plants (SSA plants) by simultaneous expression of chloroplast-targeted CuZnSOD and APX genes under the control of an oxidative stress inducible sweet potato peroxidase (SWPA2) promoter. On other hand to evaluate the possible synergistic effects of glycinebetaine (GB), SSA plants were retransformed with the GB-synthesizing choline oxidase (codA) gene targeted to chloroplasts under the control of the SWPA2 promoter (SSAC plants) (Ahmad et al 2010). Simultaneous synthesis of glycinebetaine along with the overexpression of CuZnSOD and APX rendered transgenic plants synergistically tolerant to various abiotic stresses including oxidative, salt and drought stresses. In other words it is possible to genetically transfer all of above 3 genes as a single recombinant event through direct plastid genetic engineering.
Recently HegedÅ±s et al 2008 provide the evidence that transgenic tobacco lines overexpressing alfalfa ferritin in the chloroplasts with transit peptide under the control of a Rubisco small subunit gene promoter (C3 and C8), suffered less oxidative damage in comparison to the wild-type genotype and show higher tolerance to various stress factors, generating ROS including low temperature-induced photo inhibition. Earlier integration and expression of a Delta-9 desaturase gene have also been demonstrate in potato plastids that controlls the insertion of double bonds in fatty acid chains to achieve higher content of unsaturated fatty acids, a desirable trait for stress tolerance of higher plants in addition to improved nutritional value (Gargano et al 2003).
The feasibility to use chloroplast genetic engineering for weed control has been explored in several studies that aimed at producing herbicide-tolerant plants. The most commonly used herbicide, glyphosate is a broad spectrum, nonselective, systemic herbicide known to inhibit the plant aromatic amino acid biosynthetic pathway by competitively inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synsthase (EPSPS), a nuclear-encoded chloroplast-targeted enzyme involved in the biosynthesis of aromatic amino acids (Devine and Daniell, 2004). Transgenic plants resistant to glyphosate are typically engineered to overexpress EPSPS gene.(Daniell et al 1998 and Ye et al 2001). Since the target of glyphosate resides within the chloroplast, chloroplast transgenic engineering is an ideal strategy for developing glyphosate resistance in plants. Transgenic tobacco plastids expressing EPSPS gene resulted in accumulation of over 250-fold more EPSPS enzyme when compared to nuclear transgenics (Ye et al., 2001). On the contrary, such increased levels of glyphosate-resistant EPSPS did not correlate to increased tolerance to glyphosate. One reason for this discrepancy between protein level and tolerance was that the nuclear encoded gene is expressed at a high enough level to confer resistance in the appropriate cell types, whereas the plastid transgene is not (Ye et al 2001). Similarly transplastomic tobacco plants expressing bar genes exhibited field-level tolerance to phosphinothricin (PPT), that was conferred by even the lowest levels of nuclear bar expression (Iamtham and Day, 2000; Ye et al., 2001 et al Lutz, Knapp, and Maliga, 2001)., Moreover, the bacterial bar gene, conferring phosphinothricin (glufosinate) resistance [Iamtham and Day 2000 and Lutz et al 2001] led to high-level enzyme accumulation of phosphinothricin acetyltransferase (PAT) up to ~7% of TSP [Lutz et al 2001]â€¦â€¦â€¦.plant... However, the bar gene was not found to be suitable due to early lethality of herbicide [Ye et al 2003]. On the contarary plastome engineering of 4-hydroxyphenylpyruvate dioxygenase (hppd) gene from Pseudomonas fluorescens, which is part of the biosynthetic pathway leading to plastoquinone and vitamin E for herbicide tolerance in a major agronomic crops, tobacco and soybean( Dufourmantel et al 2007) resulted in accumulation of HPPD to approximately 5% of total soluble protein in transgenic chloroplasts of both specieswith strong herbicide tolerance, performing better than nuclear transformants. In addition, the over-expression of HPPD has no significant impact on the vitamin E content of leaves or seeds (what about tubers??), quantitatively or qualitatively.