No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Stable chloroplast transformation in potato: Use of green fluorescent protein as a plastid marker
Abstract
We describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-specific plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/ Trps16::PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regeneration system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastidexpressed green fluorescent protein was used as a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.
... Expression of visual marker gene gfp in calli and shoots were visualized by inverted microscopy and the observed expression level of potato cultivars did not show any statistically significant differences and these differences were probably because of heteroplastomic status of plastome. In comparison, Sidorov et al. (1999) documented transplastomic event in potato with a single step in-vitro culture on which they reported 6.67% (1 transformant out of 15 shoots) and 2.85% (1from 35 shots) regeneration performance. These findings are much lesser than the regeneration proficiencies of four potato cultivars used in our study. ...
... However, Nguyen et al. (2005) reported PCR based evidence of integration of aadA and gfp in transplastomic potato. Adopting the same genotype and underlying regeneration protocol of Nguyen et al. (2005), Valkov et al. (2011) roughly doubled (11 shoot per 100 bombardments) the efficiency of transformation compared to previous results documented by Sidorov et al. (1999) and Nguyen et al. (2005). This finding obtained by Valkov et al. (2011) is similar to our current findings regarding the cultivars Challenger and Sante. ...
... The presence of homologous flanking sequences in a transformation event can instigate the improved frequency of transformation ) that was proven by Valkov et al. (2014) on potatoes (one transplastomic shoot per bombardment). Improved transplastomic events were recorded by several authors in tobacco (Svab and Maliga 1993;Langbecker et al. 2004) and both of these findings were similar to our current procedure and achievement, although lower efficacy of transformation in Arabidopsis, potato (Solanum tuberosum) and tomato (Lycopersicon esculentum) also reported (Sidorov et al. 1999;Ruf et al. 2001). ...
... Expression of visual marker gene gfp in calli and shoots were visualized by inverted microscopy and the observed expression level of potato cultivars did not show any statistically significant differences and these differences were probably because of heteroplastomic status of plastome. In comparison, Sidorov et al. (1999) documented transplastomic event in potato with a single step in-vitro culture on which they reported 6.67% (1 transformant out of 15 shoots) and 2.85% (1from 35 shots) regeneration performance. These findings are much lesser than the regeneration proficiencies of four potato cultivars used in our study. ...
... However, Nguyen et al. (2005) reported PCR based evidence of integration of aadA and gfp in transplastomic potato. Adopting the same genotype and underlying regeneration protocol of Nguyen et al. (2005 roughly doubled (11 shoot per 100 bombardments) the efficiency of transformation compared to previous results documented by Sidorov et al. (1999) and Nguyen et al. (2005). This finding obtained by Valkov et al. (2011) is similar to our current findings regarding the cultivars Challenger and Sante. ...
... The presence of homologous flanking sequences in a transformation event can instigate the improved frequency of transformation ) that was proven by Valkov et al. (2014) on potatoes (one transplastomic shoot per bombardment). Improved transplastomic events were recorded by several authors in tobacco (Svab and Maliga 1993;Langbecker et al. 2004) and both of these findings were similar to our current procedure and achievement, although lower efficacy of transformation in Arabidopsis, potato (Solanum tuberosum) and tomato (Lycopersicon esculentum) also reported (Sidorov et al. 1999;Ruf et al. 2001). ...
The potato chloroplast was transformed with codon optimized synthetic hybrid cry gene (SN19) to mitigate crop losses by Colorado potato beetle (CPB). The bombarded explants (leaves and internode) were cultured on MS medium supplemented with BAP (2.0 mg/l), NAA (0.2 mg/l), TDZ (2.0 mg/l) and GA3 (0.1 mg/l); spectinomycin 50 mg/l was used as a selection agent in the medium. Leaf explants of cultivar Kuroda induced highest percentage (92%) of callus where cultivar Santae produced the highest percentage (85.7%) of transplastomic shoots. Sante and Challenger showed 9.6% shoot regeneration efficiency followed by cultivar Simply Red (8.8%). PCR amplification yielded 16 postive transplastomic plantlets out of 21 spectinomycin resistant ones. Target gene integration was confirmed by PCR and Southern blot, whereas RT-qPCR was used to assess the expression level of transgene. The localization of visual marker gene gfp was tracked by laser scanning confocal microscopy which confirmed its expression in chloroplasts of leaf cells. The transplastomic plants ensured high mortality to both larvae and adult CPB. Foliage consumption and weight gain of CPB fed on transplastomic leaves were lower compared to the control plants. Sucessful implementation of current research findings can lead to a viable solution to CPB mediated potato losses globally.
... In contrast to the random nature of nuclear transformation, the gene of interest is integrated into the plastome through homologous recombination, which avoids negative effects associated with transgene insertion in transcriptionally silent regions or with the disruption of host genes or regulatory regions (Gelvin, 2017). Moreover, mechanisms of gene silencing are not present in plastids, so expression of the transgene is stable in progeny of transplastomic plants (Sidorov et al., 1999;Thanh et al., 2005). In addition, other advantages include high expression levels of transgenes and protein accumulation, the opportunity of expressing several genes in operons and the inherent confinement of transgenes and recombinant products in plastids (Valkov et al., , 2014Segretin et al., 2012). ...
... The availability of the complete chloroplast genome sequence of S. tuberosum in 2005, allowed the construction of species-specific vectors by increasing homology and the improvement of potato plastid transformation efficiency (Scotti et al., 2011;Valkov et al., 2011). Even though it has attractive advantages and potential applications in potato biotechnology, low transformation frequencies and the reduced levels of transgene expression registered in tubers limit a wider use of potato plastid transformation (Sidorov et al., 1999;Thanh et al., 2005;Segretin et al., 2012). ...
... These systems offer alternatives to the conventional ones, although their use requires further optimization. Visual markers genes such as glucuronidase, luciferase and green fluorescent protein have also been adopted, although to a lesser extent (Sidorov et al., 1999;Verhees et al., 2002;Rakosy-Tican et al., 2007). Alternatively, markerfree transformation of potato has also been reported. ...
Potato (Solanum tuberosum L.) is a crop of world importance that produces tubers of high nutritional quality. It is considered one of the promising crops to overcome the challenges of poverty and hunger worldwide. However, it is exposed to different biotic and abiotic stresses that can cause significant losses in production. Thus, potato is a candidate of special relevance for improvements through conventional breeding and biotechnology. Since conventional breeding is time-consuming and challenging, genetic engineering provides the opportunity to introduce/switch-off genes of interest without altering the allelic combination that characterize successful commercial cultivars or to induce targeted sequence modifications by New Breeding Techniques. There is a variety of methods for potato improvement via genetic transformation. Most of them incorporate genes of interest into the nuclear genome; nevertheless, the development of plastid transformation protocols broadened the available approaches for potato breeding. Although all methods have their advantages and disadvantages, Agrobacterium-mediated transformation is the most used approach. Alternative methods such as particle bombardment, protoplast transfection with polyethylene glycol and microinjection are also effective. Independently of the DNA delivery approach, critical steps for a successful transformation are a rapid and efficient regeneration protocol and a selection system. Several critical factors affect the transformation efficiency: vector type, insert size, Agrobacterium strain, explant type, composition of the subculture media, selective agent, among others. Moreover, transient or stable transformation, constitutive or inducible promoters, antibiotic/herbicide resistance or marker-free strategies can be considered. Although great efforts have been made to optimize all the parameters, potato transformation protocols are highly genotype-dependent. Genome editing technologies provide promising tools in genetic engineering allowing precise modification of targeted sequences. Interestingly, transient expression of genome editing components in potato protoplasts was reported to generate edited plants without the integration of any foreign DNA, which is a valuable aspect from both a scientific and a regulatory perspective. In this review, current challenges and opportunities concerning potato genetic engineering strategies developed to date are discussed. We describe their critical parameters and constrains, and the potential application of the available tools for functional analyses or biotechnological purposes. Public concerns and safety issues are also addressed.
... The aadA protein modifies the antibiotic by adenylation when the plastid genome is transformed with the aadA gene and prevents binding to chloroplast ribosomes that allow greening (Wirmer and Westhof, 2006;Wilson, 2014). To date, several studies have been conducted to produce stable transplastomic potato to achieve several desirable traits and different PGR combinations were applied for the in vitro regeneration (Sidorov et al., 1999;Nguyen et al., 2005;Valkov et al., 2011Valkov et al., , 2014Scotti et al., 2011Scotti et al., , 2012You et al., 2019). ...
... In comparison, Sidorov et al. (1999) documented potato plastid transformation with a single step procedure on which they used a noncommercial genotype (FL1607 breeding line) based on its responsiveness in in vitro. They reported 6.67% (1 transformant out of 15 shots) and 2.85% (1 from 35 shots) regeneration performance from two vectors (pZS197 vector and vector targeting the rps12-rrn16 intergenic region, respectively). ...
... However, Nguyen et al. (2005) reported integration of aadA and gfp in pMSK18 transplastomic potato plants by PCR as a proof of transplastomic event. Adopting the same genotype and underlying regeneration protocol previously published by Nguyen et al. ( 2005), Valkov et al. (2011) roughly doubled (11 shoot per 100 bombardments/one shoot every nine bombardments) the efficiency of transformation compared to previous results from Sidorov et al. (1999) andNguyen et al. (2005). This finding obtained by Valkov et al. (2011) is similar to our current findings regarding the cultivars Challenger and Sante. ...
In contrast with traditional nuclear gene transformation, transplastomic technology has opened a new horizon for the transgenic plant research that offers several beneficial aspects including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. However, this technology has been well adopted and established in tobacco, the introduction and adoption of cost-effective, swift, and reproducible protocol for in vitro regeneration of transplastomic potato is challenging and laborious. The present research aimed to develop such prompt and efficient protocol to instigate and revive the regeneration potential with the combinations of different plant growth regulators (PGRs). Leaves and internodal explants from four potato cultivars were transformed with chloroplast transformation vector via particle bombardment and cultured on MS media supplemented with suitable PGRs and selection agents. Leaf explants of cultivar Kuroda induced highest (92%) number of calli where cultivar Sante produced the highest (85.7%) transplastomic shoots. Thidiazuron was found more proficient (41%) for shoot regeneration. Finally, within only seven weeks, we got 21 spectinomycin resistant shoot, and 16 of those showed integration of target genes into the plastome in PCR screening.
... Non-green plastids are desirable for the expression of recombinant proteins in edible plant parts to enhance the nutritional value of tubers or fruits, or to deliver pharmaceuticals. However, plastid transgenes are expressed at extremely low levels in the amyloplasts of storage organs such as tubers [1][2][3] . Here, we report a regulatory system comprising a variant of the maize RNA-binding protein PPR10 and a cognate binding site upstream of a plastid transgene that encodes green fluorescent protein (GFP). ...
... Seed-specific expression of vaccines may also be desirable when the seed is used as fodder. Attempts to express transgenes in non-green potato plastids (amyloplasts) thus far resulted in very low levels of recombinant protein-0.02% of the total soluble protein (TSP) in tubers 2 and 0.05% in microtubers 3 . Here we report an expression system that stimulates protein expression in tuber amyloplasts with no substantial impact on the protein levels in leaves. ...
... This finding suggests that GFP accumulation is subject to the same limiting factor, possibly the level of gfp mRNA. The 1.3% TSP is substantially higher than protein levels obtained previously in potato amyloplasts: 0.02% TSP in tubers 2 and 0.05% TSP in microtubers 3 . An increase in GFP levels in the tuber was obtained without an obvious deleterious impact on vegetative growth. ...
Non-green plastids are desirable for the expression of recombinant proteins in edible plant parts to enhance the nutritional value of tubers or fruits, or to deliver pharmaceuticals. However, plastid transgenes are expressed at extremely low levels in the amyloplasts of storage organs such as tubers1–3. Here, we report a regulatory system comprising a variant of the maize RNA-binding protein PPR10 and a cognate binding site upstream of a plastid transgene that encodes green fluorescent protein (GFP). The binding site is not recognized by the resident potato PPR10 protein, restricting GFP protein accumulation to low levels in leaves. When the PPR10 variant is expressed from the tuber-specific patatin promoter, GFP accumulates up to 1.3% of the total soluble protein, a 60-fold increase compared with previous studies² (0.02%). This regulatory system enables an increase in transgene expression in non-photosynthetic plastids without interfering with chloroplast gene expression in leaves.
... 10 veces más presión de selección que en transformación nuclear. Sidorov et al. (1999) Hoja Biolístico. Partículas de oro de 0,6 µm. ...
... Estas dos secuencias homólogas suelen tener una longitud aproximada de 1 kb y generalmente se clonan de la propia especie a transformar, aunque se han propuesto vectores de transformación universales que insertan el transgén en zonas del plastoma altamente conservadas entre especies (Verma y Daniell, 2007). Esta estrategia se ha empleado en transformación plastidial de patata (Sidorov et al., 1999;Nguyen et al., 2005), tabaco (DeGray et al., 2001), Lesquerella fendleri (Skarjinskaia et al., 2003), petunia (Zubko et al., 2004) y coliflor (Nugent et al., 2006) donde obtuvieron bajas frecuencias de transformación de las cuales acaban culpando a la similitud incompleta de las zonas de recombinación. Además, la existencia de patrones distintos de edición del ARN (Corneille et al., 2000;Ruf et al., 2001) o presencia o ausencia de determinados intrones (Lelivelt et al., 2005) hace que la práctica habitual sea la del clonado de las zonas de recombinación propias de la especie a transformar. ...
... Sin embargo realizando algo similar en SSC (ndhF-rpl32/trnL) y LSC (accD/ORF184) consiguieron en ambas similares niveles de eficiencia de transformación y de expresión del transgén, si bien a nivel transcripcional se han detectado diferencias debido a la transcripción de operones (Zhang et al., 2001a). En patata por ejemplo, en dos trabajos de transformación independientes, obtienen mejores resultados con vectores que transforman en LSC que en IR (Sidorov et al., 1999;Nguyen et al., 2005). ...
Maize is one of the most important crops for the nutritional intake of humans, being such via direct utilization as food or through livestock breeding. The constant growth of human population, the increasing meat consumption in emerging economy countries, the use of grain for biofuel production or the climate change are demanding an increase in the productivity of this crop. Plant biotechnology offers diverse procedures to achieve this objective, being plastid transformation one of them. This technique brings important advantages to plant transformation such as high expression level of the gene of interest or prevent the transgene transmission through pollen to other crops due to the maternal inheritance of plastids. Achieving plastid transformation of maize would allow the transfer of those advantages to this important crop.
We have developed diverse in vitro culture procedures and worked with different maize genotypes to select the best candidates to be plastidially transformed. The best protocol tested has been the regeneration through somatic embryogenesis induced from immature embryos. We also obtained a green calli culture procedure using the peptide hormone -PSK, designed a second round of regeneration on selective media using mature somatic embryos and a protocol for culturing callus cell aggregates on liquid media as an alternative to cell suspensions.
The three selection schemes tested were antibiotic based: the gene aadA gene and streptomycin, the nptII gene and kanamycin and the Aph(4) gene and hygromycin selection. Those genes were cloned in four different plastid transformation vectors which introduced the transgenes into the following regions of the inverted repeat regions of the plastome: 16SRNAr-trnV-ORF85/ORF58 or 16SRNAr-trnI/trnA-23SRNAr. A total of 54 transformation experiments were made with 12 different types of materials and 1738 maize regenerants were obtained, 7 of which were transplastomic regenerants with a high level of heteroplasmy. Six of them were obtained in streptomycin selection and 1 with hygromycin.
It was also designed an alternative selection scheme which was based on the complementation of 3 nuclear photosynthetic albino mutants: csr1-1, crs1-2 y crs2-2. We obtained embryogenic calli from those mutants cultured in light or dark conditions from immature embryos. The mutated gene was introduced in a plastid transformation vector which introduces the gene into the 16SRNAr-trnI/trnA-23SRNAr recombination zone. No regenerants were obtained from 6 transformation experiments.
... Besides precise transgene integration by homologous recombination, plastid transformation allows for high level of transgene expression and protein accumulation, coexpression of several transgenes in operons, and transgene containment, due to maternal inheritance of cytoplasmic organelles in most crop species [4][5][6]. To date, transplastomic plants have been obtained with several crops, such as tomato, soybean, cotton, lettuce, cabbage, eggplant, sugar beet, rice, and potato [7][8][9][10][11][12][13][14][15]. The availability of the complete chloroplast genome sequences of Solanum tuberosum [16][17][18] facilitated the design of species-specific vectors, allowing for the improvement of plastid transformation in potato through a more efficient homologous recombination. ...
... In our hands, the number of transplastomic shoots increased to about one per bombardment adopting an improved regeneration protocol and transformation vectors containing potato flanking regions [19]. One transplastomic shoot every 15-35 biolistic shots had been previously reported in potato [15,20]. The use of plastid species-specific flanking sequences determined the increase of transformation frequency also in cotton, soybean, and lettuce [8][9][10]. ...
For a long time, plastid transformation has been a routine technology only in tobacco due to lack of effective selection and regeneration protocols, and, for some species, due to inefficient recombination using heterologous flanking regions in transformation vectors. Nevertheless, the availability of this technology to economically important crops offers new possibilities in plant breeding to manage pathogen resistance or improve nutritional value. Herein we describe an efficient plastid transformation protocol for potato (Solanum tuberosum subsp. tuberosum), achieved by the optimization of the tissue culture procedures and using transformation vectors carrying homologous potato flanking sequences. This protocol allowed to obtain up to one shoot per shot, an efficiency comparable to that usually accomplished in tobacco. Further, the method described in this chapter has been successfully used to regenerate potato transplastomic plants expressing recombinant GFP protein in chloroplasts and amyloplasts or long double-stranded RNAs for insect pest control.
... The first ever high-frequency plastid transformation was successfully established in Nicotiana tabacum (Svab et al. 1990) and many other reports revealed its suitability as model experimental plant system for chloroplast transformation (Gottschamel et al. 2013). Apart from tobacco, chloroplast transformation was also reported in other plants such as potato (Sidorov et al. 1999), rapeseed (Cheng et al. 2010), tomato (Ruf et al. 2001), cabbage (Liu et al. 2007), lettuce (Lelivelt et al. 2005), rice (Lee et al. 2006), Cedrela odorata (López-Ochoa et al. 2015), scoparia , and bitter melon (Narra et al. 2018). However, the development of chloroplast transformation has been extended to many other plant systems, until today, this technology remains challenging and limited to achieve due to Srinivas Kota and Raghuvardhan Lakkam contributed equally. ...
... Every time, species-specific plastid transformation vectors are not necessary to achieve plastid transformation and this could be done by use of heterologous vectors up to a possible extent in attaining plastid transformants successfully. On this point of view, successful plastid transformation in potato, tomato, and scoparia has been achieved with tobacco plastid genome vectors Ruf et al. 2001;Sidorov et al. 1999). However, there could be a chance for a drastic decrease in transformation efficiency with heterologous vectors (Lutz et al. 2007;Skarjinskaia et al. 2003). ...
In the present study, we focused on designing a species-specific chloroplast vector for Capsicum annuum L. and finding out its transformation efficiency compared to a heterologous vector. The plastid transformation vector (CaIA) was designed to target homologous regions trnA and trnI of IR region. A selectable marker gene aadA, whose expression is controlled by psbA promoter and terminator, was cloned between two flanking regions. A heterologous vector pRB95, which targets trnfM and trnG of LSC region along with aadA driven by rrn promoter and psbA terminator, was also used for developing plastid transformation in Capsicum. Cotyledonary explants were bombarded with stabilized biolistic parameters: 900 psi pressure and 9 cm flight distance, and optimized regeneration protocol (0.7 mg/L TDZ + 0.2 mg/L IAA) was used to obtain transplastomic lines on selection medium (300 mg/L spectinomycin). The aadA integration and homoplasmy were confirmed by obtaining 1.2 and 3.7 kb amplicons in CaIA transformants and subsequently verified by Southern blotting, whereas in pRB95 transformants, integration was confirmed by PCR with 1.45 kb and 255 bp amplicons corresponding to aadA integration and flanks, respectively. The transformation efficiencies attained with two plastid vectors were found to be 20%, i.e., 10 transplastomic lines in 50 bombarded plates, with CaIA and 2%, i.e., 1 transplastomic line in 50 bombarded plates, with heterologous pRB95, respectively.
... Compared with conventional transgenic technologies, plastid engineering offers several potential advantages that have been extensively reviewed [1][2][3]. Plastid transformation has mostly been confined to Nicotiana tabacum [3] and it has taken almost ten years to extend the technology to other plant species, including Solanum tuberosum [4] and Lycopersicon esculentum [5]. Plastid transformation has also been reported for other plants including Brassica napus [6], Lesquerella fendleri [7] and Oryza sativa [8]although problems with heteroplasmy and plant fertility have yet to be resolved. ...
... Two hundred and thirty-seven independent kanamycin-resistant transplastomic clones from 45 bombardments (between 1 and 19 per bombardment) were recovered and 81 out of 113 tested clones showed INT-mediated attP vector insertion into the attB site. Therefore, transformation efficiencies (up to 17 per bombardment) are roughly fourfold higher than reported previously [4,8,9] (up to five per bombardment using homologous recombination and a non-lethal selection system). Nevertheless, the homologous recombination approach eliminates the introduction of unwanted plasmid sequences, which are a potential concern with INT-mediated plastid transformation. ...
Plastid transformation offers the unique advantages of high-level transgene expression and increased trans-gene containment compared with conventional trans-genic technologies. The process relies on the homologous recombination machinery of the plastid incorporating foreign DNA into the plastome, which restricts the method to species where this type of incorporation works well. However, Pal Maliga and colleagues have recently reported a novel approach for integrating foreign DNA into the plastid genome that works independently of homologous recombination. The transgenic plastid system Plastid transformation is accomplished using a multistep process in which the transformation vectors contain a selectable marker gene and passenger gene(s) flanked by plastid targeting sequences [1]. Targeting sequences enable foreign genes to be incorporated into the predetermined location through two crossover events within the homologous flanking regions. Insertion of transgenes in intergenic regions without interference with flanking genes has been successful at 14 sites throughout the plastome [2], either by biolistic DNA delivery or by polyethylene glycol treatment. Nevertheless, interference was found at one site and knockouts have been reported for 28 plastid genes. Plastid genetic engineering is emerging as an alternative new technology in plant biotechnology. Compared with conventional transgenic technologies, plastid engineering offers several potential advantages that have been extensively reviewed [1-3]. Plastid transformation has mostly been confined to Nicotiana tabacum [3] and it has taken almost ten years to extend the technology to other plant species, including Solanum tuberosum [4] and Lycopersicon esculentum [5]. Plastid transformation has also been reported for other plants including Brassica napus [6], Lesquerella fendleri [7] and Oryza sativa [8]-although problems with heteroplasmy and plant fertility have yet to be resolved. To address these problems research was focused on the use of selectable markers, the manipulation of gene expression-control elements and the development of efficient tissue-culture systems [9]. Nevertheless, the inefficient incorporation of foreign DNA into plastomes, such as occurs in Arabidopsis thaliana [10], remains to be overcome. However, incorporating transgenes into the plastome is only the first step in obtaining a genetically stable transgenic plant because several cell generations are necessary to dilute out all wild-type plastome copies [8]. Pal Maliga and colleagues recently outlined an alternative approach to incorporate foreign DNA into the plastome efficiently. The technique uses the phiC31 Streptomyces phage site-specific inte-grase (INT) to catalyze recombination to yield stable transplastomic plants [11]. Integrase-mediated recombination system Site-specific recombination systems are common in pro-karyotes and lower eukaryotes. They participate in integrating bacteriophages into host genomes. In these systems, recombinations are catalyzed by phage-encoded recombinases. These recombinases can be grouped into two types based on limited amino acid sequence homology and catalytic residues (either serine or tyrosine). The serine recombinases are referred to as the invertase or resolvase and posses a catalytic serine residue, whereas the tyrosine group members are referred to as l integrases and use a tyrosine residue during DNA cleavage. The phiC31 Streptomyces phage-encoded integrase belongs to the serine family of recombinases. Like other family members, the phiC31 phage integrase brings two recom-bination sites (the bacterial attB and phage attachment attP sites) together in a synapse and then catalyzes a concerted four-strand staggered break in the DNA sub-strates while forming transient covalent attachments with the recessed 5 0 ends [12]. One pair of half sites rotates by 1808 relative to the other to form the recombinant configuration, followed by ligation of the DNA backbone. Thus, the integration reaction generates recombinant junctions attL and attR as products. During integration, the attB and attP sites share only 3 bp sequence identity at the crossover point and 16 bp within a span of w50 bp. The crossover point for both the attachment sites is flanked by different inverted repeats (in the region of 34 bp of attB and 39 bp of attP sites) that perhaps provide binding sites for the integrase [12]. It has been observed that some integrases function autonomously whereas others catalyze the reaction with the help of DNA-binding proteins, termed accessory factors (recombination directionality factors, RDFs [13]), to influence the choice of substrates that are recombined by their cognate recombinase. The best-studied RDF is Xis, encoded by phage l, which is required in the excision reaction between attL and attR junctions to generate attP and attB as products but inhibits integrative Corresponding author: Khan, M.S. (sarwarkhan_40@hotmail.com).
... If applicable, please provide one. Sidorov et al. (1999), Skarjinskaia et al. (2003), Yabuta et al. (2013).' are given in list but not cited in text. Please cite in text or delete them from list. ...
A protocol has been established for genetic transformation of the chloroplasts in two new cultivars of tomato (Solanum lycopersicum) grown in India and Australia: Pusa Ruby and Yellow Currant. Tomato cv. Green Pineapple was also used as a control that has previously been used for establishing chloroplast transformation by other researchers. Selected tomato cultivars were finalized from ten other tested cultivars (Green Pineapple excluded) due to their high regeneration potential and better response to chloroplast transformation experiment. This protocol was set up using a chloroplast transformation vector (pRB94) for tomatoes that is made up of a synthetic gene operon. The vector has a chimeric aadA selectable marker gene that is controlled by the rRNA operon promoter (Prrn). This makes the plant or chloroplasts resistant to spectinomycin and streptomycin. After plasmid-coated particle bombardment, leaf explants were cultured in 50 mg/L selection media. Positive explant selection from among all the dead-appearing (yellow to brown) explants was found to be the major hurdle in the study. Even though this study was able to find plastid transformants in heteroplasmic conditions, it also found important parameters and changes that could speed up the process of chloroplast transformation in tomatoes, resulting in homoplasmic plastid-transformed plants.
... The first successful plastid transformation was performed in the green unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) (Boynton et al. 1988). This technique was subsequently expanded to model plants including tobacco (Nicotiana tabacum) (Svab et al. 1990) and Arabidopsis (Arabidopsis thaliana) (Ruf et al. 2019;Sikdar et al. 1998;Yu et al. 2017), as well as crop plants including potato (Solanum tuberosum) (Sidorov et al. 1999) and tomato (S. lycopersicum) (Ruf et al. 2001). ...
The plastid is a promising target for the production of valuable biomolecules via genetic engineering. We recently developed a plastid-specific gene delivery system for leaves or seedlings using KH-AtOEP34, a functional peptide composed of the polycationic DNA-binding peptide KH and the Arabidopsis thaliana plastid-targeting peptide OEP34. Here, we established a liquid culture system for inducing multiple shoots in the model plants A. thaliana and Nicotiana benthamiana and the crop plant strawberry (Fragaria×ananassa) and tested the use of these plant materials for peptide-mediated gene delivery to plastids. Our liquid culture system efficiently induced multiple shoots that were enriched in meristems. Using these meristems, we performed KH-AtOEP34-mediated gene delivery to plastids and tested the delivery and integration of a cassette composed of the spectinomycin resistance gene aadA, the GFP reporter gene, and sequences homologous to plastid DNA. Genotyping PCR revealed the integration of the cassette DNA into plastid DNA several days after delivery in all three plants. Confocal laser scanning microscopy and immunoblotting confirmed the presence of plasmid-derived GFP in the plastids of meristems, indicating that the plasmid DNA was successfully integrated into plastid DNA and that the cassette was expressed. These results suggest the meristems developed in our liquid culture system are applicable to peptide-mediated delivery of exogeneous DNA into plastids. The multiple shoots generated in our liquid novel culture system represent promising materials for in planta peptide-mediated plastid transformation in combination with spectinomycin selection.
Fullsize Image
... Most of the activity on transformation of plant chloroplast DNA was done using the bombing method. In this way, transplastomic potatoes plants (Sidorov et al., 1999), tomatoes plants (Ruf et al., 2001), transplastomic Arabidopsis (Sikdar et al., 1998), and petunias (Zubko et al., 2004) were obtained. ...
The article is dealed with problems and prospects of experiments with biological objects directed on change of a natural hereditary basis, which include risks of creation of new unnatural living organisms that can represent biological danger to the humans and environment. We noted that the possibility of uncontrolled creation of highly pathogenic strains of microorganisms, which do not exist in nature, for humans or animals, poses a great threat. We considered that such new pathogenic strains should be dangerous to the environment and will inevitably endanger human and animal life on the planet. There are several quite convincing examples in the world illustrated the danger of invading the genetic program of a living organism, while ignoring the laws of evolution and genetics, focusing only on short-term financial benefits to individuals, companies or financial groups. We established that a key element in preventing the spread of infection from laboratories or other artificial places of consolidation is the strict implementation of standard microbiological requirements. The development of effective measures to prevent and eliminate the consequences of biological attacks, taking into account their various possible scenarios, should be based on biomedical, social, economic and legal criteria. We suggested we need to consider the cost-benefit analysis and other indices characterizing the state of ecology, economy, health, and social relations when implement the measures on biological safety. In Ukraine today there is a priority to develop a legislative position in the field of counteraction to bioterrorism. This issue concerns not only the development and adoption of special laws, but also the implementation of the policy of "efficiency" against biological threats by the relevant state authorities. This also includes the establishment of full state control over enterprises and institutions related to biological agents and toxins, and the improvement of the system of "state-scientist" to control the leakage of professional knowledge outside the country.
... Svab and Maliga (1993) successfully transformed tobacco cp (Chen et al., 2014;Yarbakht et al., 2015) using the aadA gene as a selective marker. To date, this transformation system has been applied in Arabidopsis thaliana (Sikdar et al., 1998), potato (Sidorov et al., 1999), soybean (Dufourmantel et al., 2004), rice (Lee et al., 2006), and other herbaceous plants (Liu et al., 2007;Cheng et al., 2010;Wei et al., 2011;Harada et al., 2014;Lelivelt et al., 2014;Muralikrishna et al., 2016). In the present study, the aadA gene was selected as an effective selective marker to ensure that the selected regulatory elements can perform their biological functions normally. ...
Salix floderusii is a rare alpine tree species in the Salix genus. Unfortunately, no extensive germplasm identification, molecular phylogeny, and chloroplast genomics of this plant have been conducted. We sequenced the chloroplast (cp) genome of S. floderusii for the first time using second-generation sequencing technology. The cp genome was 155,540 bp long, including a large single-copy region (LSC, 84,401 bp), a small single-copy region (SSC, 16,221 bp), and inverted repeat regions (IR, 54,918 bp). A total of 131 genes were identified, including 86 protein genes, 37 tRNA genes, and 8 rRNA genes. The S. floderusii cp genome contains 1 complement repeat, 24 forward repeats, 17 palindromic repeats, and 7 reverse repeats. Analysis of the IR borders showed that the IRa and IRb regions of S. floderusii and Salix caprea were shorter than those of Salix cinerea, which may affect plastome evolution. Furthermore, four highly variable regions were found, including the rpl22 coding region, psbM/trnD-GUC non-coding region, petA/psbJ non-coding region, and ycf1 coding region. These high variable regions can be used as candidate molecular markers and as a reference for identifying future Salix species. In addition, phylogenetic analysis indicated that the cp genome of S. floderusii is sister to Salix cupularis and belongs to the Subgenus Vetrix. Genes (Sf-trnI, Sf-PpsbA, aadA, Sf-TpsbA, Sf-trnA) obtained via cloning were inserted into the pBluescript II SK (+) to yield the cp expression vectors, which harbored the selectable marker gene aadA. The results of a spectinomycin resistance test indicated that the cp expression vector had been successfully constructed. Moreover, the aadA gene was efficiently expressed under the regulation of predicted regulatory elements. The present study provides a solid foundation for establishing subsequent S. floderusii cp transformation systems and developing strategies for the genetic improvement of S. floderusii.
... Chromoplasts in mature tomatoes are differentiated from chloroplasts, which are very active in gene expression. This makes plastid transgenic expression in tomato more successful than in other plants (Sidorov et al., 1999;Ruf et al., 2001;Wurbs et al., 2007). Non-green plastids are ideal hosts for recombinant protein expression; they are more suitable for the production and storage of PNPs than chloroplasts without affecting plant adaptability (Mellor et al., 2018). ...
Plant natural products (PNPs) are the main sources of drugs, food additives or new biofuels and have become the hotspot in synthetic biology. In the past two decades, the engineered biosynthesis of many PNPs have been achieved by constructing the microbial cell factories. Along with rapid development of plant physiology, genetics and plant genetic modification technique, the host has now expanded to complex plant system from initial single-celled microbes. Plant synthetic biology is an emerging field that combines engineering principles with plant biology. In this review, we introduced the recent advances of biosynthetic pathway elucidation of PNPs and summarized the progress of engineered PNPs biosynthesis in plant cells. Furthermore, a future vision of plant synthetic biology was prospected. Although there is still a long way to break all the bottlenecks in plant synthetic biology, the rising of this filed is expected to provide a huge opportunity for future agriculture and industry.
... Studies demonstrate a positive correlation between the rate of recombination and both the length and degree of sequence homology in prokaryotes [224,225]. Decreasing identity between transformation vectors and target plastome regions, that is, using N. tabacum flanking sequences to transform different species, yields much lower transformation efficiencies [226][227][228][229][230]. Both intra-and interfamilial variation in target sequences negatively impacted homologous recombination events and concomitantly transformation efficiency. ...
The plastid genome (plastome) has proved a valuable source of data for evaluating evolutionary relationships among angiosperms. Through basic and applied approaches, plastid transformation technology offers the potential to understand and improve plant productivity, providing food, fiber, energy, and medicines to meet the needs of a burgeoning global population. The growing genomic resources available to both phylogenetic and biotechnological investigations is allowing novel insights and expanding the scope of plastome research to encompass new species. In this chapter, we present an overview of some of the seminal and contemporary research that has contributed to our current understanding of plastome evolution and attempt to highlight the relationship between evolutionary mechanisms and the tools of plastid genetic engineering.
... Haselof et al., 1997;Rouwendal et al., 1997;Elliott et al., 1999;Molinier et al., 2000;Kaeppler et al., 2001), en la transferencia dirigida a los cloroplastos (e.g. Sidorov et al., 1999), así como para cosas tan diversas como la monitorización del movimiento de proteínas, estudios de compartimentación celular, análisis funcional de promotores y silenciamiento génico inducido por virus (Rizzuto et al., 1995;Köhler 1998;Ruiz et al., 1998). Se han empleado también como marcadores alternativos en experimentos de hibridación somática (Olivares-Fuster et al., 2002) e incluso para producir plantas ornamentales que emiten una fluorescencia brillante en las partes vegetativas o las flores (Mercuri et al., 2002). ...
Cucumber is one of the most important horticultural species at economic level. Despite its agronomic relevance, there is currently no efficient transformation method available. The problems to obtain transgenic cucumber plants are mainly due to the low morphogenetic response in explants of certain genotypes, poor adequacy of selection methods, decrease in regeneration rate as a consequence of the usual treatments in a transformation experiment and, above all, the high rate of 'escape' plants. Taking into account the existing problems, the first objective of the present work has been the evaluation of the morphogenetic response in cucumber explants and the development of regeneration methods potentially useful in transformation experiments. These studies have been carried out with two commercial cultivars, four pure lines and one line that has been frequently used in cucumber breeding programs. Different types of primary explants have been evaluated to determine which may be the most suitable in a transformation experiment. The influence of the ontogenic state of the seedling on the degree of myxoploidy in cotyledon explants has been studied. Likewise, the ploidy level of regenerated plants from these explants has been determined. Regarding the culture medium, the effect of growth regulators, as well as that of other components, such as silver nitrate or copper sulphate, has been studied. In the context of the second objective, studies on the different stages of the transformation process by co-cultivation of cucumber explants with Agrobacterium tumefaciens have been carried out. Several factors, such as the Agrobacterium strain, how to carry out the phases of inoculation and co-culture, the suitability of different marker genes in the selection process and the influence of different components of the culture medium have been analyzed. Attempts have also been made to infer the causes of the occurrence of 'escape' plants or the formation of chimeras in cucumber transformation experiments. Finally, based on the results obtained, some solutions that could help to avoid this problem in future experiments are proposed.
... However, plastid transformation was achieved in sugarcane, but low transformation efficiency and heteroplasmy remained to be resolved. The transformation efficiency resulting in one transformation event per 27 bombarded plates was found to be lower than reported in Arabidopsis [25,26], potato [27][28][29], and tomato [30,31]. However, transformation efficiency was comparable with rice [11] since target tissues in both cases were embryogenic calli. ...
We report here plastid transformation in sugarcane using biolistic transformation and embryogenesis-based regeneration approaches. Somatic embryos were developed from unfurled leaf sections, containing preprogrammed progenitor cells, to recover transformation events on antibiotic-containing regeneration medium. After developing a proficient regeneration system, the FLARES (fluorescent antibiotic resistance enzyme, spectinomycin and streptomycin) expression cassette that carries species-specific homologous sequence tails was used to transform plastids and track gene transmission and expression in sugarcane. Plants regenerated from streptomycin-resistant and genetically confirmed shoots were subjected to visual detection of the fluorescent enzyme using a fluorescent stereomicroscope, after genetic confirmation. The resultant heteroplasmic shoots remained to segregate on streptomycin-containing MS medium, referring to the unique pattern of division and sorting of cells in C4 monocotyledonous compared to C3 monocotyledonous and dicotyledonous plants since in sugarcane bundle sheath and mesophyll, cells are distinct and sort independently after division. Hence, the transformation of either mesophyll or bundle sheath cells will develop heteroplasmic transgenic plants, suggesting the transformation of both types of cells. Whilst developed transgenic sugarcane plants are heteroplasmic, and selection-based regeneration protocol envisaging the role of division and sorting of cells in the purification of transplastomic demands further improvement, the study has established many parameters that may open up exciting possibilities to express genes of agricultural or pharmaceutical importance in sugarcane.
... We anticipated that increasing the size of the target organelle may provide an advantage in transformation efficiency in potato as it has been previously demonstrated in tobacco 41 . Considering the transformation efficiencies of potato chloroplasts reported in the literature [45][46][47] , our macro-chloroplast lines can be efficiently transformed, yielding approximately 1 transplastomic plant per each plate bombarded (0.9 and 1.1 for pIR and pSSC construct, respectively, Table 1). However, the transformation efficiency of transgenic lines was not greater than the efficiency obtained in our laboratory for wild-type potato leaves. ...
Chloroplast biotechnology is a route for novel crop metabolic engineering. The potential bio-confinement of transgenes, the high protein expression and the possibility to organize genes into operons represent considerable advantages that make chloroplasts valuable targets in agricultural biotechnology. In the last 3 decades, chloroplast genomes from a few economically important crops have been successfully transformed. The main bottlenecks that prevent efficient transformation in a greater number of crops include the dearth of proven selectable marker gene-selection combinations and tissue culture methods for efficient regeneration of transplastomic plants. The prospects of increasing organelle size are attractive from several perspectives, including an increase in the surface area of potential targets. As a proof-of-concept, we generated Solanum tuberosum (potato) macro-chloroplast lines overexpressing the tubulin-like GTPase protein gene FtsZ1 from Arabidopsis thaliana. Macro-chloroplast lines exhibited delayed growth at anthesis; however, at the time of harvest there was no significant difference in height between macro-chloroplast and wild-type lines. Macro-chloroplasts were successfully transformed by biolistic DNA-delivery and efficiently regenerated into homoplasmic transplastomic lines. We also demonstrated that macro-chloroplasts accumulate the same amount of heterologous protein than wild-type organelles, confirming efficient usage in plastid engineering. Advantages and limitations of using enlarge compartments in chloroplast biotechnology are discussed.
... Boynton, Gillham, and coworkers (Boynton et al., 1988) were succeeded in plastid transformation in a unicellular alga, Chlamydomonas reinhardtii, in 1988 followed by transformation of the plastid genome in 1990 in tobacco (Svab et al, 1990) and further reached out to numerous other higher plants, such as Arabidopsis (Sikdar et al., 1998), potato (Sidorov et al., 1999), tomato (Ruf et al., 2001), rape (Hou et al., 2003), ...
For many reasons tobacco is extensively used as a model plant in transformation research and used for this study as well. Tobacco plants are cultivated in several countries on large scale. Tobacco has numerous essential traditional and modern uses in clinical field. The principle aim of this present study was to optimize the conditions for seed germination, best shooting regeneration media, optimization of antibiotics spectinomycin and kanamycin for wild type plants and in addition, to develop a protocol for efficient genetic transformation of tobacco plant cv. Petit Havana with Agrobacterium tumefaciens. Seeds of tobacco were germinated on different media without any plant growth regulators. 100% seeds germination efficiency was observed on ½ MS and best sterilization time of tobacco with ethanol was for 1 minute which also showed 100% germination efficiency. RMOP (NAA and BAP) media showed the highest shoots regeneration efficiency for both explants nodes and leaves which is nodes showed 75% and leaves showed 55% regeneration efficiency among the different media which is used for regeneration. 150 mg/L concentration of kanamycin and 500 mg/L concentration of spectinomycin for both explants (nodes and leaves) was optimized for the selection of transgenic tissues. For transformation infection time for 5 minutes and co-cultivation time period of two days showed maximum transformation efficiency of 83.33% as compared to other methods. Histochemical GUS (β-glucuronidase) assay was used to check the GUS gene expression in the nodal explants. GUS gene primers were used which amplified a 708bp fragment which also confirmed the transformation of GUS gene by Agrobacterium tumifaciens strain C58C1 containing vector p35SGUSINT. Taken together, the present study develops a tissue culture protocol and basis for Agrobacterium-mediated transformation of Nicotiana tabacum cv. Petit Havana using nodal explants.
Keywords: Tobacco, Spectinomycin, Kanamycin, Agrobacterium tumefaciens, GUS, Infection time, Co-cultivation time.
... Chlamydomonas reinhardtii, where transforming DNA is incorporated into the chloroplast genome by homologous recombination. Since then systems involving biolistics, polyethylene glycol-induced transformation of protoplasts, or microinjection, have been developed for several agronomically important higher plants (Daniell, 1999;Dix, 1999;Khan and Maliga, 1999;Knoblauch et aL, 1999;Sidorov et aL, 1999;Heifetz, 2000). ...
Plasmodium species (the causative agents of malaria), and related parasites of the phylum Apicomplexa such as Toxoplasma gondii, have a plastid organelle of endosymbiotic origin. This organelle is regarded as a potential new drug target. It contains its own genome encoding many of the proteins and RNAs required for protein synthesis, but its function is unclear. Nucleus-encoded proteins are likely to control the primary function and biogenesis of the plastid and I have studied two of these: FtsZ (involved in bacterial and plastid division), and Ycfl6 (a protein of unknown function that may interact with Ycf24, encoded on the plastid genome). First I set up a screen to check that any genes I identified were malarial in origin, and not from the mycoplasma that contaminate our cultures. Searches for ftsZ by degenerate PCR, database analysis and low-stringency hybridisation, were unsuccessful. The possibility remains that the apicomplexan plastid divides by some other mechanism, as mitochondria and some bacteria do, for example using a dynamin-related protein. A dynamin-like sequence was identified on chromosome 14 in P. falciparum, and its transcription at the appropriate stage of the erythrocytic cycle was confirmed. However, this protein could be involved in mitochondrial division. AycfI6-like sequence was found on chromosome 14 of P. falciparum and expressed in Escherichia coli for antibody production. This sequence encodes a putative plastid-targeting amino-terminal peptide; reporter protein and immunofluorescence studies will confirm whether it is localised in the plastid. To study the function of ycfl6,1 disrupted an orthologous version in the cyanobacterium Synechocystis and found it is an essential gene whose partial loss was deleterious. The δycfl6 mutant cyanobacteria were arrested in cytokinesis and resembled those found previously in our laboratory for δycf24 mutants, but the effects were less severe. I confirmed that ycf24 and ycfl6 are co-transcribed in Synechocystis, and perhaps form part of the same operon. These findings add weight to the hypothesis that Ycfl6 and Ycf24 act in the same pathway, and that the imported Ycfl6 and the endogenous plastid protein Ycf24 are both essential for plastid maintenance. Based on recent additions to the literature, I propose that Ycfl6 is involved in maturation of Fe-S cluster-containing proteins required for resistance to oxidative stress and the provision of reducing power for biosynthetic pathways that occur in the plastid of apicomplexans.
... Boynton, Gillham, and colleagues first achieved plastid transformation of Chlamydomonas with this plastid promoter in 1988 [40]; Svab, Hajukiewicz and Maliga followed, in 1990, with the first report of plastid transformation in tobacco, although they used a different fragment of chloroplast DNA in their target plasmid [50]. Plastid transformation has since been reported in many flowering plants, such as tobacco [50,51,52], Arabidopsis [53,54], potato [55], rice [56], rapeseed [57], and tomato [58]. A protocol for plastid transformation of an elite rapeseed cultivar (B. ...
In recent years, plant genetic engineering has advanced agriculture in terms of crop improvement, stress and disease resistance, and pharmaceutical biosynthesis. Cells from land plants and algae contain three organelles that harbor DNA: the nucleus, plastid, and mitochondria. Although the most common approach for many plant species is the introduction of foreign DNA into the nucleus (nuclear transformation) via Agrobacterium- or biolistics-mediated delivery of transgenes, plastid transformation offers an alternative means for plant transformation. Since there are many copies of the chloroplast genome in each cell, higher levels of protein accumulation can often be achieved from transgenes inserted in the chloroplast genome compared to the nuclear genome. Chloroplasts are therefore becoming attractive hosts for the introduction of new agronomic traits, as well as for the biosynthesis of high-value pharmaceuticals, biomaterials and industrial enzymes. This review provides a comprehensive historical and biological perspective on plastid transformation, with a focus on current and emerging approaches such as the use of single-walled carbon nanotubes (SWNTs) as DNA delivery vehicles, overexpressing morphogenic regulators to enhance regeneration ability, applying genome editing techniques to accelerate double-stranded break formation, and reconsidering protoplasts as a viable material for plastid genome engineering, even in transformation-recalcitrant species.
... However, nano-sized delivery vehicles, due to their small size and tunable chemical properties, can enable the translocation of biological barriers that are otherwise not possible by micron-sized vehicles (Fig. 1b) [52]. This enables applications of such nanocarriers for plant chloroplast transformation, which is challenging to achieve with micron-sized vehicles due to the similarly sized chloroplast organelle that limits the number of micron-sized vehicles that can be taken up into the organelle without damage or organelle rupture [53]. Notably, pH-responsive nanomaterials have been used for selective delivery of genetic material to chloroplasts [54]. ...
Biolistic delivery of biomolecular cargoes to plants with micron-scale projectiles is a well-established technique in plant biotechnology. However, the relatively large micron-scale biolistic projectiles can result in tissue damage, low regeneration efficiency, and create difficulties for the biolistic transformation of isomorphic small cells or subcellular target organelles (i.e., mitochondria and plastids). As an alternative to micron-sized carriers, nanomaterials provide a promising approach for biomolecule delivery to plants. While most studies exploring nanoscale biolistic carriers have been carried out in animal cells and tissues, which lack a cell wall, we can nonetheless extrapolate their utility for nanobiolistic delivery of biomolecules in plant targets. Specifically, nanobiolistics has shown promising results for use in animal systems, in which nanoscale projectiles yield lower levels of cell and tissue damage while maintaining similar transformation efficiencies as their micron-scale counterparts. In this chapter, we specifically discuss biolistic delivery of nanoparticles for plant genetic transformation purposes and identify the figures of merit requiring optimization for broad-scale implementation of nanobiolistics in plant genetic transformations.
... of foreign genes in to the chloroplast, similar kind leaf used as experimental tissue for the chloroplast transformation in plants potato (Sidorov et al. 1999b), cotton (Kumar et al. 2004a), tobacco (Svab and Maliga 1993) and tomato (Ruf et al. 2001). ...
The high expression level of industrial and metabolically important proteins in plants can be achieved by plastid transformation. The CaIA vector, a Capsicum-specific vector harboring aadA (spectinomycin resistance), is a selectable marker controlled by the PsbA promoter, and the terminator is flanked by the trnA and trnI regions of the inverted repeat (IR) region of the plastid. The CaIA vector can introduce foreign genes into the IR region of the plastid genome. The biolistic method was used for chloroplast transformation in Scoparia dulcis with leaf explants followed by antibiotic selection on regeneration medium. Transplastomes were successfully screened, and the transformation efficiency of 3 transgenic lines from 25 bombarded leaf explants was determined. Transplastomic lines were evaluated by PCR and Southern blotting for the confirmation of aadA insertion and its integration into the chloroplast genome. Seeds collected from transplastomes were analyzed on spectinomycin medium with wild types to determine genetic stability. The increased chloroplast transformation efficiency (3 transplastomic lines from 25 bombarded explants) would be useful for expressing therapeutically and industrially important genes in Scoparia dulcis L.
Keywords : Scoparia dulcis L., Heterologous vectors, CaIA plastid vector, Inverted repeat region, Transplastomic lines
... It has been established in several other species, including Haematococcus pluvialis [10], Dunaliella tertiolecta [11], Cyanidioschyzon merolae [12] and Phaeodactylum tricornutum [13]. Although chloroplast transformation has been reported in several higher plants species [14][15][16][17][18], the transformation of algal chloroplast offers certain advantages over higher plants. For example, the higher plants contain several thousand copies of the chloroplast genome in each cell, the transformation of each of those is quite challenging [19]. ...
In recent years, microalgae have emerged as an alternative platform for large-scale production of recombinant proteins for different commercial applications. As a production platform, it has several advantages, including rapid growth, easily scale up and ability to grow with or without the external carbon source. Genetic transformation of several species has been established. Of these, Chlamydomonas reinhardtii has become significantly attractive for its potential to express foreign proteins inexpensively. All its three genomes – nuclear, mitochondrial and chloroplastic – have been sequenced. As a result, a wealth of information about its genetic machinery, protein expression mechanism (transcription, translation and post-translational modifications) is available. Over the years, various molecular tools have been developed for the manipulation of all these genomes. Various studies show that the transformation of the chloroplast genome has several advantages over nuclear transformation from the biopharming point of view. According to a recent survey, over 100 recombinant proteins have been expressed in algal chloroplasts. However, the expression levels achieved in the algal chloroplast genome are generally lower compared to the chloroplasts of higher plants. Work is therefore needed to make the algal chloroplast transformation commercially competitive. In this review, we discuss some examples from the algal research, which could play their role in making algal chloroplast commercially successful.
... Although chloroplast transformation has been reported in several higher plant species too [25][26][27][28][29][30], the transformation of algal chloroplast offers certain advantages over higher plants. For example, higher plants contain several thousand copies of the chloroplast genome in each cell, transformation of each of those is quite challenging [31]. ...
The last few years have seen an ever‐increasing interest in the exploitation of microalgae as alternative platform to produce high‐value products, including biofuels, industrial enzymes, therapeutic proteins, including antibodies, hormones, and vaccines. Due to some unique attractive features, engineering of the chloroplast genome provides a promising platform for the production of high value targets because it allows manipulation of metabolic processes in ways that would be impossible, or at least prohibitively difficult through traditional approaches. Since its initial demonstration in 1988 in Chlamydomonas reinhardtii, genetic tools have been developed which have made it possible to produce high‐value molecules in different species. However, the commercial application of microalgae as production platform is hindered by many factors like poor biomass, low product yields and costly downstream processing methodologies. In this review, we discuss the potential of microalgae to use as an alternative production platform for high value targets using chloroplast transformation technology. This article is protected by copyright. All rights reserved
... To date, chloroplast transformation has been achieved in some plant species including tobacco, Arabidopsis, solanaceous species, lettuce, and poplar. [96][97][98][99][100][101] However, extending the species range of the chloroplast transformation technology has proven extremely difficult, in particular for major agricultural crops. [101,102] Mitochondrial transformation has been realized only in the budding yeast Saccharomyces cerevisiae and Chlamydomonas reinhardtii and is very difficult in most organisms (including plants) since there is lack of viable transformation approaches. ...
The pentatricopeptide repeat (PPR) protein family, with more than 400 members, is one of the largest and most diverse protein families in land plants. A small subset of PPR proteins contain a C‐terminal small MutS‐related (SMR) domain. Although there are relatively few PPR‐SMR proteins, they play essential roles in embryo development, chloroplast biogenesis and gene expression, and plastid‐to‐nucleus retrograde signaling. Here, recent advances in understanding the roles of PPR‐SMR proteins and the SMR domain based on a combination of genetic, biochemical, and physiological analyses are described. In addition, the potential of the PPR‐SMR protein SOT1 to serve as a tool for RNA manipulation is highlighted. Pentatricopeptide repeat‐small MutS‐related (PPR‐SMR) proteins play essential roles in embryo development, chloroplast biogenesis and gene expression, and retrograde signaling. The PPR motifs and SMR domain of the PPR‐SMR protein SOT1 confer RNA sequence specificity and endonucleolytic activity, respectively. SOT1 could be used as a tool for RNA manipulation, particularly in studies of chloroplast and mitochondrial biology and RNA viral defense.
... The relevant use of specific vector was best studied in targeting inverted repeat region (IR) trnI/trnA of Brassica napus (Cheng et al. 2010) and Oryza sativa ) and large single copy region (LSC) rbcL/accD genes of Lactuca sativa L (Kanamoto et al. 2006). In contrast, the use of heterologous vectors in plastidal transformation attained appreciable frequency rates in species that are closely related at genome level, including that vectors used for transformation of the tobacco plastid genome have been successfully used for potato (rbcL/accD), tomato (trnfM/trnG) and scoparia (trnR/trnN) plastid transformation (Sidorov et al. 1999;Ruf et al. 2001;Muralikrishna et al. 2016). As well, the vectors employed for chloroplast transformation of tobacco and lesquerella (rrn16S/trnV) contained the flanking sequences from petunia, lettuce or arabidopsis (DeGray et al. 2001;Skarjinskaia et al. 2003;Ruhlman et al. 2010). ...
... Plastid genome transformation can be carried out as known in the art, see e.g. Sidorov VA et al. 1999, Plant J.19: 209-216 or Lutz KA et al. 2004 (6):906-13. ...
The present invention relates to the use of a nucleic acid sequence for the generation of transgenic plants having enhanced drought tolerance, and to a method for the generation of transgenic plants with enhanced drought tolerance.
... However, extending the species range of the technology has proven extremely difficult, and over the past 25 years, only very few species could be added to the list of transformable plants. These include a few solanaceous species 18,19 , lettuce 20 and poplar 21 . What all these plants have in common is that they display favourable properties in in vitro culture and are relatively easy to regenerate. ...
The development of technologies for the stable genetic transformation of plastid (chloroplast) genomes has been a boon to both basic and applied research. However, extension of the transplastomic technology to major crops and model plants has proven extremely challenging, and the species range of plastid transformation is still very much limited in that most species currently remain recalcitrant to plastid genome engineering. Here, we report an efficient plastid transformation technology for the model plant Arabidopsis thaliana that relies on root-derived microcalli as a source tissue for biolistic transformation. The method produces fertile transplastomic plants at high frequency when combined with a clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9)-generated knockout allele of a nuclear locus that enhances sensitivity to the selection agent used for isolation of transplastomic events. Our work makes the model organism of plant biology amenable to routine engineering of the plastid genome, facilitates the combination of plastid engineering with the power of Arabidopsis nuclear genetics, and informs the future development of plastid transformation protocols for other recalcitrant species. © 2019, The Author(s), under exclusive licence to Springer Nature Limited.
... Once the construct is synthesized, depending on the need it could undergo in vitro testing in a suitable model system or can be directly transferred into the plants using either the transient systems or stable organelle or nuclear transformation ( Fig. 3.1) [58]. Plastid transformation methods have been successfully used for the production of pharmaceuticals and synthetic fusion proteins [59,60], metabolic pathway, or pest resistance engineering in various crops such as soybean [61], potato [62], tobacco [63,64], sugar beet [65], eggplant [66], oilseed rape, etc. [67]. Large synthetic DNA has been successfully inserted into plant nuclear genome by using routine somatic embryogenesis and recent methods such as virus-mediated transfer [68], multiple-round in vivo site-specific assemblyeassisted transfer [69], binary bacterial artificial chromosomes [70], use of transformationcompetent artificial chromosomes [71], and plant artificial chromosomeeassisted transformations [72,73]. ...
... The distinct characteristics of chloroplast transformation such as site-specific transgene integration (Daniell, 2006), the absence of epigenetic gene silencing and position effects (Daniell et al., 2001;Rigano et al., 2009;Verma et al., 2010), stacking of transgenes into operons (Bock, 2013) and the excellent biosafety profile of transplastomic plants offer great potential in plant biotechnology. Significant interest in producing recombinant proteins in plastids of crop species with edible parts has resulted in transplastomic carrots (Kumar et al., 2004), tomato (Ruf et al., 2001;Zhou et al., 2008), potato (Sidorov et al., 1999), soybean (Dufourmantel et al., 2004;Moravec et al., 2007), cauliflower (Nugent et al., 2006), eggplant , cabbage and sugar beet (De Marchis et al., 2009). Edible crops offer the potential of oral delivery of therapeutical proteins, resulting in much reduced downstream protein processing costs (Kwon et al., 2013;Streatfield, 2006). ...
Dengue fever is a mosquito (Aedes aegypti) ‐transmitted viral disease that is endemic in more than 125 countries around the world. There are four serotypes of the dengue virus (DENV 1‐4) and a safe and effective dengue vaccine must provide protection against all four serotypes. To date, the first vaccine, Dengvaxia (CYD‐TDV), is available after many decades’ efforts, but only has moderate efficacy. More effective and affordable vaccines are hence required. Plants offer promising vaccine production platforms and food crops offer additional advantages for the production of edible human and animal vaccines, thus eliminating the need for expensive fermentation, purification, cold storage and sterile delivery. Oral vaccines can elicit humoral and cellular immunity via both the mucosal and humoral immune systems.
Here, we report the production of tetravalent EDIII antigen (EDIII‐1‐4) in stably transformed lettuce chloroplasts. Transplastomic EDIII‐1‐4‐expressing lettuce lines were obtained and homoplasmy was verified by Southern blot analysis. Expression of EDIII‐1‐4 antigens was demonstrated by immunoblotting, with the EDIII‐1‐4 antigen accumulating to 3.45% of the total protein content. Immunological assays in rabbits showed immunogenicity of EDIII‐1‐4. Our in vitro gastrointestinal digestion analysis revealed that EDIII‐1‐4 antigens are well protected when passing through the oral and gastric digestion phases but underwent degradation during the intestinal phase. Our results demonstrate that lettuce chloroplast engineering is a promising approach for future production of an affordable oral dengue vaccine.
This article is protected by copyright. All rights reserved.
... The relevant use of specific vector was best studied in targeting inverted repeat region (IR) trnI/trnA of Brassica napus (Cheng et al. 2010) and Oryza sativa ) and large single copy region (LSC) rbcL/accD genes of Lactuca sativa L (Kanamoto et al. 2006). In contrast, the use of heterologous vectors in plastidal transformation attained appreciable frequency rates in species that are closely related at genome level, including that vectors used for transformation of the tobacco plastid genome have been successfully used for potato (rbcL/accD), tomato (trnfM/trnG) and scoparia (trnR/trnN) plastid transformation (Sidorov et al. 1999;Ruf et al. 2001;Muralikrishna et al. 2016). As well, the vectors employed for chloroplast transformation of tobacco and lesquerella (rrn16S/trnV) contained the flanking sequences from petunia, lettuce or arabidopsis (DeGray et al. 2001;Skarjinskaia et al. 2003;Ruhlman et al. 2010). ...
The plastid transformation is used for high level expression of certain metabolically and industrially important recombinant proteins in plants. The vector, pFaadAII, a tobacco based vector system, harbouring a chimeric gene consisting of aadA coding region from Escherichia coli with 5′ 16S rDNA promoter and 3′ untranslated transcript region (UTR) of chlamydomonas rbcL gene, located in between the intergenic regions of rp132 and trnL genes. This vector used for transformation of plastids targets the foreign sequences to the small single-copy region of the plastome. Biolistic mode of approach for chloroplast transformation in Scoparia dulcis L., was achieved by bombarding the leaf explants and spectinomycin based selection system was used for regeneration of transformed plants. Transplastomic lines have been successfully established with overall efficiency of two transgenic lines for twenty-five bombarded explants. Integration of aadA in selection based regenerants was characterized by PCR and protein accumulation analysis along with seedlings experiment obtained from selfing. The chloroplast transformation developed in this plant system will provide scope for research in plastid based metabolic engineering pathways.
... Biolistics, also known as particle bombardment or the "Gene Gun" approach, remains the most widely used technique [123] . This method, once optimised, has an efficiency of 1-10 transformants per Petri dish, and has proven to function in the largest variety of species, including crops such as rice [124] , tomato [125] , potato [126] , eggplant [127] , soybean [128] , and wheat [129] . Recently an efficient transformation protocol was reported for the model species Arabidopsis thaliana [130] . ...
Plastids are subcellular organelles which perform photosynthesis in plants and algae. They are descended from free-living photosynthetic bacteria which were engulfed by early eukaryotes ~1.5 billion years ago. Nearly all plastids retain a small genome separate from the nuclear genome of the plants in which they reside, which is termed the plastome (plastid genome). This genome encodes many of the proteins required for photosynthesis, as well as bacteria-like transcriptional and translational machinery. Biotechnological techniques for making precise modifications to the plastome have been available for three decades, nearly as long as the equivalent techniques for the plant nuclear genome. However, while nuclear genetic engineering quickly moved from a laboratory novelty to a tool used for the improvement of crop plants planted on over a billion acres, plastome engineering remains largely confined to research use. The primary reasons for this lack of application are the species restrictions and technical difficulty of the transformation process, which until recently was only possible in a few species.
In 2007, a protocol for plastid transformation was reported for Marchantia polymorpha, a thalloid liverwort classically used as a model species. Marchantia offers rapid generation time, small size, simple genetics, and asexual reproduction by means of gemmae, small disks of tissue which provide a powerful platform for live-tissue microscopy. In this thesis, tools for Marchantia plastid transformation are systematically improved by the generation of the first plastome sequence assembly for a widely used laboratory strain of Marchantia, optimisation of the transformation protocol itself, and a comparison of the in vivo activity of plastid regulatory elements through a fluorescent marker and quantitative microscopy. The highly conserved nature of the land plant plastome suggests the improvements to plastid transformation developed in Marchantia will translate to other species.
... In addition to introduction of the DNA molecules into the nuclear genome, they can be transferred to the genomes of the organelles, chloroplast and mitochondria. For instance, Sidorov et al. [38] described the stable chloroplast transformation for potato. The gene expression in chloroplast has advantages such as reduced gene dispersal due to the maternal inheritance, high copy number of the plastids, simultaneous expression of several genes under single promoter, and position effect and gene silencing are avoided by homologous recombination [39]. ...
The vaccines are very effective vehicles for the prevention of various diseases. The cost of production and the need of cold-chain limit the vaccination programs especially in low-income countries. Hence, the plants offer a suitable platform to produce the immunogenic proteins with reduced cost without a requirement for cold-chain. The transgenic plants expressing antigens might be either used for the purification of the recombinant proteins or consumed as a food for the oral administration of the plant-based vaccine. A variety of plants have been used as the expression system, such as tobacco, rice, maize, carrot, soybean, potato and barley, each plant possessing pros and cons. Agrobacterium-mediated transformation, biolistics or electroporation techniques are used for the transformation of the plants, and the transgenic plants can be utilized for the development of vaccines against bacterial pathogens such as Mycobacterium tuberculosis, Bacillus anthracis, and viral diseases like Human Papillomavirus, Human Immunodeficiency Virus, as well as the diseases like cancer, synucleopathies or atherosclerosis.
... In addition to introduction of the DNA molecules into the nuclear genome, they can be transferred to the genomes of the organelles, chloroplast and mitochondria. For instance, Sidorov et al. [38] described the stable chloroplast transformation for potato. The gene expression in chloroplast has advantages such as reduced gene dispersal due to the maternal inheritance, high copy number of the plastids, simultaneous expression of several genes under single promoter, and position effect and gene silencing are avoided by homologous recombination [39]. ...
The vaccines are very effective vehicles for the prevention of various diseases. The cost of production and the need of cold-chain limit the vaccination programs especially in low-income countries. Hence, the plants offer a suitable platform to produce the immunogenic proteins with reduced cost without a requirement for cold-chain. The transgenic plants expressing antigens might be either used for the purification of the recombinant proteins or consumed as a food for the oral administration of the plant-based vaccine. A variety of plants have been used as the expression system, such as tobacco, rice, maize, carrot, soybean, potato and barley, each plant possessing pros and cons. Agrobacterium-mediated transformation, biolistics or electroporation techniques are used for the transformation of the plants, and the transgenic plants can be utilized for the development of vaccines against bacterial pathogens such as Mycobacterium tuberculosis, Bacillus anthracis, and viral diseases like Human Papillomavirus, Human Immunodeficiency Virus, as well as the diseases like cancer, synucleopathies or atherosclerosis.
... This number of genomes allows a greater number of transgenes per cell, resulting in more transcription and translation of transgenes versus nuclear transformation. In combination with absent or rare silencing phenomena (Wani et al. 2010;Sidorov et al. 1999), this property generally leads to higher protein levels (Maliga 1993;Wakasugi et al. 2001;Staub et al. 2000;Guda et al. 2000). Other advantages of chloroplast transformation include the feasibility of expressing multiple proteins through polycistronic mRNAs (Daniell and Dhingra 2002), and gene containment due to the maternal inheritance of chloroplast (Daniell 2002(Daniell , 2007Daniell and Parkinson 2003;Ruf et al. 2001). ...
In this chapter, a general outlook on the plant transformation approaches is provided with emphasis in applications related to molecular farming. The rationale of nuclear, chloroplast, and transient expressions mediated by viral vectors are reviewed. Implications of such technologies in terms of protein yields, posttranslational modifications, scalability, and production time scale are critically analyzed. New trends in plant genetic engineering are also identified and perspectives on how these technologies might influence the molecular farming field are provided.
Chloroplast transformation has emerged as a promising platform for the development of modified transplastomic plants expressing many useful products including pharmaceutical drugs, enzymes, biomaterials, and products related to the agriculture industry. Due to high copy number of chloroplasts in green plants, it is an attractive technology for the high mass production of expressed foreign proteins. Moreover, multiple genes can be expressed into plants in a single transformation event without any epigenetic effects because of operon system of chloroplasts like prokaryotes. Plastid transformations solve the problem of expressed transgene containment, gene silencing, and minimum pleiotropic effects, which are predominant in nuclear transformation. In this chapter, we have discussed the benefits of chloroplast transformation in plant molecular farming, different methods used for plastid transformation, application of chloroplast bioengineering in difference fields of science, and advantages and limitations of chloroplast transformation for plant molecular farming.
The disciplines of evolutionary biology and plant and animal breeding have been intertwined throughout their development, with responses to artificial selection yielding insights into the action of natural selection and evolutionary biology providing statistical and conceptual guidance for modern breeding. Here we offer an evolutionary perspective on a grand challenge of the 21st century - feeding humanity in the face of climate change. We first highlight promising strategies currently underway to adapt crops to current and future climate change. These include methods to match crop varieties with current and predicted environments and to optimize breeding goals, management practices, and crop microbiomes to enhance yield and sustainable production. We also describe the promise of crop wild relatives and recent technological innovations such as speed breeding, genomic selection, and genome editing for improving environmental resilience of existing crop varieties or for developing new crops. Next, we discuss how methods and theory from evolutionary biology can enhance these existing strategies and suggest novel approaches. We focus initially on methods for reconstructing the evolutionary history of crops and their pests and symbionts, because such historical information provides an overall framework for crop improvement efforts. We then describe how evolutionary approaches can be used to detect and mitigate the accumulation of deleterious mutations in crop genomes, identify alleles and mutations that underlie adaptation (and maladaptation) to agricultural environments, mitigate evolutionary trade-offs, and improve critical proteins. Continuing feedback between the evolution and crop biology communities will ensure optimal design of strategies for adapting crops to climate change.
Chloroplast engineering has matured considerably in recent years. It is emerging as a promising tool to address the challenges related to food security, drug production, and sustainable energy posed by an ever-growing world population. Chloroplasts have proven their potential by efficiently expressing transgenes, encapsulating recombinant proteins, and protecting them from cellular machinery, making it possible to obtain highly functional proteins. This quality has also been exploited by interfering RNA technology. In addition to the practical attributes offered by chloroplast transformation, such as the elimination of position effects, polycistronic expression, and massive protein production, the technique represents an advance in biosafety terms; however, even if its great biotechnological potential, crops that have efficiently transformed are still a proof of concept. Despite efforts, other essential crops have remained recalcitrant to chloroplast transformation, which has limited their expansion. In this chapter, we address the most recent advances in this area and the challenges that must be solved to extend the transformation to other crops and become the de facto tool in plant biotechnology.
Chloroplast is a discrete, highly structured, and semi-autonomous cellular organelle. The small genome of chloroplast makes it an up-and-coming platform for synthetic biology. As a special means of synthetic biology, chloroplast genetic engineering shows excellent potential in reconstructing various sophisticated metabolic pathways within the plants for specific purposes, such as improving crop photosynthetic capacity, enhancing plant stress resistance, and synthesizing new drugs and vaccines. However, many plant species exhibit limited efficiency or inability in chloroplast genetic transformation. Hence, new transformation technologies and tools are being constantly developed. In order to further expand and facilitate the application of chloroplast genetic engineering, this review summarizes the new technologies in chloroplast genetic transformation in recent years and discusses the choice of appropriate synthetic biological elements for the construction of efficient chloroplast transformation vectors.
Vaccines represent one of the most promising strategies for mitigating economic losses imposed by infectious diseases to global aquaculture. While the majority of commercial vaccines employ injection as the main route of delivery, a substantial body of research explores other avenues for the stimulation of protective immunity. Oral vaccines, representing the most favourable and convenient solution for the aquaculture industry, have been at the forefront of scientific interest for the last couple of decades. The orchestrated effort resulted in identifying the main roadblocks on the path to an ideal edible vaccine and provided several ingenious solutions improving the activation of immunity, ensuring the stability of administered antigens and their uptake upon the passage through the stomach. In the presented review, we describe advances in the available knowledge of the processes prerequisite for developing protective mucosal vaccines and focus readers' attention on the untapped potential of plant‐based production systems in this effort. We propose that these approaches not only meet production demands but also fulfil all requirements of an oral vaccine, addressing all the obstacles, previously solved via separate strategies, in one platform. Thus, combined with the available knowledge of molecular adjuvants and produced for a fraction of the cost, plant‐based production, so‐called molecular farming, is an ideal candidate for vaccine development, paving the road to sustainable aquaculture.
Petunia hybrida is a commercial ornamental plant and is also an important model species for genetic analysis and transgenic research. Here we describe the steps required to isolate stable plastid transformants in P. hybrida using the commercial Pink Wave cultivar. Wave cultivars are popular spreading Petunias sold as ground cover and potted plants. Transgenes introduced into P. hybrida plastids exhibit stable expression over many generations. The development of plastid transformation in P. hybrida provides an enabling technology to bring the benefits of plastid engineering, including maternal inheritance and stable expression of performance-enhancing trait genes, to the important floriculture and horticulture industries.
Chloroplast biotechnology has emerged as a promissory platform for the development of modified plants to express products aimed mainly at the pharmaceutical, agricultural, and energy industries. This technology’s high value is due to its high capacity for the mass production of proteins. Moreover, the interest in chloroplasts has increased because of the possibility of expressing multiple genes in a single transformation event without the risk of epigenetic effects. Although this technology solves several problems caused by nuclear genetic engineering, such as turning plants into safe bio-factories, some issues must still be addressed in relation to the optimization of regulatory regions for efficient gene expression, cereal transformation, gene expression in non-green tissues, and low transformation efficiency. In this article, we provide information on the transformation of plastids and discuss the most recent achievements in chloroplast bioengineering and its impact on the biopharmaceutical and agricultural industries; we also discuss new tools that can be used to solve current challenges for their successful establishment in recalcitrant crops such as monocots.
How to cite: Quintín Rascón-Cruz Q, González-Barriga CD, Iglesias-Figueroa BF, et al. Plastid transformation: Advances and challenges for its implementation in agricultural crops. Electron J Biotechnol 2021;51. https://doi.org/10.1016/j.ejbt.2021.03.005
The sections in this article are
Introduction
Historical Aspects
Unique Features of Chloroplast Genetic Engineering
Maternal Inheritance and Gene Containment
Crop Species Stably Transformed via the Plastid Genome
Agronomic Traits Conferred via the Plastid Genome
Transgenic Plastids as Bioreactors
Biomaterials, Enzymes, and Amino Acids
Conclusions
Acknowledgements
With distribution of genetic materials and advance molecular characteristics,
the chloroplast is prokaryotic compartments within the eukaryotic plants that
have turned into a crucial source for the genetic engineering and transplastomic
plants are becoming more popular means of agricultural development with elevated
crop yield. To address global agricultural problems, genetic modification of crop
plants is a rapid and promising solution to adapt the environment-friendly and well-controlled
farming system. The transplastomic plant with high accumulation of foreign
proteins (up to 45–46% TSP) and stable transgene expression with gene
containment can be a unique choice for the agricultural innovation of coming centuries.
Although the transplastomic plants still facing encumber to ensure the full
potential exploitation and expansion as an economical means, the removal of hardness
and obstacles of this technology and commercialization can contribute for the
sustainable development of future agriculture. In this book chapter, we intend to
recapitulate the up to date development and achievement of transplastomic plant
including gene transfer procedures in plastid genomes, regulable expression of plastid
transgenes, plant trait improvement by foreign gene expression, biopharmaceuticals
production, engineering of metabolic pathways in plant, study of transformation
mediated RNA editing technologies, bio-safety issues and public concerns on transplastomic plants and overall beneficial aspects. We believe that the utilization of
transplastomic plants will ensure an eco-
Plastome (plastid genome) engineering has grown up and got smarter for the transgene expression. Plastid transformation has profound benefits over nuclear transformation, includes a higher level of transgene expression, integration via homologous recombination, transgene containment, lack of gene silencing, and position effect. Substantial and fruitful progress has been achieved in plastome engineering of vegetable crops through the use of improved regeneration/selection procedures, plastid transformation vectors with efficient promoters, and 3/, 5/regulatory sequences. Plastid transformation technology developed for vegetable crops being used as a platform for the production of industrially important proteins and some of the genes of agronomic importance has been stably integrated and expressed in plastome. Although great progress has been accomplished in the plastid transformation of vegetable crops, still it is restricted to few species because of the unavailability of whole plastome sequencing. In this review, the author focus on the technology, progress, and advancements in plastid transformation of vegetable plants such as lettuce, tomato, potato, cabbage, cauliflower, eggplant, carrot, soybean, and bitter melon are reviewed. The conclusions, future prospects, and expansion of plastid transformation technology to other vegetable crops for genetic improvement and production of edible vaccines are proposed.
The genetic transformation of plants is an important biotechnological tool used for crop improvement for many decades. The present study was focussed to investigate various factors affecting genetic transformation of potato cultivar ‘Kufri Chipsona 1’. It was observed that explants pre-cultured for 2 days on MS2 medium (MS medium containing 10 µM silver nitrate, 10 µM BA, 15 µM GA3), injured with a surgical blade and co-cultivated with Agrobacterium tumefaciens strain EHA105 [O.D600 (0.6)] for 2 days results in maximum transient β-glucuronidase (GUS) expression. The addition of 100 µM acetosyringone in MS2 medium also increased rate of transient GUS expression in both the explants. Clumps of putative transgenic shoots were regenerated using the optimised culture conditions from leaf and internodal explants. The stable integration of T-DNA was established using histochemical staining for GUS and amplification of DNA fragment specific to nptII and uidA genes. Within the clumps, around 67.85% of shoots showed uniform GUS expression in all the tissues and about 32.15% shoots show intermittent GUS expression establishing chimeric nature. Uniform GUS staining of the tissue was used as initial marker of non-chimeric transgenic shoots. Quantitative expression of nptII transgene was found to be directly proportional to uniformity of GUS staining in transgenic shoots. The present investigation indicated that manipulation of culture conditions and the medium composition may help to get transgenic shoots with uniform expression of transgene in all the tissues of potato cultivar ‘Kufri Chipsona 1’.
A procedure for cybrid production, based on double treatment of donor protoplasts by physical and afterwards chemical mutagens at superlethal doses (γ-irradiation at a dose of 1000 Gy was applied for the inactivation of nuclei; 3-5 mMN-nitroso-N-methylurea was used for the efficient induction of plastome mutation) and the rescuing of mutant plastids after fusion with untreated recipient protoplasts, was developed. For identification of mutant donor-type plastids in fusion products a selection for streptomycin was performed. In two sets of experiments, in whichS. tuberosum served as the recipient of foreign cytoplasm with the wild tuber-bearing speciesS. bulbocastanum andS. pinnatisectum as donors, a total of about 40 streptomycin-resistant colonies was isolated. Eight regenerants from theS. tuberosum+S. bulbacastanum fusion combination and four fromS. tuberosum+S. pinnatisectum were further investigated using chromosome counting, analysis of esterase isoenzymes, restriction analysis of organelle DNA, and blot hybridization. All but one plant from both combinations were characterised as potato cybrids possessing exclusively foreign plastids and retaining a morphology typical of the recipient. Only in one line was rearranged mtDNA detected. The availability of potato cybrids facilitates the analysis of plastome-encoded breeding traits and the identification of the most valuable source of cytoplasm among the wild potato species. The described system for producing cybrids without genetic selectable markers in the parental material offers the possibility for the rescue of cytoplasmic mutations which are impossible to isolate by conventional approaches.
An engineered green fluorescent protein (GFP) from the jellyfish Aequora victoria was used to develop a facile and rapid rice transformation system using particle bombardment of immature embryos. The mgfp4 gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright-green fluorescence easily detectable and screenable in rice tissue 12–22
days after bombardment. Visual screening of transformed rice tissue, associated with a low level of antibiotic selection,
drastically reduced the quantity of tissue to be handled and the time required for the recovery of transformed plants. GFP
expression was observed in primary transformed rice plants (T0) and their progeny (T1). We describe various techniques to observe GFP in vitro and in vivo. The advantages of this new screenable marker in rice
genetic engineering programmes are discussed.
Interest in genetic transformation of plant cells has increased the need for molecular analysis of DNA from small samples of plant tissue. The ideal method for extracting this DNA would be simple and rapid to reduce the number of steps required (and to allow a large number of extractions to be run at a time). High, reproducible yields would allow extractions from a minimum mass of tissue enabling one to assay small tissue fragments or single colonies. The ability to assay a minimum amount of tissue could save considerable time otherwise spent waiting for the treated tissue to grow to a large enough size for conventional extraction methods to be used. Finally, the DNA produced needs to be sufficiently pure to allow restriction endonuclease digestion and genomic blot analysis. The procedure outlined in this report was specifically designed to reduce the number of required steps and maintain an efficient yield of high molecular weight DNA suitable for analysis. It is derived from a combination and modification of published procedures for CsCI purification of plant DNA (Bendich et al., 1979) and miniprep of bacterial plasmids (Birboim and Doly, 1979). The basic extraction procedure consists of three stpes; 1) homogenization, 2) phenol wash, and 3) ethanol precipitation. Since the buffer volumes are minimized as well as the number of extraction steps, the DNA yield is sufficient Inquiries to." lrvin J. Mettler, Stauffer Chemical Company, 1200 So. 47th Street, Richmond, California 94804.
We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of beta-glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.
Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.
A synthetic green fluorescent protein (GFP) gene (pgfp) was constructed to improve GFP expression in plants. Corn and tobacco protoplast transient assays showed that pgfp gave about 20-fold brighter fluorescence than the wild-type gene (gfp). Replacement of the serine at position 65 with a threonine (S65Tpgfp) or a cysteine (S65Cpgfp) yielded 100- to 120-fold brighter fluorescence than wild-type gfp upon excitation with 490-nm light. Incorporation of a plant intron into the coding region yielded an additional 1.4-fold improvement, for a cumulative improvement of about 150-fold in fluorescence at 490-nm excitation. Various versions of pgfp were also stably introduced into corn, wheat, tobacco, and Arabidopsis plants. Bright-green fluorescence was observed with a fluorescence microscope in virtually all examined tissues of transgenic monocots and dicots. In the case of Arabidopsis, expression of the pgfp gene under the enhanced 355 promoter of the cauliflower mosaic virus produced green fluorescence that was readily detectable by eye using a hand-held, long-wave ultraviolet lamp and/or a black-light source.
Individual plastids of vascular plants have generally been considered to be discrete autonomous entities that do not directly
communicate with each other. However, in transgenic plants in which the plastid stroma was labeled with green fluorescent
protein (GFP), thin tubular projections emanated from individual plastids and sometimes connected to other plastids. Flow
of GFP between interconnected plastids could be observed when a single plastid or an interconnecting plastid tubule was photobleached
and the loss of green fluorescence by both plastids was seen. These tubules allow the exchange of molecules within an interplastid
communication system, which may facilitate the coordination of plastid activities.
Glyphosate is a potent herbicide. It works by competitive inhibition of the enzyme 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS), which catalyzes an essential step in the aromatic amino acid biosynthetic pathway. We report the genetic engineering of herbicide resistance by stable integration of the petunia EPSPS gene into the tobacco chloroplast genome using the tobacco or universal vector. Southern blot analysis confirms stable integration of the EPSPS gene into all of the chloroplast genomes (5000-10,000 copies per cell) of transgenic plants. Seeds obtained after the first self-cross of transgenic plants germinated and grew normally in the presence of the selectable marker, whereas the control seedlings were bleached. While control plants were extremely sensitive to glyphosate, transgenic plants survived sprays of high concentrations of glyphosate. Chloroplast transformation provides containment of foreign genes because plastid transgenes are not transmitted by pollen. The escape of foreign genes via pollen is a serious environmental concern in nuclear transgenic plants because of the high rates of gene flow from crops to wild weedy relatives.
Pollen-mediated movement of transgenes from transplastomic oilseed rape (Brassica napus) into wild relatives will be avoided if chloroplasts are maternally transmitted. We assess the probability of chloroplast exchange between conventional oilseed rape and wild Brassica rapa to model the future behavior of transplastomic cultivars. Primers specific to cpDNA were used to demonstrate maternal inheritance of chloroplasts in 47 natural hybrids between cultivated B. napus and wild B. rapa. We conclude that there will be no or negligible pollen-mediated chloroplast dispersal from oilseed rape. Transgene introgression could still occur in mixed populations, however, if B. napus acted as the recurrent female parent. Rate of transfer would then depend on the abundance of mixed populations, their persistence as mixtures, and hybridization frequency within stands. A low incidence of sympatry (0.6-0.7%) between wild B. rapa and cultivated B. napus along the river Thames, UK, in 1997 and 1998, suggests mixed stands will form only rarely. Eighteen feral populations of B. napus also showed a strong tendency toward rapid decline in plant number, seed return, and ultimately, extinction within 3 years. Conversely, hybrid production is significant in mixed stands, and the absence of control practices means that oilseed rape will have slightly greater persistence. We infer that some introgression from transplastomic B. napus into B. rapa is inevitable in mixed populations even though such populations will occur infrequently and will tend to lose B. napus plants relatively quickly. Chloroplast exchange will be extremely rare and scattered.
The plastid rRNA (rrn) operon in chloroplasts of tobacco (Nicotiana tabacum), maize, and pea is transcribed by the plastid-encoded plastid RNA polymerase from a ς⁷⁰-type promoter (P1). In contrast, therrn operon in spinach (Spinacia oleracea) and mustard chloroplasts is transcribed from the distinct Pc promoter, probably also by the plastid-encoded plastid RNA polymerase. Primer-extension analysis reported here indicates that in Arabidopsis both promoters may be active. To understand promoter selection in the plastid rrn operon in the different species, we have tested transcription from the spinach rrn promoter in transplastomic tobacco and from the tobacco rrn promoter in transplastomic Arabidopsis. Our data suggest that transcription of the rrn operon depends on species-specific factors that facilitate transcription initiation by the general transcription machinery.
A synthetic green fluorescent protein (GFP) gene (pgfp) was constructed to improve GFP expression in plants. Corn and tobacco protoplast transient assays showed that pgfp gave about 20-fold brighter fluorescence than the wild-type gene (gfp). Replacement of the serine at position 65 with a threonine (S65Tpgfp) or a cysteine (S65Cpgfp) yielded 100- to 120-fold brighter fluorescence than wild-type gfp upon excitation with 490-nm light. Incorporation of a plant intron into the coding region yielded an additional 1.4-fold improvement, for a cumulative improvement of about 150-fold in fluorescence at 490-nm excitation. Various versions of pgfp were also stably introduced into corn, wheat, tobacco, and Arabidopsis plants. Bright-green fluorescence was observed with a fluorescence microscope in virtually all examined tissues of transgenic monocots and dicots. In the case of Arabidopsis, expression of the pgfp gene under the enhanced 35S promoter of the cauliflower mosaic virus produced green fluorescence that was readily detectable by eye using a hand-held, long-wave ultraviolet lamp and/or a black-light source.
Plastid engineering currently relies on DNA delivery by the biolistic process. We report here stable plastid transformation in tobacco by an alternate direct transformation protocol that is based on polyethylene glycol (PEG) treatment of leaf protoplasts in the presence of the transforming DNA. Clones with transformed plastid genomes were selected by spectinomycin resistance encoded by a mutant 16S ribosomal RNA gene. Incorporation of the transforming DNA into the plastid genome was confirmed by two unselected markers, streptomycin resistance and a novel PstI site that flank the spectinomycin resistance mutation in plasmid pZS148. Our simple and inexpensive protocol eliminates the dependence on the particle gun for chloroplast transformation and should facilitate applications of plastome engineering in crops.
The green fluorescent protein gene (gfp) is a widely used reporter in both animals and plants. Fusions between the plastidrrnpromoter or theEscherichia coli trcpromoter and thegfpcoding region have been delivered to chloroplasts using gold or tungsten microprojectiles, and fluorescence from GFP was visible in individual tobacco chloroplasts and in the abnormally large chloroplasts of thearc6 mutant ofArabidopsis thaliana2–4 days after bombardment. The fusion of thegfpcoding region to the bacterialtrcpromoter demonstrated that a bacterial promoter is active in chloroplastsin vivo. GFP was also detectable in amyloplasts of potato tubers and in chromoplasts of marigold petals, carrot roots and pepper fruits 4 days after bombardment. This demonstrates 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.
Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.
Plastid transformation is reported in Arabidopsis thaliana following biolistic delivery of transforming DNA into leaf cells. Transforming plasmid pGS31A carries a spectinomycin resistance
(aadA) gene flanked by plastid DNA sequences to target its insertion between trnV and the rps12/7 operon. Integration of aadA by two homologous recombination events via the flanking ptDNA sequences and selective amplification of the transplastomes
on spectinomycin medium yielded resistant cell lines and regenerated plants in which the plastid genome copies have been uniformly
altered. The efficiency of plastid transformation was low: 2 in 201 bombarded leaf samples. None of the 98 plants regenerated
from the two lines were fertile.
A procedure is described for the rapid and efficient production of transgenic potato (Solanum tuberosum L.) plants using Agrobacterium-based vectors. The key factors are the genotype and the level of kanamycin sulfate used for selection. Optimized culture conditions easily and routinely permitted the isolation of hundreds of individual regenerates expressing β-glucuronidase (GUS) transcriptionally fused to promoters from cauliflower mosaic virus (CaMV 35S) or a class-I patatin gene.
The plastid genome of higher plants is relatively small, 120–230-kb in size, and present in up to 10,000 copies per cell. Standard protocols for the introduction of transforming DNA employ biolistic DNA delivery or polyethylene glycol treatment. Genetically stable, transgenic plants are obtained by modification of the plastid genome by homologous recombination, followed by selection for the transformed genome copy by the expression of marker genes that protect the cells from selective agents. Commonly used selective agents are antibiotics, including spectinomycin, streptomycin, kanamycin and chloramphenicol. Selection for resistance to amino acid analogues has also been successful. The types of plastid genome manipulations include gene deletion, gene insertion, and gene replacement, facilitated by specially designed transformation vectors. Methods are also available for post-transformation removal of marker genes. The model species for plastid genetic manipulation is Nicotiana tabacum, in which most protocols have been tested. Plastid transformation is also available in several solanaceous crops (tomato, potato, eggplant) and ornamental species (petunia, Nicotiana sylvestris). Significant progress has been made with Brasssicaceae including cabbage, oilseed rape and Arabidopsis. Recent additions to the crops in which plastid transformation is reproducibly obtained are lettuce, soybean and sugar beet. The monocots are a taxonomic group recalcitrant to plastid transformation; initial inroads have been made only in rice.
Potato virus X (PVX) and potato virus Y (PVY) infection in potato may result in the loss of certification of seed potatoes and affect quality and yield of potatoes in commercial production. We transformed a major commercial cultivar of potato, Russet Burbank, with the coat protein genes of PVX and PVY. Transgenic plants that expressed both CP genes were resistant to infection by PVX and PVY by mechanical inoculation. One line was also resistant when PVY was inoculated with viruliferous green peach aphids. These experiments demonstrate that CP protection is effective against mixed infection by two different viruses and against mechanical and aphid transmission of PVY.
Nicotiana tabacum lines carrying maternally inherited resistance to spectinomycin were obtained by selection for green callus in cultures bleached by spectinomycin. Two levels of resistance was found. SPC1 and SPC2 seedlings are resistant to high levels (500 micrograms/ml), SPC23 seedlings are resistant to low levels (50 micrograms/ml) of spectinomycin. Lines SPC2 and SPC23 are derivatives of the SR1 streptomycin-resistant plastome mutant. Spectinomycin resistance is due to mutations in the plastid 16S ribosomal RNA: SPC1, an A to C change at position 1138; SPC2, a C to U change at position 1139; SPC23, a G to A change at position 1333. Mutations similar to those in the SPC1 and SPC2 lines have been previously described, and disrupt a conserved 16S ribosomal RNA stem structure. The mutation in the SPC23 line is the first reported case of a mutation close to the region of the 16S rRNA involved in the formation of the initiation complex. The new mutants provide markers for selecting plastid transformants.
The very high genome copy number in cytoplasmic organelles is a puzzling fact in cell biology. It is proposed here that high copy number reflects an increased need for organellar ribosomes that can only be satisfied by the increased ribosomal RNA gene number that results from genome multiplication.
Polycistronic mRNAs are the predominant form of plastid primary transcripts. To determine if there is internal initiation of translation of promoter-distal open reading frames, a promoterless uidA reporter gene was integrated into the tobacco plastid genome downstream of the rbcL gene. Monocistronic uidA mRNA does not accumulate from the promoterless uidA construct. However, due to inefficient rbcL transcription termination, a polycistronic transcription unit is created that contains the uidA gene as the second cistron. Numerous stop codons in all three reading frames between the rbcL and uidA coding regions ensure that translation of uidA initiates only from the correct start codon. The encoded reporter gene product, beta-glucuronidase (GUS) accumulates to high levels in the transplastomic plants indicating that promoter-distal cistrons can be efficiently translated in plastids.
The pPRV plasmids are vectors for targeted insertion of foreign genes into the tobacco plastid genome ptDNA). The vectors
are based on the pUC119 plasmid which replicates in E.coli but not in plastids. The spectinomycin resistance (aadA) gene and a multiple cloning site (MCS) are flanked by 1.8-kb and 1.2- ptDNA sequences. Biolistic delivery of vector DNA,
followed by spectinomycin selection, yields plastid transformants at a reproducible frequency, ∼ 1 transplastomic line per
bombarded sample. The selected aadA gene and linked non-selectable genes cloned into the MCS are incorporated into the ptDNA by homologous recombination events
via the flanking ptDNA sequences. The transplastomes thus generated are stable, and are maternally transmitted to the seed
progeny. The pPRV vector series targets insertions between the divergently transcribed trnV gene and rps12/7 operon. The lack of readthrough transcription of appropriately oriented transgenes makes the vectors an ideal choice for
the study of transgene promoter activity.
The plastid psbA gene encodes the 32 kDa D1 polypeptide of photosystem II. It has previously been shown that the initiation of psbA mRNA translation in tobacco is regulated by sequences outside of the coding region. To identify the cis-acting regulatory sequences involved in the translational control, a series of chimeric uidA genes, encoding the beta-glucuronidase (GUS) reporter enzyme, were introduced into the plastid genome. GUS accumulation in response to the light (135- to 200-fold), and the arrest of uidA mRNA translation in light-grown seedlings following transfer for 2 h to the dark, was observed only if the transgenes contained the psbA 5'-untranslated leader region (UTR). Changes in GUS accumulation were accompanied by little or no changes in the uidA mRNA levels. The data indicate that the initiation of D1 translation in tobacco plastids is controlled via the psbA 5'-UTR.
We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 micrograms/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 micrograms/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10,000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.
We report here a 100-fold increased frequency of plastid transformation in tobacco by selection for a chimeric aadA gene encoding aminoglycoside 3"-adenylyltransferase, as compared with that obtained with mutant 16S rRNA genes. Expression of aadA confers resistance to spectinomycin and streptomycin. In transforming plasmid pZS197, a chimeric aadA is cloned between rbcL and open reading frame ORF512 plastid gene sequences. Selection was for spectinomycin resistance after biolistic delivery of pZS197 DNA into leaf cells. DNA gel-blot analysis confirmed incorporation of the chimeric aadA gene into the plastid genome by two homologous recombination events via the flanking plastid gene sequences. The chimeric gene became homoplasmic in the recipient cells and is uniformly transmitted to the maternal seed progeny. The ability to transform routinely plastids of land plants opens the way to manipulate the process of photosynthesis and to incorporate novel genes into the plastid genome of crops.
Nicotiana plumbaginifolia protoplasts were directly transformed by PEG treatment with a cloned 16S rRNA gene isolated from a double antibiotic-resistant Nicotiana tabacum plastid mutant. Putative plastid transformants were selected in cell culture by their spectinomycin resistance and identified by their unselected streptomycin resistance. Alternatively, cell lines were selected in the presence of both antibiotics. The cell line (and its regenerated plants) selected solely for spectinomycin resistance demonstrated an extensive segregation of streptomycin resistance in subsequent tests, while the double-selected line showed stable resistance for both antibiotics. The resistance markers were inherited maternally. In the putative plastid transformants the origin of the resistance mutations was identified by the absence of an AatII site, missing in the donor N. tabacum plastid gene (spectinomycin resistance site) but present in that of wild-type N. plumbaginifolia, and a sequence analysis of the particular nucleotide changes in both resistance sites. Restriction enzyme analysis of total plastid DNA (ptDNA), and the recloning and full sequencing of the fragment introduced, investigated in one of the plastid transformants, showed no DNA rearrangements accompanied with the integration process. Sequence analysis indicated a targeted, homologous integration of the DNA fragment introduced but an unexpectedly complete homology of the parental ptDNA sequences in this region prevented the location of borders. Although the frequency of plastid transformant colonies (2 x 10(-5)) should still be improved, this method for stable chloroplast DNA transformation is comparable with or more efficient than the particle bombardment techniques.
Russet Burbank potato plants have been genetically improved to resist insect attack and damage by Colorado potato beetles (Leptinotarsa decemlineata (Say)) by the insertion of a cryIIIA gene encoding the insect control protein of Bacillus thuringiensis var. tenebrionis. A modified gene that dramatically improved plant expression of this protein was utilized. Its expression in Russet Burbank potato plants resulted in protection from damage by all insect stages in the laboratory and in dramatic levels of protection at multiple field locations. Analysis of these genetically modified potatoes indicated that they conform to the standards for Russet Burbank potatoes in terms of agronomic and quality characteristics including taste.
To determine how to utilize the green fluorescent protein (GFP) as a marker for subcellular localization and as a label for plant mitochondria in vivo, transgenic suspension cells and tobacco plants expressing GFP with and without a mitochondrial localization signal were generated. The first GFP form used, GFP1, is easily observable in cells with low autofluorescence, such as suspension cells or trichomes, but masked in green tissue. For the visualization of GFP in cells and tissues with high autofluorescence, such as leaf, the use of a very strong promoter (35S35SAMV), a highly expressed modified mGFP4 coding region and a brighter mutant form of GFP (S65T) was necessary. Confocal or two-photon laser scanning microscopy reveal a distinct subcellular localization of the fluorescence in cells expressing GFP or coxIVGFP. In cells expressing untargeted GFP, fluorescence accumulates in the nucleoplasm but is also distributed throughout the cytoplasm. It is excluded from vacuoles, nucleoli and from round bodies that are likely to be leucoplasts. In contrast, fluorescence is localized specifically to mitochondria in cells expressing coxIVGFP fusion protein as shown by co-localization with a mitochondrial-specific dye. This permits the direct observation of mitochondria and mitochondrial movements in living plant cells and tissues throughout plant development. Three-dimensional reconstruction of individual cells can give additional information about the distribution and numbers of mitochondria.
The Bacillus thuringiensis (Bt) crystal toxins are safe biological insecticides, but have short persistance and are poorly effective against pests that feed inside plant tissues. Production of effective levels of these proteins in plants has required resynthesis of the genes encoding them. We report that amplification of an unmodified crylA(c) coding sequence in chloroplasts up to approximately 10,000 copies per cell resulted in the accumulation of an unprecedented 3-5% of the soluble protein in tobacco leaves as protoxin. The plants were extremely toxic to larvae of Heliothis virescens, Helicoverpa zea, and Spodoptera exigua. Since the plastid transgenes are not transmitted by pollen, this report has implications for containment of Bt genes in crop plants. Furthermore, accumulation of insecticidal protein at a high level will facilitate improvement in the management of Bt resistant insect populations.
The plastid rRNA (rrn) operon in chloroplasts of tobacco (Nicotiana tabacum), maize, and pea is transcribed by the plastid-encoded plastid RNA polymerase from a sigma70-type promoter (P1). In contrast, the rrn operon in spinach (Spinacia oleracea) and mustard chloroplasts is transcribed from the distinct Pc promoter, probably also by the plastid-encoded plastid RNA polymerase. Primer-extension analysis reported here indicates that in Arabidopsis both promoters may be active. To understand promoter selection in the plastid rrn operon in the different species, we have tested transcription from the spinach rrn promoter in transplastomic tobacco and from the tobacco rrn promoter in transplastomic Arabidopsis. Our data suggest that transcription of the rrn operon depends on species-specific factors that facilitate transcription initiation by the general transcription machinery.
Businesses based on new technology and upscale taste—Microsoft and Amazon.com, Starbucks, and Nordstrom—have infused the Pacific Northwest with wealth and entrepreneurial energy.
Stable genetic transformation of the plastid genome is reported in a higher plant, Nicotiana tabacum. Plastid transformation was obtained after bombardment of leaves with tungsten particles coated with pZS148 plasmid DNA. Plasmid pZS148 (9.6 kilobases) contains a 3.7-kilobase plastid DNA fragment encoding the 16S rRNA. In the 16S rRNA-encoding DNA (rDNA) a spectinomycin resistance mutation is flanked on the 5' side by a streptomycin resistance mutation and on the 3' side by a Pst I site generated by ligating an oligonucleotide in the intergenic region. Transgenic lines were selected by spectinomycin resistance and distinguished from spontaneous mutants by the flanking, cotransformed streptomycin resistance and Pst I markers. Regenerated plants are homoplasmic for the spectinomycin resistance and the Pst I markers and heteroplasmic for the unselected streptomycin resistance trait. Transgenic plastid traits are transmitted to the seed progeny. The transgenic plastid genomes are products of a multistep process, involving DNA recombination, copy correction, and sorting out of plastid DNA copies.
Starch, a major storage metabolite in plants, positively affects the agricultural yield of a number of crops. Its biosynthetic
reactions use adenosine diphosphate glucose (ADPGlc) as a substrate; ADPGlc pyrophosphorylase, the enzyme involved in ADPGlc
formation, is regulated by allosteric effectors. Evidence that this plastidial enzyme catalyzes a rate-limiting reaction in
starch biosynthesis was derived by expression in plants of a gene that encodes a regulatory variant of this enzyme. Allosteric
regulation was demonstrated to be the major physiological mechanism that controls starch biosynthesis. Thus, plant and bacterial
systems for starch and glycogen biosynthesis are similar and distinct from yeast and mammalian systems, wherein glycogen synthase
has been demonstrated to be the rate-limiting regulatory step.
The uses of GFP in plants
- J Haseloff
- K R Siemering