Efficient and Stable Transformation of Lactuca sativa L. cv. Cisco (lettuce) Plastids

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to ~36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.     

Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts

Plastid transformation offers several unique advantages compared with nuclear genome transformation, such as high level of transgene expression within plastids, expressing multiple transgenes as operons, lack of position effect due to site-specific transgene integration, and reducing risks of gene flow via pollen due to maternal inheritance of the plastid genome. Plastid transformation has been applied to several herbal species, but as yet there are no applications to tree species. We report here the first successful plastid transformation in a tree species, Populus alba. A vector for plastid transformation of poplar (Populus alba) was constructed, which carried the spectinomycin resistance gene and the green fluorescence protein gene as marker genes. In the regenerated shoots, the site-specific integration of foreign genes and the establishment of a high homoplastomic state were confirmed. Immunoblot analysis and histological observations corroborated the accumulation of green fluorescence protein in chloroplasts. The establishment of a plastid transformation system in poplar provides a novel tool for tree biotechnology.     

Advances in chloroplast engineering

The chloroplast is a pivotal organelle in plant cells and eukaryotic algae to carry out photosynthesis, which provides the primary source of the world＇s food. The expression of foreign genes in chloroplasts offers several advantages over their expression in the nucleus： high-level expression, transgene stacking in operons and a lack of epigenetic interference allowing stable transgene expression. In addition, transgenic chloroplasts are generally not transmitted through pollen grains because of the cytoplasmic localization. In the past two decades, great progress in chloroplast engineering has been made. In this paper, we review and highlight recent studies of chloroplast engineering, including chloroplast transformation procedures, controlled expression of plastid transgenes in plants, the expression of foreign genes for improvement of plant traits, the production of biopharmaceuticals, metabolic pathway engineering in plants, plastid transformation to study RNA editing, and marker gene excision system.     

Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome

Applications of chloroplast engineering in agriculture and biotechnology will depend critically on success in extending the crop range of chloroplast transformation, and on the feasibility of expressing transgenes in edible organs (such as tubers and fruits), which often are not green and thus are much less active in chloroplast gene expression. We have improved a recently developed chloroplast-transformation system for tomato plants and applied it to engineering one of the central metabolic pathways in fruits: carotenoid biosynthesis. We report that plastid expression of a bacterial lycopene beta-cyclase gene results in herbicide resistance and triggers conversion of lycopene, the main storage carotenoid of tomatoes, to beta-carotene, resulting in fourfold enhanced pro-vitamin A content of the fruits. Our results demonstrate the feasibility of engineering nutritionally important biochemical pathways in non-green plastids by transformation of the chloroplast genome.     

High‐level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes

Transgene expression from the plant's plastid genome represents a promising strategy in molecular farming because of the plastid's potential to accumulate foreign proteins to high levels and the increased biosafety provided by the maternal mode of organelle inheritance. In this article, we explore the potential of transplastomic plants to produce human immunodeficiency virus (HIV) antigens as potential components of an acquired immunodeficiency syndrome (AIDS) vaccine. It is shown that the HIV antigens p24 (the major target of T‐cell‐mediated immune responses in HIV‐positive individuals) and Nef can be expressed to high levels in plastids of tobacco, a non‐food crop, and tomato, a food crop with an edible fruit. Optimized p24‐Nef fusion gene cassettes trigger antigen protein accumulation to up to approximately 40% of the plant's total protein, demonstrating the great potential of transgenic plastids to produce AIDS vaccine components at low cost and high yield.     

Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny

The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stable plastid transformation in rice. We produced fertile transplastomic rice plants and demonstrated transmission of the plastid-expressed green fluorescent protein (GFP) and aminoglycoside 3'-adenylyltransferase genes to the progeny of these plants. Transgenic chloroplasts were determined to have stably expressed the GFP, which was confirmed by both confocal microscopy and Western blot analyses. Although the produced rice plastid transformants were found to be heteroplastomic, and the transformation efficiency requires further improvement, this study has established a variety of parameters for the use of plastid transformation technology in cereal crops.     

Genetic engineering of the chloroplast

Transformation of the plastid genome has a number of inherent advantages for the engineering of gene expression in plants. These advantages include: 10-50 times higher transgene expression levels; the absence of gene silencing and position effect variation; the ability to express polycistronic messages from a single promoter; uniparental plastid gene inheritance in most crop plants that prevents pollen transmission of foreign DNA; integration via a homologous recombination process that facilitates targeted gene replacement and precise transgene control; and sequestration of foreign proteins in the organelle which prevents adverse interactions with the cytoplasmic environment. It is now 12 years since the first conclusive demonstration of stable introduction of cloned DNA into the Chlamydomonas chloroplast by the Boynton and Gillham laboratory, and 10 years since the laboratory of Pal Maliga successfully extended these approaches to tobacco. Since then, technical developments in plastid transformation and advances in our understanding of the rules of plastid gene expression have facilitated tremendous progress towards the goal of establishing the chloroplast as a feasible platform for genetic modification of plants.     

Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance

Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. We report here high-level expression of betaine aldehyde dehydrogenase (BADH) in cultured cells, roots, and leaves of carrot (Daucus carota) via plastid genetic engineering. Homoplasmic transgenic plants exhibiting high levels of salt tolerance were regenerated from bombarded cell cultures via somatic embryogenesis. Transformation efficiency of carrot somatic embryos was very high, with one transgenic event per approximately seven bombarded plates under optimal conditions. In vitro transgenic carrot cells transformed with the badh transgene were visually green in color when compared to untransformed carrot cells, and this offered a visual selection for transgenic lines. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7-fold more, and accumulated 50- to 54-fold more betaine (93-101 micromol g(-1) dry weight of beta-Ala betaine and Gly betaine) than untransformed cells grown in liquid medium containing 100 mm NaCl. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mm), the highest level of salt tolerance reported so far among genetically modified crop plants. BADH expression was 74.8% in non-green edible parts (carrots) containing chromoplasts, and 53% in proplastids of cultured cells when compared to chloroplasts (100%) in leaves. Demonstration of plastid transformation via somatic embryogenesis utilizing non-green tissues as recipients of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants.     

Post‐harvest light treatment increases expression levels of recombinant proteins in transformed plastids of potato tubers

Plastid genetic engineering represents an attractive system for the production of foreign proteins in plants. Although high expression levels can be achieved in leaf chloroplasts, the results for non‐photosynthetic plastids are generally discouraging. Here, we report the expression of two thioredoxin genes (trx f and trx m) from the potato plastid genome to study transgene expression in amyloplasts. As expected, the highest transgene expression was detected in the leaf (up to 4.2% of TSP). The Trx protein content in the tuber was approximately two to three orders of magnitude lower than in the leaf. However, we demonstrate that a simple post‐harvest light treatment of microtubers developed in vitro or soil‐grown tubers induces up to 55 times higher accumulation of the recombinant protein in just seven to ten days. After the applied treatment, the Trx f levels in microtubers and soil‐grown tubers increased to 0.14% and 0.11% of TSP, respectively. Moreover, tubers stored for eight months maintained the capacity of increasing the foreign protein levels after the light treatment. Post‐harvest cold induction (up to five times) at 4°C was also detected in microtubers. We conclude that plastid transformation and post‐harvest light treatment could be an interesting approach for the production of foreign proteins in potato.     

Plastid biotechnology: prospects for herbicide and insect resistance, metabolic engineering and molecular farming

Transgene expression from the chloroplast (plastid) genome offers several attractions to plant biotechnologists, including high-level accumulation of foreign proteins, transgene stacking in operons and a lack of epigenetic interference with the stability of transgene expression. In addition, the technology provides an environmentally benign method of plant genetic engineering, because plastids and their genetic information are maternally inherited in most crops and thus are largely excluded from pollen transmission. During the past few years, researchers in both the public and private sectors have begun to explore possible areas of application of plastid transformation in plant biotechnology as a viable alternative to conventional nuclear transgenic technologies. Recent proof-of-concept studies highlight the potential of plastid genome engineering for the expression of resistance traits, the production of biopharmaceuticals and metabolic pathway engineering in plants.     

The suffulta mutation in tomato reveals a novel method of plastid replication during fruit ripening

Mutant alleles at the suffulta locus of tomato dramatically affect the pattern of plastid division throughout the plant, resulting in few, greatly enlarged chloroplasts in leaf and stem cells. suffulta plants are compromised in growth and have distinctly pale stems. The green developing tomato fruit are generally paler compared with the wild type, but ripe red fruit are much more similar in colour and pigment content. By using plastid-targeted green fluorescent protein, the underlying plastid phenotypes in the ripening suffulta fruit reveal that enlarged chlorophyll-containing chloroplasts degenerate and give rise to a wild type-like population of chromoplasts in ripe fruit by a process of plastid budding and fragmentation, resulting in a heterogeneous population of plastid-derived structures which eventually become chromoplasts. In stomatal guard cells, plastid-derived structures lacking chlorophyll, but containing GFP, are also observed, especially in guard cells which completely lack normal chloroplasts. How this novel 'replication' process in suffulta relates to conventional plastid division and stromule formation is discussed.     

Transgenic plastids in basic research and plant biotechnology

Facile methods of genetic transformation are of outstanding importance for both basic and applied research. For many years, transgenic technologies for plants were restricted to manipulations of the nuclear genome. More recently, a second genome of the plant cell has become amenable to genetic engineering: the prokaryotically organized circular genome of the chloroplast. The possibility to directly manipulate chloroplast genome-encoded information has paved the way to detailed in vivo studies of virtually all aspects of plastid gene expression. Moreover, plastid transformation technologies have been intensely used in functional genomics by performing gene knockouts and site-directed mutageneses of plastid genes. These studies have contributed greatly to our understanding of the physiology and biochemistry of biogenergetic processes inside the plastid compartment. Plastid transformation technologies have also stirred considerable excitement among plant biotechnologists, since transgene expression from the plastid genome offers a number of most attractive advantages, including high-level foreign protein expression and transgene containment due to lack of pollen transmission. This review describes the generation of plants with transgenic plastids, summarizes our current understanding of the transformation process and highlights selected applications of transplastomic technologies in basic and applied research.     

Optimization of the expression of the HIV fusion inhibitor cyanovirin-N from the tobacco plastid genome

Plants with transgenic plastid (chloroplast) genomes represent a promising production platform in molecular farming, mainly because of the plastids' potential to accumulate foreign proteins to very high levels and the increased biosafety conferred by the maternal mode of plastid inheritance. Although some transgenes can be expressed to extraordinarily high levels, the expression of others has been unsuccessful. Lack of detectable transgene expression is usually attributable to either RNA instability or protein instability. Here, we have investigated the possibilities to improve the production of a pharmaceutical protein that is difficult to express in chloroplasts: the HIV-1 fusion inhibitor cyanovirin-N (CV-N). Testing various N-terminal and C-terminal fusions to peptide sequences from two proteins known to accumulate to high levels in transgenic plastids (GFP and the protein antibiotic PlyGBS), we show that both low mRNA stability and low protein stability contribute to the lack of detectable CV-N expression in chloroplasts. Both problems can be alleviated by N-terminal fusions to the CV-N coding region, thus highlighting a suitable strategy for optimization of plastid transgene expression.     

Expression of thermostable microbial cellulases in the chloroplasts of nicotine-free tobacco

An inexpensive source of active cellulases is critical to efficient and cost-effective conversion of lignocellulosic biomass to ethanol. Transgenic plants expressing foreign cellulases are potential sources of cellulases for biomass conversion. A number of foreign proteins have been reported to accumulate to high levels when the transgene is incorporated into the chloroplast genome rather than into the nuclear genome. We developed plastid transformation vectors carrying two Thermobifida fusca thermostable cellulases, Cel6A and Cel6B, and expressed them in nicotine-free or nicotine-containing tobacco varieties following chloroplast transformation. We obtained homoplasmic tobacco plants expressing Cel6A or Cel6B. Maximum estimates of expression levels ranged from 2 to 4% of total soluble protein. Enzyme assays indicated that both Cel6A and Cel6B expressed in transplastomic tobacco were active in hydrolyzing crystalline cellulose. With further optimization, it may be feasible to produce bacterial cellulases in tobacco chloroplasts in large quantities.     

Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts

Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-labile toxin. LTK63 is devoid of any toxic activity, but still retains its mucosal adjuvanticity. The LTK63 was cloned into chloroplast targeting vector and transformed to tobacco chloroplasts by particle bombardment. PCR and Southern blot analyses confirmed stable homologous recombination of the LTK63 gene into the chloroplast genome. The amount of LTK63 protein detected in tobacco chloroplasts was approximately 3.7% of the total soluble protein. The GM1-ganglioside binding assay confirmed that chloroplast-synthesized LTB of LTK63 binds to the intestinal membrane GM1-ganglioside receptor. Thus, the expression of LTK63 in chloroplasts provides a potential route toward the development of a plant-based edible vaccine for high expression system and environmentally friendly approach.     

A novel chloroplast transformation vector compatible with the Gateway® recombination cloning technology

To analyze the suitability of Gateway^® vectors for transformation of chloroplasts, we converted a standard plastid transformation vector into a Gateway^® destination vector containing the necessary recombination sites attR1 and attR2. Insertion of the green fluorescent protein (GFP) coding sequence with associated T7g10 ribosome binding site into this destination vector created the expression vector for transformation of tobacco chloroplasts with the biolistic method. Correct integration of the transgene into the plastid genome was verified by PCR and the homoplasmic nature of the transformed plants was confirmed by Southern Blot analysis. Expression of the GFP reporter protein was monitored by confocal laser scanning microscopy (CLSM) and quantification by western blot analysis showed a GFP accumulation level of 3 % total soluble protein (TSP). The presented results clearly demonstrate that the Gateway^® recombination sites are compatible with all steps of plastid transformation, from generation of transplastomic plants to expression of GFP. This is the first report of a plastid transformation vector made by the Gateway^® recombinant cloning technology, which proves the suitability of this system for use in chloroplasts.     

Expression of the truncated tissue plasminogen activator (K2S) gene in tobacco chloroplast

As because the plant plastid genome is highly polyploid, the transformation of chloroplasts permits the introduction of thousands of copies of foreign genes per plant cell and generates extraordinarily high levels of recombinant protein. Human tissue-type plasminogen activator is one of the most important pharmaceutical proteins involved in the breakdown of blood clots in brain and heart blood vessels. We report the introduction and expression of the truncated human tissue plasminogen activator (K2S) gene in tobacco chloroplasts. The K2S-containing vector pKCZK2S was successfully transferred to tobacco plastomes using the biolistic delivery procedure. Transplastomic plants were selected on RMOP medium containing spectinomycin (500 mg/l). In order to achieve homoplasmy, several rounds of selection and regeneration were performed. The presence, site-specific integration, homoplasmy, expression and activity assay of the transgene were confirmed in the transplastomic plants by PCR, Southern-blot, RT-PCR, SDS-PAGE, ELISA, Dot-blot, Western-blot and zymography analysis. Our results show that the tissue plasminogen activator (K2S form) protein to be expressed in tobacco chloroplasts in active form.