Progress 01/15/02 to 07/24/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This project is aligned with NP 302, Plant Biological and Molecular Processes. a) Rubisco replacement Yield increases in major US crops are beginning to plateau. One of the most evident ways to further increase plant productivity is to alter the CO2 fixing enzyme, Rubisco, to increase its activity and selectivity for CO2 versus O2. However, engineering this enzyme is complicated by the location of its genes in both the nuclear and chloroplast genomes, the occurrence of posttranslational modifications of unknown significance, the involvement of molecular chaperones in its assembly, and the regulation of its activity by the activase protein, which can exhibit specificity towards its Rubisco target. Thus facile technologies need to be developed for chloroplast and possibly nuclear
transformation directed specifically at allowing the replacement of the endogenous protein with foreign or genetically engineered forms. The downstream consequences of expressing foreign or extensively modified Rubisco proteins may then also need to be addressed. b) Transgene containment Genetic engineering of plants has tremendous potential to create crops with new metabolic capabilities, such as the ability to accumulate pharmaceuticals or other high value specialty materials. In addition to the metabolic engineering itself, however, there are some obstacles to be overcome before these technologies can be commercialized. One is the potential for spread of transgenes from the engineered plants to conventional fields of the same crops, largely by dispersal of pollen. An attractive approach to the problem of gene dispersal is to genetically engineer the chloroplast genome of crop plants, which is maternally inherited and is not transmitted through pollen. Potential for gene flow also
exists via transgenic seeds. Both of these could be avoided by harvesting foreign proteins from vegetative organs (leaves) before the onset of flowers or by engineering plants that are biennial, in which vegetative organs (like carrots containing therapeutic proteins) are harvested the first year when they do not flower. As a further safeguard, engineering cytoplasmic male sterility has potential to also provide transgene containment. Because gene flow via pollen and seeds is such a potentially serious problem, a number of government and private agencies are in search of solutions that will allow transgenic corps to be developed for commercial purposes, especially when the production of pharmaceutical proteins is being considered. c) Engineer plants to survive salt stress via the chloroplast genome Salt stress is a major abiotic stress in plant agriculture. The problem of soil salinity has been compounded by irrigation and excessive use of fertilizers. About 20% of the world's
irrigated lands are affected by salinity. Currently, high salinity limits crop production in 30% of the irrigated land in the United States and 20 million hectares globally. Carrot is classified as a salt sensitive plant and there is 7% growth reduction for every 10 mM increment in salinity above 20 mM salt. Therefore carrot is an ideal candidate for genetic manipulation for increased salt tolerance. d) Engineering the cotton chloroplast genome Cotton (Gossypium hirsutum L.) is an excellent natural source of textile fiber in the world and one of the world's most important commercial crops. The U.S. accounts for over 40% of total world fiber production ($ 6.1 billions annual sales) and is one of leading exporter in global trade of raw cotton. Products like cotton lint and cottonseed are among the top 20 major agricultural products of the United States by value as reported by the FAO in 2003 [www.fao.org/]. The annual business revenue stimulated by cotton in the U.S. economy is about
$120 billion each year, making cotton America's number one high value crop. However, cotton is particularly challenging to manipulate in-vitro due to the difficulties encountered in plant regeneration through somatic embryogenesis. In 2002/03 insect and herbicide resistant transgenic cotton was planted on 13% of the total area of world after soybean (63%) and corn (19%) and on 77% of the total area in the USA, modified via the nuclear genome, compared to 81% of GM soybean and 40% GM corn [http://www.ers.usda.gov]. So far, nuclear transgenic cotton is practiced only in restricted areas of the world. For instance, Upland cotton, Gossypium hirsutum, has the potential to hybridize with Hawaiian cotton, G. tomentosum, and feral populations of G. hirsutum in the Florida Keys, and of G. hirsutum / G. barbadense on the U.S. Virgin Islands and Puerto Rico. For these reasons, restrictions on field plot experimental use permits and commercial planting of Bt-cotton has been instituted in these
areas. Similarly, GM cotton is now planted only in regions of the world where there are no wild relatives, in order to avoid potential outcross with related weeds. Dispersal of pollen from transgenic cotton plants to surrounding non- transgenic plants has been reported. Umbeck et al. [J. Econ. Entomol. 84: 1943-1950] investigated pollen dispersal from transgenic cotton embedded in a field of conventional cotton in the United States and observed up to 5.7% out-crossing rates inspite of buffer rows. Transgene escape could be avoided via chloroplast genetic engineering because of maternal inheritance of transgenes in cotton. Another concern about GM crops expressing Bt toxins is that suboptimal production of toxins might result in an increased risk of pests developing Bt resistance. Nuclear engineered Bt cotton due to low expression of transgenes is not fully protected from the insect-pest attack and it needs several sprays of pesticides on crop fields to minimize the yield loss. e)
Low-cost production of vaccines and biopharmaceuticals Another major problem is the high cost of biopharmaceuticals. Globally, about 170 million people are infected with hepatitis C virus, with 3-4 million new infections each year (WHO fact sheet 164, October 2000). WHO Department of Communicable Disease Surveillance and Response reports that more than one third of world population is infected with Hepatitis B. In Asia, the prevalence of chronic hepatitis B and C is very high (about 110 million infected by HCV and 150 million infected by HBV). A large majority of Hepatitis C infected patients have severe liver cirrhosis and currently there is no vaccine available for this disease. The annual requirement of Insulin like Growth Factor I, per cirrhotic patient is 600 mg (1.5-2 mg per day) and the cost of IGF-1 per mg is $30,000. Current annual cost of interferon therapy for viral hepatitis is $26,000 per year (Cowley & Geoffrey, Newsweek, April 22, 2002). Therefore, agricultural
scale production of therapeutic proteins and vaccines (especially for agents of bioterrrorism) is necessary to meet such large demand at a reasonable cost. However, this should be achieved without contaminating our food supply and harming the environment or other life forms. Agricultural production of therapeutic proteins should dramatically lower cost for consumers. There is an urgent need for oral delivery of therapeutic proteins and vaccine antigens to dramatically reduce their production, purification, storage and transportation costs and minimize complications associated with intravenous delivery. Oral delivery of therapeutic proteins via plant cells (like carrots) should lower the production cost by 70-90% compared to intravenous delivery. 2. List by year the currently approved milestones (indicators of research progress) 1. Determine if the RbcS gene can be successfully expressed from the chloroplast genome of tobacco. (FY 2002-04) 2. Engineer the chloroplast genome of
economically important crops, including Cotton and Carrot. (FY 2007-09) 3. Express vaccine antigens and other human therapeutic proteins via the tobacco chloroplast genome for purification. (FY 2003-08) 4. Modify the tobacco chloroplast genome to introduce a glycosylation pathway. (FY 2007-09) 5. Express vaccine antigens and other human therapeutic proteins via the carrot chloroplast genome for oral delivery. (FY 2006-2009) 6. Determine if RNAi can be used to specifically limit native RbcS gene expression while allowing the expression of a foreign RbcS gene. (FY 2006- 2008) 7. Determine the DNA sequence of the chloroplast genome of economically important crops. (FY 2006-2007) 8. Develop new methods to improve transgene containment. (FY 2004-2007) 9. Successfully replace the endogenous tobacco Rubisco with a completely foreign protein and maintain or actually improve photosynthesis. (FY 2007- 09) 4a List the single most significant research accomplishment during FY 2006. Successful
chloroplast expression of both genes encoding Rubisco. This accomplishment is aligned with NP302 Plant Biological and Molecular Processes, Component III Biological Processes that Improve Crop Productivity and Quality (Problem 3A: Improving and Assessing Genetic Engineering Technology). We demonstrated that the gene encoding the small subunit of Rubisco, which is normally located in the nucleus, could be expressed from the chloroplast genome to achieve near normal rates of photosynthesis. Previous research indicated that expression/assembly of Rubisco using a small subunit gene located in the chloroplast was low. Rubisco levels and photosynthesis were restored by chloroplast transformation with a small subunit gene at a highly active region for transcription, using a previously transformed (nuclear) tobacco plant in which Rubisco expression is severely reduced by the incorporation of an antisense construct against the small subunit gene family in the tobacco nuclear genome. The
research opens a potential avenue for using chloroplast engineering for the facile evaluation of foreign Rubisco genes in planta. 5. Describe the major accomplishments to date and their predicted or actual impact. This project was initiated as a result of the FY 2002 (continued for 2003- 2006) Appropriations Bill passed by Congress and signed by the President for research on "means of genetically engineering chloroplasts(s) to increase efficiency of photosynthesis as a key component of agricultural production and to reduce the spread of transgenes via pollen flow." A. Engineering the carrot chloroplast genome to confer enhanced salt tolerance. This accomplishment is aligned with NP302 Plant Biological and Molecular Processes, Component III Biological Processes that Improve Crop Productivity and Quality (Problem 3A: Improving and Assessing Genetic Engineering Technology). Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop
productivity and quality. Salt stress is a major abiotic stress in plant agriculture. The problem of soil salinity has been compounded by irrigation and excessive use of fertilizers. About 20% of the worlds irrigated lands are affected by salinity. Currently, high salinity limits crop production in 30% of the irrigated land in the United States and 20 million hectares globally. Carrot is classified as a salt sensitive plant and there is 7% growth reduction for every 10 mM increment in salinity above 20 mM salt. Therefore carrot is an ideal candidate for genetic manipulation for increased salt tolerance. We achieved high-level expression of betaine aldehyde dehydrogenase (BADH in cultured cells, roots and leaves of carrot via plastid genetic engineering. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7 fold more and accumulated 50-54 fold more betaine (93-101 mmol g-1 DW of alanine betaine and glycine 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, 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 recipient of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants. B. Stable transformation of the cotton plastid genome and maternal inheritance of transgenes achieved. This accomplishment is aligned with NP302 Plant Biological and Molecular Processes, Component III Biological Processes that Improve Crop Productivity and Quality (Problem 3A: Improving and Assessing Genetic Engineering Technology). Chloroplast genetic
engineering overcomes many concerns of gene containment, low levels of transgene expression, gene silencing, positional and pleiotropic effects or presence of vector sequences in transformed genomes. Several therapeutic proteins and agronomic traits have been highly expressed via the tobacco chloroplast genome but extending this concept to important crops has been a major challenge; lack of 100% homologous species-specific chloroplast transformation vectors containing suitable selectable markers, ability to regulate transgene expression in developing plastids and inadequate tissue culture systems via somatic embryogenesis are major challenges. We have employed a Double Barrel plastid transformation vector that harbors two selectable marker genes (aphA-6 and nptII) to detoxify the same antibiotic by two enzymes, irrespective of the type of tissues or plastids; by combining this with efficient regeneration system via somatic embryogenesis, cotton plastid transformation was achieved for
the first time. Chloroplast transgenic lines were fertile, flowered and set seeds similar to untransformed plants. Transgenes stably integrated into the cotton chloroplast genome were maternally inherited and were not transmitted via pollen when out- crossed with untransformed female plants. Cotton is one of the most important genetically modified crops ($ 120 billion U.S. annual economy). Successful transformation of the chloroplast genome should address concerns about transgene escape, insects developing resistance, inadequate insect control and promote public acceptance of genetically modified cotton. C. Sequencing of the soybean chloroplast genome accomplished. This accomplishment is aligned with NP302 Plant Biological and Molecular Processes, Component III Biological Processes that Improve Crop Productivity and Quality (Problem 3A: Improving and Assessing Genetic Engineering Technology). Lack of complete chloroplast genome sequences remains as one of the major limitations to
extending chloroplast genetic engineering technology to useful crops. Addressing this problem, the soybean chloroplast genome was sequenced and compared it to the other completely sequenced legumes, Lotus and Medicago. The chloroplast genome of soybean is 152,218 basepairs (bp) in length, including a pair of inverted repeats of 25,574 bp of identical sequence separated by a small single copy region of 17,895 bp and a large single copy region of 83,175 bp. The genome contains 111 unique genes, and 19 of these are duplicated in the inverted repeat (IR). Detailed analyses of repeated sequences indicate that many of the soybean repeats that are located in the intergenic spacer regions and introns occur in the same location in the other legumes and in Arabidopsis, suggesting that they may play some functional role. This work defines common features among Legume plastid genomes which will allow plastid transformation development across this group. D. Production of Anthrax Vaccine by
chloroplast transformation. This accomplishment is aligned with NP302 Plant Biological and Molecular Processes, Component III Biological Processes that Improve Crop Productivity and Quality (Problem 3A: Improving and Assessing Genetic Engineering Technology). The currently available human vaccine for anthrax is derived from the culture supernatant of the bacteria Bacillus anthracis. In addition to the protective antigen (PA), the vaccine contains traces of the lethal and harmful factors and these may contribute to adverse side effects associated with this vaccine. Therefore, an effective expression system that can provide a clean, safe and efficacious vaccine is required. In this work expressed PA was expressed in transgenic tobacco chloroplasts by inserting the gene into the chloroplast genome, in an effort to produce anthrax vaccine in large quantities and free of extraneous bacterial contaminants. Mature leaves grown under continuous illumination contained PA up to 14.2% of
the total soluble protein which was then purified from leaf extracts and tested for its ability to induce protective immunity in mice. An average yield of about 150 mg of PA per plant suggests that 360 million doses of a purified vaccine free of bacterial toxins could be produced from one acre of land. Such high expression levels without using fermenters, the strong immune response, and the immunoprotection offered by the chloroplast- derived PA should facilitate development of a cleaner and safer anthrax vaccine at a lower production cost. This is the first report of immunogenic and immunoprotective properties of plant-derived anthrax vaccine antigen. E. Multigene engineering of the chloroplast genome. This accomplishment is aligned with NP302 Plant Biological and Molecular Processes, Component III Biological Processes that Improve Crop Productivity and Quality (Problem 3A: Improving and Assessing Genetic Engineering Technology).Work was conducted demonstrating the feasibility of
multigene engineering via the chloroplast genome. Large amounts of foreign protein accumulation in transformed chloroplasts indicating that several genes could be introduced simultaneously and processed to make their protein products. These results show that the chloroplast posttranscriptional machinery can indeed detect and translate multigenic sequences that are not of chloroplast origin. Multigene engineering is need to introduce foreign synthetic pathways into plants. F. Cytoplasmic male sterility engineered in plants. This accomplishment is aligned with NP302 Plant Biological and Molecular Processes, Component III Biological Processes that Improve Crop Productivity and Quality (Problem 3A: Improving and Assessing Genetic Engineering Technology). Naturally occurring cytoplasmic male sterility (CMS) have been known for over 100 years. CMS systems are used to produce commercial F1 hybrid lines. Work was conducted producing the first engineered cytoplasmic male sterility system in
plants. The transgenic lines showed an accelerated pattern of anther development, affecting their maturation and resulted in aberrant tissue patterns. Reversibility of the male sterile phenotype was observed under continuous illumination, resulting in viable pollen and copious amount of seeds. This study offers a new tool for transgene containment for both nuclear and organelle genomes and provides an expedient mechanism for F1 hybrid seed production. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Because of unique advantages of the chloroplast genetic engineering technology, a new biotech company has been formed for commercial development of chloroplast transformation technology. Despite recent failures of several plant made pharmaceutical
companies and down turn in economy, this company has attracted multi-million dollar investment. The company is now conducting field trials of transgenic plants for large- scale production and purification of therapeutic proteins is in progress. Establishment of this company has already created high tech jobs in Florida, South Carolina and Missouri. In addition, this company is in the process of licensing dominant patents awarded to researchers to several major biotechnology companies. Therefore several crop plants will be genetically modified using chloroplast technology, in an environmentally friendly manner to confer much needed agronomic traits or to clean up the environment (phytoremediation of toxic metals) or produce high value biomaterials (such as biodegradable polymers). Major constrains are the cost of clinical trials and public acceptance of genetically modified crops, especially in Europe. 7. List your most important publications in the popular press and presentations to
organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Daniell, H., Lee, S.B., Grevich, J., Saski, C., Quesada-Vargas, T., Guda, C., Tomkins, J., Jansen, R.K. 2006. Complete chloroplast genome sequences of Solanum bulboscastanum, Solanum lycopersicum and comparative analyses with other Solanaceae genomes. Theoretical and Applied Genetics. 112:1503-1518. Jansen, R.K., Kaittanis, C., Saski, C., Lee, S-B., Tomkins, J., Alverson, A.J., Daniell, H. 2006. Phylogenetic analyses of Vitis (Vitaceae) based on complete chloroplast genome sequences: effects of taxon sampling and phylogenetic methods on resolving relationships among rosids. BMC Evolutionary Biology. 6:32-46. Lee S-B., Kaittanis C., Hostetler J., Town C., Jansen R.K., Daniell H. 2006. The complete chloroplast genome sequence of Gossypium hirsutum: organization and phylogenetic relationships to other angiosperms. BMC Genomics. 7: 61-77.
Impacts
(N/A)
Publications
- Dhingra, A., Portis Jr., A.R., Daniell, H. 2004. Enhanced translation of a chloroplast-expressed RbcS gene restores small subunit levels and photosynthesis in nuclear RbcS antisense plants. Proceedings of the National Academy of Sciences. 101:6315-6320.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? a) Rubisco replacement Yield increases in major US crops are beginning to plateau. One of the most evident ways to further increase plant productivity is to alter the CO2 fixing enzyme, Rubisco, to increase its activity and selectivity for CO2 versus O2. However engineering this enzyme is complicated by the location of its genes in both the nuclear and chloroplast genomes, the occurrence of posttranslational modifications of unknown significance, the involvement of molecular chaperones in its assembly, and the regulation of its activity by the activase protein, which can exhibit specificity towards its Rubisco target. Thus facile technologies need to be developed for chloroplast and possibly nuclear transformation directed specifically at allowing the replacement of the endogenous protein with foreign
or genetically engineered forms. The downstream consequences of expressing foreign or extensively modified Rubisco proteins may then also need to be addressed. b) Transgene containment Genetic engineering of plants has tremendous potential to create crops with new metabolic capabilities, such as the ability to accumulate pharmaceuticals or other high value specialty materials. In addition to the metabolic engineering itself, however, there are some obstacles to be overcome before these technologies can be commercialized. One is the potential for spread of transgenes from the engineered plants to conventional fields of the same crops, largely by dispersal of pollen. An attractive approach to the problem of gene dispersal is to genetically engineer the chloroplast genome of crop plants, which is maternally inherited and is not transmitted through pollen. Potential for gene flow also exists via transgenic seeds. Both of these could be avoided by harvesting foreign proteins from
vegetative organs (leaves) before the onset of flowers or by engineering plants that are biennial, in which vegetative organs (like carrots containing therapeutic proteins) are harvested the first year when they do not flower. As a further safeguard, engineering cytoplasmic male sterility has potential to also provide transgene containment. Because gene flow via pollen and seeds is such a potentially serious problem, a number of government and private agencies are in search of solutions that will allow transgenic corps to be developed for commercial purposes, especially when the production of pharmaceutical proteins is being considered. c) Engineer plants to survive salt stress via the chloroplast genome Salt stress is a major abiotic stress in plant agriculture. The problem of soil salinity has been compounded by irrigation and excessive use of fertilizers. About 20% of the worlds irrigated lands are affected by salinity. Currently, high salinity limits crop production in 30% of the
irrigated land in the United States and 20 million hectares globally. Carrot is classified as a salt sensitive plant and there is 7% growth reduction for every 10 mM increment in salinity above 20 mM salt. Therefore carrot is an ideal candidate for genetic manipulation for increased salt tolerance. d) Engineering the cotton chloroplast genome Cotton (Gossypium hirsutum L.) is an excellent natural source of textile fiber in the world and one of the worlds most important commercial crops. The U.S. accounts for over 40% of total world fiber production ($ 6.1 billions annual sales) and is one of leading exporter in global trade of raw cotton. Products like cotton lint and cottonseed are among the top 20 major agricultural products of the United States by value as reported by the FAO in 2003 [www.fao.org/]. The annual business revenue stimulated by cotton in the U.S. economy is about $120 billion each year, making cotton Americas number one high value crop. However, cotton is particularly
challenging to manipulate in-vitro due to the difficulties encountered in plant regeneration through somatic embryogenesis. In 2002/03 insect and herbicide resistant transgenic cotton was planted on 13% of the total area of world after soybean (63%) and corn (19%) and on 77% of the total area in the USA, modified via the nuclear genome, compared to 81% of GM soybean and 40% GM corn [http://www.ers.usda.gov]. So far, nuclear transgenic cotton is practiced only in restricted areas of the world. For instance, Upland cotton, Gossypium hirsutum, has the potential to hybridize with Hawaiian cotton, G. tomentosum, and feral populations of G. hirsutum in the Florida Keys, and of G. hirsutum / G. barbadense on the U.S. Virgin Islands and Puerto Rico. For these reasons, restrictions on field plot experimental use permits and commercial planting of Bt-cotton has been instituted in these areas. Similarly, GM cotton is now planted only in regions of the world where there are no wild relatives, in
order to avoid potential outcross with related weeds. Dispersal of pollen from transgenic cotton plants to surrounding non- transgenic plants has been reported. Umbeck et al. [J. Econ. Entomol. 84: 1943-1950] investigated pollen dispersal from transgenic cotton embedded in a field of conventional cotton in the United States and observed up to 5.7% out-crossing rates inspite of buffer rows. Transgene escape could be avoided via chloroplast genetic engineering because of maternal inheritance of transgenes in cotton. Another concern about GM crops expressing Bt toxins is that suboptimal production of toxins might result in an increased risk of pests developing Bt resistance. Nuclear engineered Bt cotton due to low expression of transgenes is not fully protected from the insect-pest attack and it needs several sprays of pesticides on crop fields to minimize the yield loss. e) Low-cost production of vaccines and biopharmaceuticals Another major problem is the high cost of
biopharmaceuticals. Globally, about 170 million people are infected with hepatitis C virus, with 3-4 million new infections each year (WHO fact sheet 164, October 2000). WHO Department of Communicable Disease Surveillance and Response reports that more than one third of world population is infected with Hepatitis B. In Asia, the prevalence of chronic hepatitis B and C is very high (about 110 million infected by HCV and 150 million infected by HBV). A large majority of Hepatitis C infected patients have severe liver cirrhosis and currently there is no vaccine available for this disease. The annual requirement of Insulin like Growth Factor I, per cirrhotic patient is 600 mg (1.5-2 mg per day) and the cost of IGF-1 per mg is $30,000. Current annual cost of interferon therapy for viral hepatitis is $26,000 per year (Cowley & Geoffrey, Newsweek, April 22, 2002). Therefore, agricultural scale production of therapeutic proteins and vaccines (especially for agents of bioterrrorism) is
necessary to meet such large demand at a reasonable cost. However, this should be achieved without contaminating our food supply and harming the environment or other life forms. Agricultural production of therapeutic proteins should dramatically lower cost for consumers. There is an urgent need for oral delivery of therapeutic proteins and vaccine antigens to dramatically reduce their production, purification, storage and transportation costs and minimize complications associated with intravenous delivery. Oral delivery of therapeutic proteins via plant cells (like carrots) should lower the production cost by 70-90% compared to intravenous delivery. 2. List the milestones (indicators of progress) from your Project Plan. 1. Determine if the RbcS gene can be successfully expressed from the chloroplast genome of tobacco. 2. Engineer the chloroplast genome of economically important crops, including Cotton, Carrot 3. Express vaccine antigens and other human therapeutic proteins via
the tobacco chloroplast genome for purification 4. Modify the tobacco chloroplast genome to introduce a glycosylation pathway 5. Express vaccine antigens and other human therapeutic proteins via the carrot chloroplast genome for oral delivery 6. Determine if RNAi can be used to specifically limit native RbcS gene expression while allowing the expression of a foreign RbcS gene. 7. Determine the DNA sequence of the chloroplast genome of economically important crops. 8. Develop new methods to improve transgene containment. 9. Successfully replace the endogenous tobacco Rubisco with a completely foreign protein and maintain or actually improve photosynthesis. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Determine if RNAi can be used to specifically limit native RbcS gene expression while allowing the expression of a foreign RbcS gene Milestone
Substantially Met 2. Express vaccine antigens and other human therapeutic proteins via the tobacco chloroplast genome for purification. Milestone Substantially Met 3. Determine the DNA sequence of the chloroplast genome of economically important crops. Milestone Substantially Met 4. Develop new methods to improve transgene containment. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Express vaccine antigens and other human therapeutic proteins via the tobacco chloroplast genome for purification and study their functionality using suitable in vitro systems and animal model systems (2006-2009. Express vaccine antigens and other human therapeutic proteins via the chloroplast genome of edible crops and study their oral delivery using animal models (2006-2009). Determine the DNA sequence of the chloroplast genome of
economically important crops (FY 2006-2009). Understand the mechanism of oral delivery of foreign proteins (GFP) expressed in plants using animal models (2006-2009). Engineer chloroplast genome of economically important crops (2006-2009). Determine if RNAi can be used to specifically limit native RbcS gene expression while allowing the expression of a foreign RbcS gene (FY2006- 2007). Successfully replace the endogenous tobacco Rubisco with a completely foreign protein and maintain or actually improve photosynthesis (FY2006-9) . 4a What was the single most significant accomplishment this past year? Naturally occurring cytoplasmic male sterility (CMS) have been known for over 100 years. CMS systems are used to produce commercial F1 hybrid lines. Dr. Daniells group reported the first engineered cytoplasmic male sterility system in plants. They studied the effect of light regulation of the phaA gene coding for kethiolase engineered via the chloroplast genome. The phaA gene was
efficiently transcribed and translated in all tissue types examined, including leaves, flowers and anthers. The transgenic lines were normal except for the male sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Transgenic lines showed an accelerated pattern of anther development, affecting their maturation and resulted in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium and vacuolation, decreased the inner space of the locules, affected pollen grain and resulted in the irregular shape or collapsed phenotype. Reversibility of the male sterile phenotype was observed under continuous illumination, resulting in viable pollen and copious amount of seeds. This study offers a new tool for transgene containment for both nuclear and organelle genomes and provides an expedient mechanism for F1 hybrid seed production. 4b List other significant accomplishments, if any. Lack of complete
chloroplast genome sequences is still one of the major limitations to extending chloroplast genetic engineering technology to useful crops. Dr. Daniellss group sequenced the soybean chloroplast genome and compared it to the other completely sequenced legumes, Lotus and Medicago. The chloroplast genome of Glycine is 152,218 basepairs (bp) in length, including a pair of inverted repeats of 25,574 bp of identical sequence separated by a small single copy region of 17,895 bp and a large single copy region of 83,175 bp. The genome contains 111 unique genes, and 19 of these are duplicated in the inverted repeat (IR). Comparisons of the Glycine, Lotus and Medicago confirm organization of legume chloroplast genomes based on previous studies. Gene content of the three legumes is nearly identical. The rpl22 gene is missing from all three legumes, and Medicago is missing rps16 and one copy of the IR. Gene order in Glycine, Lotus, and Medicago differs from the usual gene order for angiosperm
chloroplast genomes by the presence of a single, large inversion of 51 kilobases (kb). Detailed analyses of repeated sequences indicate that many of the Glycine repeats that are located in the intergenic spacer regions and introns occur in the same location in the other legumes and in Arabidopsis, suggesting that they may play some functional role. The presence of small repeats of psbA and rbcL in legumes that have lost one copy of the IR indicate that this loss has only occurred once during the evolutionary history of legumes The currently available human vaccine for anthrax is derived from the culture supernatant of Bacillus anthracis. In addition to the protective antigen (PA), the vaccine contains traces of the lethal and edema factors and these may contribute to adverse side effects associated with this vaccine. Therefore, an effective expression system that can provide a clean, safe and efficacious vaccine is required. Therefore, Dr. Daniells group expressed PA in transgenic
tobacco chloroplasts by inserting the pagA gene into the chloroplast genome, in an effort to produce anthrax vaccine in large quantities and free of extraneous bacterial contaminants. Mature leaves grown under continuous illumination contained PA up to 14. 2% of the total soluble protein. PA was purified from leaf extracts and tested for its ability to induce protective immunity in mice. Cytotoxity measurements in macrophage lysis assays showed that chloroplast-derived PA was equal in potency to PA produced in B. anthracis. An average yield of about 150 mg of PA per plant should produce 360 million doses of a purified vaccine free of bacterial toxins EF and LF from one acre of land. This yield could be further increased 18-fold by using a commercial cultivar. Such high expression levels without using fermenters, the strong immune response, and the immunoprotection offered by the chloroplast-derived PA should facilitate development of a cleaner and safer anthrax vaccine at a lower
production cost. This is the first report of immunogenic and immunoprotective properties of plant-derived anthrax vaccine antigen. Dr. Daniells group demonstrated the feasibility of multigene engineering via the chloroplast genome. Northern blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons revealed that polycistronic mRNA was the predominant transcript produced. Despite the lack of processing of such polycistrons, large amounts of foreign protein accumulation was observed in these transgenic lines, indicating abundant translation of polycistrons. These results show that the chloroplast posttranscriptional machinery can indeed detect and translate multigenic sequences that are not of chloroplast origin. In contrast to native transcripts, processed and unprocessed heterologous polycistrons were stable, even in the absence of 3 untranslated regions (UTRs). Addressing questions about polycistrons, and the sequences required for their
processing and transcript stability are essential in chloroplast metabolic engineering. Knowledge of such factors would enable engineering of foreign pathways independent of the chloroplast complex post-transcriptional regulatory machinery. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project was initiated as a result of the FY 2002 (continued for 2003- 2005) Appropriations Bill passed by Congress and signed by the President for research on "means of genetically engineering chloroplasts(s) to increase efficiency of photosynthesis as a key component of agricultural production and to reduce the spread of transgenes via pollen flow." Production of Anthrax Vaccine (vaccine tested at NIH) CDC lists Bacillus anthracis as a category A agent and estimates the cost of an anthrax attack to exceed $26 billion per 100,000 exposed individuals. Concerns regarding vaccine purity, requirement for multiple injections, and a
limited supply of the protective antigen (PA), underscore the urgent need for an improved vaccine. Therefore, the recombinant PA was produced in transgenic tobacco plants; this should eliminate any inadvertent contamination of food/feed supply. Chloroplast- derived PA bound to anthrax toxin receptor, heptamerized, and bound to lethal factor, resulting in macrophage lysis; up to 25 g functional PA/ml crude extract was observed. With an average yield of 172mg of PA per plant, 400 million doses of vaccine (free of contaminants) could be produced per acre of transgenic tobacco, using an experimental cultivar in a greenhouse, which could be further enhanced 18.17 fold using a commercial cultivar in the field. Engineering the carrot chloroplast genome to confers enhanced salt tolerance Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. Salt stress is a major abiotic stress in plant agriculture. The
problem of soil salinity has been compounded by irrigation and excessive use of fertilizers. About 20% of the worlds irrigated lands are affected by salinity. Currently, high salinity limits crop production in 30% of the irrigated land in the United States and 20 million hectares globally. Carrot is classified as a salt sensitive plant and there is 7% growth reduction for every 10 mM increment in salinity above 20 mM salt. Therefore carrot is an ideal candidate for genetic manipulation for increased salt tolerance. We achieved high-level expression of betaine aldehyde dehydrogenase in cultured cells, roots and leaves of carrot via plastid genetic engineering. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7 fold more and accumulated 50-54 fold more betaine (93-101 mmol g-1 DW of alanine betaine and glycine 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, 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 recipient of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants. Stable transformation of the cotton plastid genome and maternal inheritance of transgenes Chloroplast genetic engineering overcomes concerns of gene containment, low levels of transgene expression, gene silencing, positional and pleiotropic effects or presence of vector sequences in transformed genomes. Several therapeutic proteins and agronomic traits have been highly expressed via the tobacco chloroplast genome but extending this concept to
important crops has been a major challenge; lack of 100% homologous species-specific chloroplast transformation vectors containing suitable selectable markers, ability to regulate transgene expression in developing plastids and inadequate tissue culture systems via somatic embryogenesis are major challenges. We have employed a Double Barrel plastid transformation vector that harbors two selectable marker genes (aphA-6 and nptII) to detoxify the same antibiotic by two enzymes, irrespective of the type of tissues or plastids; by combining this with efficient regeneration system via somatic embryogenesis, cotton plastid transformation was achieved for the first time. Chloroplast transgenic lines were fertile, flowered and set seeds similar to untransformed plants. Transgenes stably integrated into the cotton chloroplast genome were maternally inherited and were not transmitted via pollen when out- crossed with untransformed female plants. Cotton is one of the most important genetically
modified crops ($ 120 billion U.S. annual economy). Successful transformation of the chloroplast genome should address concerns about transgene escape, insects developing resistance, inadequate insect control and promote public acceptance of genetically modified cotton. Successful chloroplast expression of both genes encoding Rubisco We demonstrated that the gene encoding the small subunit of Rubisco, which is normally located in the nucleus, could be expressed from the chloroplast genome to achieve near normal rates of photosynthesis. Previous research indicated that expression/assembly of Rubisco using a small subunit gene located in the chloroplast was low. The research was conducted in the laboratory of Dr. Henry Daniell, University of Central Florida, using plants, materials and advice provided by Dr. Portis, in the Photosynthesis Research Unit, Urbana, IL. Rubisco levels and photosynthesis were restored by chloroplast transformation with a small subunit gene at a highly active
region for transcription, using a previously transformed (nuclear) tobacco plant in which Rubisco expression is severely reduced by the incorporation of an antisense construct against the small subunit gene family in the tobacco nuclear genome. The research opens a potential avenue for using chloroplast engineering for the facile evaluation of foreign Rubisco genes in planta. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Because of unique advantages of the chloroplast genetic engineering technology, a new biotech company has been formed for commercial development of chloroplast transformation technology. Despite recent failures of several plant made pharmaceutical companies and down turn in economy, this company has attracted multi-million dollar
investment. The company is now conducting field trials of transgenic plants for large- scale production and purification of therapeutic proteins is in progress. Establishment of this company has already created high tech jobs in Florida, South Carolina and Missouri. In addition, this company is in the process of licensing dominant patents awarded to Daniell to several major biotechnology companies. Therefore several crop plants will be genetically modified using chloroplast technology, in an environmentally friendly manner to confer much needed agronomic traits or to clean up the environment (phytoremediation of toxic metals) or produce high value biomaterials (such as biodegradable polymers). Major constrains are the cost of clinical trials and public acceptance of genetically modified crops, especially in Europe. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed
publications below). Ruiz, O. and Daniell, H. (2005) Engineering cytoplasmic male sterility via the chloroplast genome. Plant Physiology, 138: 1232-1246, featured on the cover. Quesada-Vargas, T., Ruiz, O.N., Daniell, H. (2005) Characterization of heterologous multigene operons in transgenic chloroplasts: transcription, processing and translation. Plant Physiology, 138: 1746-1762. H. Daniell, S. Kumar and N. Duformantel (2005) Breakthrough in chloroplast genetic engineering of agronomically important crops. Trends in Biotechnology, 23: 238-245. Alpeter, F., Baisakh, N., Beachy, R., Bock, R., Capell, T., Christou, P., Daniell, H. et al. (2005) Particle bombardment and genetic enhancement of crops: myths and realities. Molecular Breeding, 15: 305-327. Grevich, J. and Daniell, H. (2005) Chloroplast genetic engineering: Recent advances and perspectives. Critical Reviews in Plant Sciences, 24: 1-25. H. Daniell, S. Chebolu, S. Kumar, M. Singleton, R. Falconer (2005) Chloroplast- derived
Vaccine antigens and other Therapeutic proteins. Vaccine, 23: 1779-1783.
Impacts
(N/A)
Publications
- Dhingra, A., Portis Jr., A.R., Daniell, H. 2004. Enhanced translation of a chloroplast-expressed RbcS gene restores small subunit levels and photosynthesis in nuclear RbcS antisense plants. Proceedings of the National Academy of Sciences. 101:6315-6320.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? a) Rubisco replacement Yield increases in major US crops are beginning to plateau. One of the most evident ways to further increase plant productivity is to alter the CO2 fixing enzyme, Rubisco, to increase its activity and selectivity for CO2 versus O2. However engineering this enzyme is complicated by the location of its genes in both the nuclear and chloroplast genomes, the occurrence of posttranslational modifications of unknown significance, the involvement of molecular chaperones in its assembly, and the regulation of its activity by the activase protein, which can exhibit specificity towards its Rubisco target. Thus facile technologies need to be developed for chloroplast and possibly nuclear transformation directed specifically at allowing the replacement of the endogenous protein with foreign
or genetically engineered forms. The downstream consequences of expressing foreign or extensively modified Rubisco proteins may then also need to be addressed. b) Transgene containment Genetic engineering of plants has tremendous potential to create crops with new metabolic capabilities, such as the ability to accumulate pharmaceuticals or other high value specialty materials. In addition to the metabolic engineering itself, however, there are some obstacles to be overcome before these technologies can be commercialized. One is the potential for spread of transgenes from the engineered plants to conventional fields of the same crops, largely by dispersal of pollen. An attractive approach to the problem of gene dispersal is to genetically engineer the chloroplast genome of crop plants, which is maternally inherited and is not transmitted through pollen. Potential for gene flow also exists via transgenic seeds. Both of these could be avoided by harvesting foreign proteins from
vegetative organs (leaves) before the onset of flowers or by engineering plants that are biennial, in which vegetative organs (like carrots containing therapeutic proteins) are harvested the first year when they do not flower. Because gene flow via pollen and seeds is potentially a serious problem, a number of government and private agencies are in search of solutions that will allow transgenic crops to be developed for commercial purposes, especially when the production of pharmaceutical proteins is being considered. c) Engineer plants to survive salt stress via the chloroplast genome Salt stress is a major abiotic stress in plant agriculture. The problem of soil salinity has been compounded by irrigation and excessive use of fertilizers. About 20% of the world's irrigated lands are affected by salinity. Currently, high salinity limits crop production in 30% of the irrigated land in the United States and 20 million hectares globally. Carrot is classified as a salt sensitive plant and
there is 7% growth reduction for every 10 mM increment in salinity above 20 mM salt. Therefore carrot is an ideal candidate for genetic manipulation for increased salt tolerance. d) Engineering the cotton chloroplast genome Cotton (Gossypium hirsutum L.) is an excellent natural source of textile fiber in the world and one of the world's most important commercial crops. The U.S. accounts for over 40% of total world fiber production ($ 6.1 billions annual sales) and is one of leading exporter in global trade of raw cotton. Products like cotton lint and cottonseed are among the top 20 major agricultural products of the United States by value as reported by the FAO in 2003 [www.fao.org/]. The annual business revenue stimulated by cotton in the U.S. economy is about $120 billion each year, making cotton America's number one high value crop. However, cotton is particularly challenging to manipulate in-vitro due to the difficulties encountered in plant regeneration through somatic
embryogenesis. In 2002/03 insect and herbicide resistant transgenic cotton was planted on 13% of the total area of world after soybean (63%) and corn (19%) and on 77% of the total area in the USA, modified via the nuclear genome, compared to 81% of GM soybean and 40% GM corn [http://www.ers.usda.gov]. So far, nuclear transgenic cotton is practiced only in restricted areas of the world. For instance, Upland cotton, Gossypium hirsutum, has the potential to hybridize with Hawaiian cotton, G. tomentosum, and feral populations of G. hirsutum in the Florida Keys, and of G. hirsutum / G. barbadense on the U.S. Virgin Islands and Puerto Rico. For these reasons, restrictions on field plot experimental use permits and commercial planting of Bt-cotton has been instituted in these areas. Similarly, GM cotton is now planted only in regions of the world where there are no wild relatives, in order to avoid potential outcross with related weeds. Dispersal of pollen from transgenic cotton plants to
surrounding non- transgenic plants has been reported. Umbeck et al. [J. Econ. Entomol. 84: 1943-1950] investigated pollen dispersal from transgenic cotton embedded in a field of conventional cotton in the United States and observed up to 5.7% out-crossing rates inspite of buffer rows. Transgene escape could be avoided via chloroplast genetic engineering because of maternal inheritance of transgenes in cotton. Another concern about GM crops expressing Bt toxins is that suboptimal production of toxins might result in an increased risk of pests developing Bt resistance. Nuclear engineered Bt cotton due to low expression of transgenes is not fully protected from the insect-pest attack and it needs several sprays of pesticides on crop fields to minimize the yield loss. e) Low-cost production of vaccines and biopharmaceuticals Another major problem is the high cost of biopharmaceuticals. Globally, about 170 million people are infected with hepatitis C virus, with 3-4 million new infections
each year (WHO fact sheet 164, October 2000). WHO Department of Communicable Disease Surveillance and Response reports that more than one third of world population is infected with Hepatitis B. In Asia, the prevalence of chronic hepatitis B and C is very high (about 110 million infected by HCV and 150 million infected by HBV). A large majority of Hepatitis C infected patients have severe liver cirrhosis and currently there is no vaccine available for this disease. The annual requirement of Insulin like Growth Factor I, per cirrhotic patient is 600 mg (1.5-2 mg per day) and the cost of IGF-1 per mg is $30,000. Current annual cost of interferon therapy for viral hepatitis is $26,000 per year (Cowley & Geoffrey, Newsweek, April 22, 2002). Therefore, agricultural scale production of therapeutic proteins and vaccines (especially for agents of bioterrrorism) is necessary to meet such large demand at a reasonable cost. However, this should be achieved without contaminating our food
supply and harming the environment or other life forms. Agricultural production of therapeutic proteins should dramatically lower cost for consumers. There is an urgent need for oral delivery of therapeutic proteins and vaccine antigens to dramatically reduce their production, purification, storage and transportation costs and minimize complications associated with intravenous delivery. Oral delivery of therapeutic proteins via plant cells (like carrots) should lower the production cost by 70-90% compared to intravenous delivery. 2. List the milestones (indicators of progress) from your Project Plan. 1. Determine if the RbcS gene can be successfully expressed from the chloroplast genome of tobacco. 2. Engineer the chloroplast genome of economically important crops, including Cotton, Carrot 3. Express vaccine antigens and other human therapeutic proteins via the tobacco chloroplast genome for purification 4. Modify the tobacco chloroplast genome to introduce a glycosylation
pathway 5. Express vaccine antigens and other human therapeutic proteins via the carrot chloroplast genome for oral delivery 6. Determine if RNAi can be used to specifically limit native RbcS gene expression while allowing the expression of a foreign RbcS gene. 7. Determine the DNA sequence of the chloroplast genome of economically important crops. 8. Develop new methods to improve transgene containment. 9. Successfully replace the endogenous tobacco Rubisco with a completely foreign protein and maintain or actually improve photosynthesis. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and lyour plans to do so. 1. Determine if the RbcS gene can be successfully expressed from the chloroplast genome of tobacco. 2. Engineer the chloroplast genome of economically important crops, including
Cotton, Carrot. 3. Express vaccine antigens and other human therapeutic proteins via the tobacco chloroplast genome for purification. All listed milestone for FY 2004 were substantially accomplished. B. List the milestone (from the list in Question 2) that you expect to address over the next 3 years (2005, 2006, 2007)? What do you expect to accomplish, year by year, over the next 3 years under each milestone? 3. Express vaccine antigens and other human therapeutic proteins via the tobacco chloroplast genome for purification (FY 2005-2007) 4. Modify the tobacco chloroplast genome to introduce a glycosylation pathway (FY 2005-2006) 5. Express vaccine antigens and other human therapeutic proteins via the carrot chloroplast genome for oral delivery (FY 2005-2007) 6. Determine if RNAi can be used to specifically limit native RbcS gene expression while allowing the expression of a foreign RbcS gene (FY2005- 2006) 7. Determine the DNA sequence of the chloroplast genome of
economically important crops (FY 2004-2008). 8. Develop new methods to improve transgene containment (FY 2004-2007) 9. Successfully replace the endogenous tobacco Rubisco with a completely foreign protein and maintain or actually improve photosynthesis (FY2006-7) . The residual native small subunit gene expression using the current antisense technology may hinder successful expression/assembly when foreign genes are introduced into the chloroplast genome and a completely successful Rubisco replacement may require maintenance of the current gene locations. We will determine if RNAi technology has the potential to specifically and completely suppress gene expression from the native small subunit gene family, allowing a foreign small unit gene to be located in either the chloroplast or nuclear genome. The first goal of developing chloroplast genetic engineering in carrot plants to facilitate oral delivery of vaccines and therapeutic proteins has been achieved. This will be followed by
production of vaccines for oral delivery in carrots, for infectious diseases and agents of bioterrrorism, including Anthrax, Plague and Amoebiasis. Oral delivery is expected to reduce the cost of therapeutic proteins by 70-90%. In addition, the tobacco chloroplast genome will be modified to include glycosylation pathways to facilitate expression of several human therapeutic proteins, including antiviral proteins and monoclonal antibodies. In order for chloroplast genetic engineering technology to be extended to several major crops, the chloroplast genomes will be sequenced. New and novel method to contain transgenes including cytoplasmic male sterility will be developed. Utilizing knowledge gained from the RNAi studies and introduction of a foreign, but closely related, large and small gene set into the small subunit antisense tobacco plants, attempts will be made to replace the endogenous protein with a foreign protein possessing kinetic characteristics that are expected to result
in increased photosynthetic potential. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment During FY 2004: We demonstrated that the gene encoding the small subunit of Rubisco, which is normally located in the nucleus, could be expressed from the chloroplast genome to achieve near normal rates of photosynthesis. Previous research indicated that expression/assembly of Rubisco using a small subunit gene located in the chloroplast was low. The research was conducted in the laboratory of Dr. Henry Daniell, University of Central Florida, using plants, materials and advice provided by Dr. Portis, in the Photosynthesis Research Unit, Urbana, IL. Rubisco levels and photosynthesis were restored by chloroplast transformation with a small subunit gene at a highly active region for transcription, using a previously transformed (nuclear) tobacco plant in which Rubisco expression is severely reduced by the incorporation of an antisense
construct against the small subunit gene family in the tobacco nuclear genome. The research opens a potential avenue for using chloroplast engineering for the facile evaluation of foreign Rubisco genes in planta. B. Other Significant Accomplishment(s), if any: Dr. Daniell's group has successfully modified the carrot chloroplast genome using the BADH gene to confer very high levels of salt tolerance (up to 400 mM). Transgenic plants were shown to accumulate betaine (osmoprotection agent) as high as in halophytes. Also, high levels of foreign gene expression were achieved in the edible part (carrot roots). The accomplishment could facilitate using chloroplast transformation of this species for oral delivery of vaccines and other therapeutic proteins. Dr. Daniell's group has successfully modified the cotton chloroplast genome using novel chloroplast vector designs and demonstrated maternal inheritance patterns of the foreign genes. Chloroplast transformation of cotton, a major US crop
species, had not been reported previously. Successful transformation of the chloroplast genome should address concerns about transgene escape and inadequate insect control in this species and thereby promote public acceptance of genetically modified cotton. Dr. Daniell's group has expressed several human therapeutic proteins in transgenic plants, including vaccines and pharmaceuticals. High-level expression of these proteins in the chloroplast has potential to significantly lower their production cost. A new biotechnology company (Chlorogen Inc.) has been formed based on this technology and this has attracted multi-million dollar investment. See below for more details. C. Significant Activities that Support Special Target Populations None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project was initiated as a result of the FY 2002 (continued for 2003 & 2004) Appropriations Bill passed by Congress and signed by
the President for research on "means of genetically engineering chloroplasts(s) to increase efficiency of photosynthesis as a key component of agricultural production and to reduce the spread of transgenes via pollen flow." Production of Anthrax Vaccine (vaccine tested at NIH) CDC lists Bacillus anthracis as a category A agent and estimates the cost of an anthrax attack to exceed $26 billion per 100,000 exposed individuals. Concerns regarding vaccine purity, requirement for multiple injections, and a limited supply of the protective antigen (PA), underscore the urgent need for an improved vaccine. Therefore, the recombinant PA was produced in transgenic tobacco plants; this should eliminate any inadvertent contamination of food/feed supply. Chloroplast- derived PA bound to anthrax toxin receptor, heptamerized, and bound to lethal factor, resulting in macrophage lysis; up to 25 mg functional PA/ml crude extract was observed. With an average yield of 172mg of PA per plant, 400 million
doses of vaccine (free of contaminants) could be produced per acre of transgenic tobacco, using an experimental cultivar in a greenhouse, which could be further enhanced 18.17 fold using a commercial cultivar in the field. Engineering the carrot chloroplast genome to confers enhanced salt tolerance Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. Salt stress is a major abiotic stress in plant agriculture. The problem of soil salinity has been compounded by irrigation and excessive use of fertilizers. About 20% of the world's irrigated lands are affected by salinity. Currently, high salinity limits crop production in 30% of the irrigated land in the United States and 20 million hectares globally. Carrot is classified as a salt sensitive plant and there is 7% growth reduction for every 10 mM increment in salinity above 20 mM salt. Therefore carrot is an ideal candidate for genetic manipulation for
increased salt tolerance. We achieved high-level expression of betaine aldehyde dehydrogenase in cultured cells, roots and leaves of carrot via plastid genetic engineering. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7 fold more and accumulated 50-54 fold more betaine (93-101 mmol g-1 DW of ss-alanine betaine and glycine 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, 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 recipient of foreign DNA for the first time overcomes two of the major obstacles in
extending this technology to important crop plants. Stable transformation of the cotton plastid genome and maternal inheritance of transgenes Chloroplast genetic engineering overcomes concerns of gene containment, low levels of transgene expression, gene silencing, positional and pleiotropic effects or presence of vector sequences in transformed genomes. Several therapeutic proteins and agronomic traits have been highly expressed via the tobacco chloroplast genome but extending this concept to important crops has been a major challenge; lack of 100% homologous species-specific chloroplast transformation vectors containing suitable selectable markers, ability to regulate transgene expression in developing plastids and inadequate tissue culture systems via somatic embryogenesis are major challenges. We have employed a 'Double Barrel' plastid transformation vector that harbors two selectable marker genes (aphA-6 and nptII) to detoxify the same antibiotic by two enzymes, irrespective of
the type of tissues or plastids; by combining this with efficient regeneration system via somatic embryogenesis, cotton plastid transformation was achieved for the first time. Chloroplast transgenic lines were fertile, flowered and set seeds similar to untransformed plants. Transgenes stably integrated into the cotton chloroplast genome were maternally inherited and were not transmitted via pollen when out- crossed with untransformed female plants. Cotton is one of the most important genetically modified crops ($ 120 billion U.S. annual economy). Successful transformation of the chloroplast genome should address concerns about transgene escape, insects developing resistance, inadequate insect control and promote public acceptance of genetically modified cotton. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if
known, to the adoption and durability of the technology products? Because of unique advantages of the chloroplast genetic engineering technology, a new biotech company (Chlorogen Inc.) has been formed for commercial development of chloroplast transformation technology. Despite recent failures of several plant made pharmaceutical companies and down turn in economy, Chlorogen Inc (www.chlorogen.com) has attracted multi- million dollar investment. Chlorogen Inc. is now conducting field trials of transgenic plants for large-scale production and purification of therapeutic proteins is in progress. Establishment of Chlorogen Inc. has already created high tech jobs in Florida, South Carolina and Missouri. In addition, Chlorogen is in the process of licensing dominant patents awarded to Daniell to several major biotechnology companies. Therefore several crop plants will be genetically modified using chloroplast technology, in an environmentally friendly manner to confer much needed
agronomic traits or to clean up the environment (phytoremediation of toxic metals) or produce high value biomaterials (such as biodegradable polymers). Major constrains are the cost of clinical trials and public acceptance of genetically modified crops, especially in Europe. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Research by the group of collaborators (Dr. Daniell) was selected by General Electric in a world-wide search for cutting edge technologies on the future of medicine, displayed at WIRED NEXTFEST, San Francisco for an audience of 18,000, including 4000 high school students in San Francisco, May 2004. Daniell, H. 2004. Medical Molecular Pharming. In: Goodman, R.M. Encyclopedia of Plant and Crop Science. New York. Marcel Dekker. p. 705- 710. Molina, A., Hervas-Stubbs, S., Daniell, H., Mingo-Castel, A., Veramendi, J. 2004. High-yield expression of a viral peptide animal vaccine in
transgenic tobacco chloroplasts. Plant Biotechnology Journal. 2(2):141- 153. Daniell, H., McFadden, B.A. 2004. Chloroplast genetic engineering. U.S. Patent 6,680,426. Daniell, H., Chase, C.D. 2004. Introduction to Molecular Biology & Biotechnology of Chloroplasts and Mitochondria. In: Daniell, H., Chase, C. D., editors. Molecular Biology and Biotechnology of Plant Organelles. The Netherlands: Kluwer Academic Publishers. p. 1-12. Kumar, S., Daniell, H. 2004. Chloroplast genetic engineering for medical molecular farming. Methods in Molecular Biology. 267:365-384. Dhingra, A., Daniell, H. 2004. Engineering Herbicide Resistance Pathways in Plastids. In: Daniell, H. and Chase, C.D., editors. Molecular Biology and Biotechnology of Plant Organelles. The Netherlands: Kluwer Academic Publishers. p. 485-506. Daniell, H., Carmona-Sanchez, O., Burns, B. 2004. Chloroplast derived antibodies, biopharmaceuticals and edible vaccines. In: Schillberg, S., editor. Molecular Farming. Germany: Wiley VCH
Verlag. p. 113-133. Daniell, H., Cohill, P.R., Kumar, S., Dufourmantel, N., Dubald, M. 2004. Chloroplast genetic engineering. In: Daniell, H. and Chase, C.D., editors. Molecular Biology and Biotechnology of Plant Organelles. The Netherlands: Kluwer Academic Publishers. p. 437-484. Daniell, H., Dhingra, A., Ruiz, O.N. 2004. Chloroplast genetic engineering to confer desired plant traits. Methods in Molecular Biology. 286:111-137 Devine, A., Daniell, H. 2004. Chloroplast Genetic Engineering in Plastids. In: Moller, S., editor. United Kingdom: Blackwell Publishers. p. 283-320. Daniell, H., Parkinson, C. 2003. Jumping genes and containment. Nature Biotechnology. 21: 374-375. Daniell, H. 2003. Medical Molecular Pharming: Expression of antibodies, biopharmaceuticals and edible vaccines in plants. In: Vasil, I.K., editor. Plant Biotechnology 2002 and Beyond. London: Kluwer Academic Publishers. p. 371-388. Lee, S.B., Byun, M.O., Daniell, H. 2003. Accumulation of trehalose within transgenic
chloroplasts confers drought tolerance. Molecular Breeding. 11(1):1-13. Fernandez-San Millan, Mingeo-Castel, A., Miller, M., Daniell, H. 2003. A chloroplast transgenic approach to hyper-express and purify Human Serum Albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnology Journal. 1(2):71-79. Ruiz, O.N., Hussein, H., Terry, N., Daniell, H. 2003. Phytoremediation of organomercurial compounds via chloroplast genetic engineering. Plant Physiology. 132:1344-1352. Daniell, H., Khan, M.S. 2003. Engineering the chloroplast genome for biotechnology applications. In: Steward, N., editor. Transgenic Plant: Current Innovations and Future Trends. United Kingdom: Horizon Scientific Press. p. 83-111. Daniell, H., McFadden, B.A. 2003. Chloroplast genetic engineering. U.S. Patent 6,642,053.
Impacts
(N/A)
Publications
- Dhingra, A., Portis Jr, A.R., Daniell, H. 2004. Enhanced translation of a cholorplast expressed Rbcs gene restores SSU levels and photosynthesis in nuclear antisense Rbcs plants [abstract]. American Society of Plant Biologists Annual Meeting. Available: http://abstracts.aspb. org/pb2004/public/P40/7109.html.
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