SBIR-STTR Award

Although laboratory-scale advances in molecular and genetic engineering bring the promise of one day utilizing plants to produce human therapeutic proteins (such as insulin) there is a concomitant requirement for technological advances in plant biomass production that will convey the future products of plant molecular engineering to the marketplace in a safe, secure, reliable and economically feasible manner. The proposed work will determine the feasibility of combining a) existing state of the art controlled environment agriculture (CEA) techniques to produce large quantities of biomass with b) optimization of the environmental growth parameters to increase yield and uniformity of a specific protein in a transgenic plant, namely Green Fluorescent Protein from transgenic tobacco as a model system. OBJECTIVES: The Phase I project technical objective is to determine whether CEA environmental control parameters can be optimized to affect production of both biomass AND transgenic protein in a model system, namely, the expression of Green Fluorescent Protein (GFP) in transgenic tobacco plants. GFP is a suitable protein because amounts can be readily quantified by Western blots using commercially available antibodies and commercially available purified GFP as a standard. Amounts of properly folded fluorescent GFP can also be determined by fluorometric assay. We have chosen tobacco expressing GFP for two reasons. First, at present tobacco is the only plant species in which chloroplast genome transformation can be performed routinely. There is considerable interest in expressing transgenes from the chloroplast genome because of the high yields of protein that are possible (Maliga, 2003; Heifetz, 2001). The chloroplast genomes of a few other species, such as potato and tomato, have been transformed, but often only by much more effort and time than is required for tobacco transformation. Thus, optimizing tobacco CEA for transgenic plant production will be applicable to production of other valuable proteins. Second, seeds of tobacco transgenic lines containing GFP expressed from the chloroplast or from the nuclear genome are available from a local collaborator, Dr. Maureen Hanson (Molecular Biology and Genetics, Cornell). Western blot assays will be performed in the laboratories of Dr. Sushen Gan and Dr. Larry Walker. Given the short time-frame of this project-8 months-we think it is essential to use materials that are immediately available. APPROACH: The 4 primary factors for investigation are air temperature, solution temperature, light and nutrient solution concentration. For the Phase I research, these factors will first be studied in environmentally controlled growth chambers so that multiple tests can be run simultaneously. Plant biomass and GFP protein production will be quantified. Light will be supplied by standard cool white fluorescent lamps. Seeded trays will be moved to a 24 oC growth chamber. Water and nutrient solution will be supplied by ebb-and-flood benches. This will be modeled after our existing system for our lettuce production. We will develop an early growth curve for the crop. When seedlings begin to shade one another, re-spacing in needed. However root development is also a critical factor in determining the timing of re-spacing. Seedlings must be re-spaced before roots become entangled, leading to damage and transplant. Root and shoot development will be closely monitored to determine the optimal time for re-spacing. It is critical to determine the best time to harvest the crop. For our purposes, the best time to harvest would be indicated when and if the GFP production curve peaks. This may be determined by developmental stage, or through a controlled-input environmental stress. However, other inputs (such as space) may become limiting and be a factor in the optimal harvest time. Comparison of GFP protein production with existing (non-CEA) greenhouse data. After completion of the above growth-chamber analyses, the optimal conditions for GFP production would be combined in a CEA prototype greenhouse test. Optimal temperature, light intensity, and nutrient solution nitrate content will be provided to the test plants to determine: Determination of GFP Content We will use two Petit Havana tobacco transgenic lines. Tobacco line 35S-ER-GFP is a nuclear transformant that contains a fluorescence-enhanced GFP coding region whose expression is controlled by the 35S promoter and that carries an ER targeting signal, which has been shown to enhance accumulation of the GFP (Haseloff et al., 1997). Seedlings of the homoplasmic Cp-Prrn-GFP line grown in the greenhouse were found to contain GFP at 5% of the total soluble protein, as quantified by Western blots comparing the transgenic plant protein to purified GFP (Reed et al., 2001). We will assay GFP from plants grown under different regimes to determine the optimum conditions for yield of the transgenic proteins, using Western blots. We will also assay the level of GFP that is properly folded to give green fluorescence in a spectrofluorimeter. Some containment practices to be observed are, for example, access to the research area will be restricted to required persons only. During the Phase I research, secondary containment will be required for movement of materials into/out of the growth chamber/greenhouse areas. At the end of experiments, un-used biologically active experimental plant tissues and equipment will be autoclaved or decontaminated. Protective clothing will be worn to minimize dissemination and hands will be washed before leaving the facility

Genomic research has identified many proteins encoded by DNA sequences, ranging from industrial enzymes to pharmaceuticals to nanostructures valuable to industry and medicine. Plants produce proteins at lower cost and with flexibility of scale, options often lacking in mammalian cell cultures and microbial fermentation. Plant cells have more chloroplasts than nuclei, making chloroplast (plastid) transformants more productive. Additionally, plastids are also present in roots, possibly increasing yield (and they are harvestable only in hydroponic systems). Plant environments influence biomass and protein production, and specific protein production can be guided using refined, accurate environment controls. Hydroponics permits separate and independent root and shoot environment control. Our purpose in this two year effort is to use a tobacco transformant and a representative protein (cellulase) to define how root and aerial temperatures and daily light integrals influence biomass and protein production, and the potential for changing the relative expression of the protein of interest. Concomitantly, transformants containing a pharmaceutical gene will be developed so a combination of more valuable protein and optimized plant production system will be ready for commercialization in tandem. OBJECTIVES: The primary goal of the experimental efforts of Phase 2 is to develop tobacco as a vehicle for future production of target proteins (TPs) of various kinds in controlled environments. This work will move us closer to prototype-scale commercial production of a pharmaceutical TP and beyond in Phase 3. In light of our Phase 1 market research, we no longer anticipate large-scale production of industrial-quality cellulase taking place in the greenhouse, because it can be more economically produced in other ways, but the cellulase transformants we have prepared will continue to serve well as example TPs. Laboratory-quality production in a greenhouse remains a potentially viable opportunity. We are investing our efforts in tobacco as a vehicle crop because, for the time being, it is the species in which chloroplast transformants are most easily created. We are primarily interested in chloroplast-transformants because target protein expression in this type of transformant is typically several times greater than in nuclear transformants. As part of Phase 1, we leveraged the Phase 1 USDA funding to obtain additional funding to prepare chloroplast transforms expressing cellulase for use in Phase 2. For these transformants we used Samsun, an excellent cultivar used in Phase 1, and a new nicotine-free line designated 22X. During Phase 2 we intend to produce transformants containing a pharmaceutical product (to be selected as part of the Phase 2 effort) for use in Phase 3. In the long-run, the best commercial prospects for CEA production of transgenic products will be those products that require containment and cannot be grown in the field where production costs typically would be less. Pharmaceuticals fall into this category. To find optimal environmental set points and cultural methods to produce target proteins (TPs) in a greenhouse at minimum production cost, we will need to repeat the same process needed to perfect production of any commercial greenhouse crop. However, in this case and because of the high value of the TP, we should include consideration of more expensive inputs than usual, and more elaborate cultural techniques if they result in enhanced expression or accumulation of the transgene products. Finding optimal environmental set points in controlled environments is a challenge because, in contrast to field production, many environmental parameters can be controlled and there is a myriad of options. Not every variable that can be manipulated should, to be practical, be systematically varied with every other variable; the number of combinations is too great. Our focus in the planned experiments will be long-term environmental set-points. A final objective for Phase 2 is to demonstrate optimized continuous production of transgenic biomass in a realistic small-scale production system, using the results of our research. APPROACH: In the growth chamber research, we intend to perform three main experiments, each of which will be replicated three times. Repeats in time will provide replication and also allow counterbalancing for chamber effects. The first set of experiments will have three levels of air temperature (provided by chambers) and three levels of root temperature (provided by production systems within chambers). Locations of production systems within chambers will be randomized. When all three repeats are complete we will conduct a 3-way ANOVA, with chamber as a blocking factor, and air temperature and root temperature as the independent variables, each with three levels. The second set of experiments will have three levels of daily light integral in a fixed photosynthetic period of 18 hours, with three levels of root temperature and the analysis will be similar. The final set of experiments will have three levels of photosynthetic period with a fixed daily light integral. Depending on the outcome of the earlier sets of experiments, instead of root temperature being examined, additional cultivars may be included. The greenhouse research program is intended to follow the chamber research and confirm the findings in the somewhat different conditions of the greenhouse. The experiments will focus on the two levels of the main independent variables shown to be of greatest interest (air temperature, daily light integral and photosynthetic period), and each experiment will be repeated twice. Three root temperatures will be available. To simultaneously fix photosynthetic period and daily light integral, done in the second set of chamber experiments, will be more difficult, but can be approximated reasonably well with the use of supplemental lighting, shade cloth and occasional manual intervention. To achieve these objectives we will establish experimental hydroponic plant production systems in three walk-in growth chambers, and two greenhouse sections. Each location will have three hydroponic production systems, and all production systems will be completely independent of one another in the root zone so root temperature can be varied as required. In the root zone there will be a positive circulation system and continuous aeration; root zone temperature will be computer controlled and continuously logged. Temperature will be adjusted up through use of submersed heaters, and lowered through use of a cold finger with circulated water chilled external to the units. Aerial temperatures and light intensities will be continuously logged in both greenhouse and growth chambers. Throughout all experiments, elemental analysis of the nutrient solution will be performed every two weeks, and appropriate corrections made to restore original concentrations of all ions. A fourth growth chamber will be equipped with an ebb-and-flood bench on which seedlings will be produced for all experiments until they reach transplant size. The temperature of the reservoir of nutrient solution in the ebb-and-flood bench will be controlled and continuously logged, as will aerial temperature and light level.