Progress 09/01/07 to 08/31/09
Outputs
OUTPUTS: Our research partner was Cornell University, as sub grantee. During Phase I and early Phase II, we worked in collaboration with the laboratories of Drs. Maureen Hanson and Beth Ahner, also of Cornell University, whose work was focused on developing more productive transformants, and who supplied us the transformed plant materials. Shortly after the start of Phase II, they succeeded in creating a chloroplast transformant of Samsun tobacco designated Cel6A, expressing and accumulating high levels of a cellulase enzyme. It was this transformant and its untransformed parent that we used as example crops in Phase II. We believe that this sharing of research products was mutually beneficial, both as to research techniques and to specific plant products. Our outputs include data and knowledge gained by working with the research materials. The data has been shared with the Cornell CEA researchers, staff and students and is expected to impact ongoing and future plant research, particularly in controlled system environments such as growth chambers and greenhouses. The researchers attended conferences and shared the general research subject matter with other conference attendees. PARTICIPANTS: Dr. Louis Albright, as principal investigator led the team of researchers and worked closely with the sub grantee, Cornell University. Additional researchers include Melissa Brechner, post doc associate; David de Villiers, PhD, Research Associate, Phase II PI for Cornell, and lead researcher for the sub grantee; Timothy J. Shelford, PhD, Growth chamber set up, and control program programmer; Colin Meeks, BSc, Chief Research Grower; Beth A. Ahner, Professor, Genetic Transformation Specialist, Cornell University; Ben Gray, PhD Grad Student, Developer of Cel6A; Huijun Yang, PhD, Post-doc, Developer of Interleukin transformant, tissue analysis; Maureen Hanson, Liberty Hyde Bailey Professor, Molecular Geneticist, Cornell University and Long Xi, PhD, Post-Doc. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Tobacco is a major field crop, but a new greenhouse crop. The opportunities for enhancing productivity in controlled environments such as greenhouses, growth chambers and plant factories, are far greater than exist in the field. Plants can be re-spaced, irrigation and fertilization can be precisely managed, temperature can be continuously controlled, and supplementary light and CO2 can be supplied as needed. However, until the advent of bio-molecular farming, no one had reason to optimize greenhouse production of tobacco. In Phase I of this SBIR project, we explored the effect of temperature and daily light integral on expression of green fluorescent protein (GFP) in two different genetic lines of tobacco that had been transformed to include the GFP gene either in the nuclear or the chloroplast genome. We found evidence that these environmental factors affected biomass production and target enzyme as a proportion of total soluble protein (TSP) in different ways in the nuclear transformants, so that optimization of production of the target chemical might not be simply a matter of maximizing biomass productivity. We found the chloroplast transformant to be far superior to the nuclear transformants in terms of enzyme tissue content as percent of TSP; in the case of the chloroplast transformant, different light integral levels did not affect percent TSP.
Publications
- No publications reported this period
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: The stated intent in the proposal for funding of the Phase II was to use chloroplast-transformed tobacco cultivars expressing cellulase as model crops with which to determine how to produce pharmaceuticals in controlled environments (CEs) most cost effectively. It was thought unlikely that cellulase enzymes would be a major target chemical for commercial CE production, but it was expected knowledge of the factors affecting the performance of chloroplast transformants expressing cellulase would generalize to transformants expressing other chemicals requiring/ benefiting from greenhouse containment/production, that would be commercially viable. A second objective of our Phase II proposal was to identify a commercially promising target protein (TP) and transform a suitable vehicle plant (not necessarily tobacco) for its commercial production in Phase III and beyond. (Note: a small profitable industry may be possible for production of specialty types of cellulase in tobacco in CEs, in small quantities for experimental purposes.) Our Phase I research showed that a target protein, green fluorescent protein (GFP), was recoverable in much higher amounts in chloroplast than in nuclear transformants, as expected, and also that a number of environmental and cultural factors strongly influence both overall biomass production, and percentage of target protein (in this case GFP) found in total soluble protein and thus in the tissue in general. But levels of environmental parameters optimal for biomass production did not necessarily result in highest content of TP in the tissue, suggesting some subtlety of approach is called for in optimizing productivity of TP. Our research showed that GFP increased in leaves as they got older, up to a point, and suggested that the balance between production, accumulation, and degradation of TP in the leaf was important. Plant age at time of harvest was also shown to be a dominant factor in TP productivity on a whole-plant basis, corroborating the individual leaf analysis. In our Phase II application we proposed a systematic evaluation of the effects of temperature and various light parameters in the aerial environment, and temperature in the root environment, on absolute productivity of target protein (i.e. g m-2 d-1 of cellulase). In practice, we will determine fresh and dry mass productivity, total soluble protein productivity, and target protein as a percentage of total soluble protein, separately, and compute target protein productivity from these data. We will develop these data longitudinally in order to determine optimal timing of harvest. In the above work the whole plant will be sampled. We also proposed a subset of experiments in which each leaf of the plant will be systematically analyzed, and this done longitudinally, to better understand overall plant TP productivity. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
With the pilot studies completed satisfactorily, and decisions made on how concretely to proceed, the formal experiments are about to begin, and will commence during the latter part of September 2008 with seeding of the first rep of the photosynthetic period experiment. Student help has been enlisted for day-to-day management of the experiments. Thereafter experiments will proceed continuously until the work is done. Tissue analysis will commence at the beginning of 2009, working off a backlog of stored tissue. At present no obstacles to successful completion of the work are foreseen, although there may be a small time overrun. We will consider and timely request an extension, if appropriate.
Publications
- No publications reported this period
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