Grantee Research Project Results
2007 Progress Report: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
EPA Grant Number: R829479Center: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Center Director: Schumacher, Dorin
Title: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Investigators: Schumacher, Dorin , Sadowsky, Michael J. , Wackett, Lawrence P. , Samac, Deborah A. , Vance, Carroll P. , Cheng, Zong-Ming , Weeks, Donald P. , Pantalone, Vincent R , Doty, Sharon , Palli, Subba Reddy Reddy , Collins, Glenn , Scholthof, Herman B , Scholthof, Karen-Beth G
Current Investigators: Schumacher, Dorin
Institution: The Consortium for Plant Biotechnology Research, Inc
EPA Project Officer: Aja, Hayley
Project Period: November 1, 2001 through October 31, 2006 (Extended to December 31, 2007)
Project Period Covered by this Report: November 1, 2006 through October 31,2007
Project Amount: $2,620,600
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research
Objective:
1. Transgenic plants for bioremediation of atrazine and related herbicides
- Produce transgenic tall fescue, switchgrass and ryegrass that have the ability to degrade and detoxify atrazine, by incorporation of the p-atzA gene using Agrobacterium and biolistic transformation strategies.
- Modify initial vectors used for plant transformation by inclusion of a 5’untranslated region of alcohol dehydrogenase gene, which has been shown to act as an enhancer of translation.
- Quantify plant growth and atrazine degradation ability by transformed plant lines growing in soil.
2. Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Biding Proteins.
- To construct the transformation vectors with the already cloned metallohistin cDNA agNt84 under control by constitutive promoter dual CaMV35S.
- To transfer the genes into a woody plant, a Populus hybrid, and annual plants, Brassica napus and B. juncea, and to confirm transformation by polymerase chain reaction and Southern blot.
- To determine levels of RNA and protein expression in different tissues.
- To characterize the transgenic plants for their metal accumulation capacity and phytoremediation potential.
- To determine the short and long term effects of the overexpression of metallohistin on growth and development of the transgenic plants.
3. Development of herbicide resistant energy and biomass crops
The goal of this project was to use a genetically engineered version of the dicamba monooxygenase (DMO) gene to produce energy and biomass crops that are resistant to treatment with the herbicide dicamba and, in so doing, provide efficient, low-cost and environmentallyfriendly control of broadleaf weeds in target crops.
4. Environmentally Superior Soybean Genome Development
The long term objective is to develop a commercially acceptable, superior quality, high yielding soybean variety with low seed phytate. The specific aim of this proposed research is to utilize SSR molecular genetic markers distributed among all 20 soybean linkage groups to facilitate genome recovery, and to use low-phytate markers Satt237 and Satt561 for dual marker assisted selection for gene transfer of the low phytate trait to a superior quality, high yielding conventional soybean variety, ‘5601T’.
5. CPBR Fellowship: Optimizing willow transformation for enhanced phytoremediation and biofuel production
There are no published reports of willow transformation, so the primary aim of this project is to develop effective transformation protocols. The specific aims were to: Propagate sterile explants of the willow clones in tissue culture; begin embryonic callus culture; optimize transformation and regeneration methods; verify transgene presence and expression in transformed willow; and evaluate transformation frequencies from the different protocols. The genes we will introduce to enhance biofuel production and phytoremediation are an antisense 4CL-1 gene cloned from willow and a cytochrome P450 2E1 gene, respectively.
6. Development of tightly regulated ecdysone receptor-based gene switches for use in agriculture
- Develop EcR gene switches that can support tight regulation of transgene expression in tobacco plants.
- Test the utility of EcR gene switch in functional genomics studies. Hypothesis
7. Engineered plant virus proteins for biotechnology
The overall aim of the project is to determine conditions that improve the performance and stability of virus vectors for optimum expression of foreign proteins in plants. The objectives are:
- Test the protective effect of the coat protein of satellite panicum mosaic virus on the stability of plant virus-derived gene expression vectors.
- Test the effect of RNA silencing suppressors on the performance of plant virus-derived gene expression vectors.
- Determine the effect of host species background on the performance and stability of plant virus-derived gene expression vectors.
Progress Summary:
1. Transgenic plants for bioremediation of atrazine and related herbicides
Our research goal is to produce transgenic plants that bioremediate atrazine contaminated soil and soil water, and prevent atrazine movement into waterways to begin with. In our initial first and second year studies, we transformed alfalfa, Arabidopsis and tobacco plants with a bacterial atrazine chlorohydrolase gene and showed that transformants had the ability to degrade atrazine. In this last year, we extended or studies and transformed tall fescue, switchgrass and ryegrass with a modified bacterial atzA (p-atzA) gene and evaluated transformants for their ability to degrade atrazine. These plants were chosen as they have significantly larger root area indices, and hence greater surface area for adsorption, atrazine uptake and degradation, than the nongrass species we previously examined. We also re-transformed alfalfa with a new vector system to increase expression of atzA in planta. To achieve our goals, we created a series of new vectors for plant transformations, pSAM1a, pSAM1b, pUAM1a, pUAM1b, and pPW1Plus. The uidA gene of pScBV-3m vector (Tzafrir et al., 1998), which is under transcriptional control of the sugarcane bacilliform badnavirus (ScBV) promoter and maize alcohol dehydrogenase 1 first intron was replaced by the p-atzA gene (Wang et al., 2005), and the resulting vector was designated pSAM1b. The uidA gene of pAHC25 (Christensen and Quail, 1996), which is under transcriptional control of the maize ubiquitin promoter and its first intron was also replaced by the p-atzA gene, and the resulting vector was designated pUAM1b. To construct a binary vector, pPW1Plus, 5’-untranslated region of tobacco alcohol dehydrogenase gene was removed from ADH NF construct (Satoh et al., 2004) and inserted between the cassava vein mosaic virus (CsVMV) promoter and the p-atzA gene of pPW1 (Wang et al., 2005). For the binary vectors pSAM1a and pUAM1a for Agrobacterium-mediated grass plant transformation, expression cassettes for p-atzA gene were removed from two different vectors, pSAM1b and pUAM1b, respectively, which were constructed for biolistic transformation previously, and then inserted into the multicloning site of the T-DNA region of pCAMBIA1305.1 (CAMBIA, Canberra, Australia), respectively. The vectors pSAM1b and pUAM1b were transformed into tall fescue by biolistic bombardment, vectors pSAM1a and pUAM1a were transferred into tall fescue, switchgrass, and ryegrass by Agrobacterium mediated transformation, and pPW1Plus was transferred into alfalfa by Agrobacterium mediated transformation.
Following transformation and plant regeneration, we obtained 7 lines of tall fescue (Festuca arundinacea Schreb. cv. Kentuky31), 2 lines of perennial ryegrass (Lolium perenne L.), 34 lines of switchgrass (Panicum virgatum L. cv. Alamo), and 28 lines of alfalfa (Medicago sativa L. cv. Regen-SY). Regenerated plants were evaluated for successful transformation by using the polymerase chain reaction (PCR) technique and PCR primers specific for the modified atzA gene. Plant lines were also tested for production of patzA mRNA by using RT-PCR.
Functionally active AtzA enzyme in transgenic plants was evaluated by examining the hydrolytic dechlorination of 14C-UL-ring-labeled atrazine into hydroxyatrazine in vitro and in vivo using TLC analysis (Wang et al. 2005). We also evaluated transgenic plants for their ability to degrade varying concentrations of atrazine in agar, under hydroponic growth conditions, and soil.
PCR analyses indicated that 4 of 7 (57%) transgenic tall fescue (TF) lines, 2 of 2 (100%) transgenic ryegrass (PR) lines, 17 of 23 (74%) transgenic switchgrass (SG) lines, and 11 of 14 (79%) of transgenic alfalfa (AF) lines contained the p-atzA gene. RT-PCR analyses indicated that 4 of 6 (67%) TF lines, 2 of 2 (100%) PR lines, 17 of 23 (74%) SG lines, and 11 of 14 (79%) AF lines contained transcripts for p-atzA mRNA (RT-PCR+), indicating the transgenes were expressed in planta.
We also evaluated 1 TF, 3 SG, and 1 AF transgenic plant lines for in vitro degradation of atrazine. This was done by incubating extracted plant root, leaf, and stem tissues with 14Catrazine and analyzing the products via TLC analyses. Results of this analysis indicated that all 5 plant lines produced functionally active atrazine chlorohydrolase, transforming atrazine to hydroxyatrazine. The one transgenic TF line was also evaluated for in vivo degradation of atrazine using 14C atrazine and hydroponic growth conditions. Plants were evaluated 23 days post herbicide addition. Results of this study indicated that the transgenic TF line dechlorinated atrazine to hydroxyatrazine in planta in the leaves, stems, and roots of tall fescue.
Hydroponic studies and transpiration assays were also used to evaluate 1 TF, 4 SG, and 3 AF transgenic lines for tolerance to atrazine. These studies indicated that the alfalfa, tall fescue and switchgrass transgenic lines were resistant to 1 ppm, 6.5 ppm, and 25 ppm atrazine, respectively, and had greater growth and transpiration, compared to the wild-type parent lines.
Lastly, we evaluated 3 transgenic AF lines for there ability to degrade atrazine in soil. Results of these studies are ongoing. Initial analyses indicated that the 3 transgenic alfalfa lines were able to grow in the presence of 0.18 and 0.39 ppm atrazine in soil, while 1 of the 3 AF transgenic lines had an increased reduction in the initial amount of atrazine compared to the other 2 lines, wild-type parent, and soil control.
2. Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Biding Proteins.
One of our goals has been to identify a vascularspecific promoter in order to express metallohistins specifically in vascular tissue. A Cg164pro::GUS fusion was made and was tested in Lotus japonicus. A typical GUS staining pattern resulting from that construct is shown to the left (Figure 1). In progress is the construction of Cg164pro::AgNt84 which will then be used to drive metallohistin production in select transgenic species.
Figure 1
When expressed transiently in tobacco or onion cells 35S::AgNt84-GFP fusions clearly show that the fusion proteins are directed to the ER, and further that the protein is located at attachment sites between the plasma membrane and cell wall (Fig. 2).
Figure 2
Again using transient expression of onion epidermal cells, we attempted to determine whether there was a correlation between ectopic expression of metallohistin protein (Fig. 3 A) and metal uptake as determined by dithizone staining of tissue incubated in the presence of divalent cations. (Fig 2B). We were unable to demonstrate such a correlation perhaps due to low levels of metallohistin-GFP fusion protein. Fig. 4B shows the predicted location of dithizone staining.
Figure 3. Creation of AgNt84 fragment vectors.
To assess the influence of the 5’ and 3’ untranslated regions (UTRs) on translation efficiency three fragments of the AgNt84 cDNA have been transformed into Nicotiana tabacum cv Xanthi. The three fragments are: 1) only the coding region; 2) the 5’ UTR and the coding region, and 3) the 5’ UTR, the coding region, and the 3’ UTR (Fig. 4). These three fragments of the AgNt84 cDNA were cloned into pMDC32 (Curtis and Grossnilaus 2003) using Gateway cloning (Invitrogen).
Figure 4. The three proposed AgNt84 sequences. (Top) AgNt84 gene with only the coding area inserted into the clone. (Middle) AgNt84 with the 5’ region and the coding area inserted into the clone. (Bottom) AgNt84 with the 5’ region, coding area, and the 3’ untranslated region inserted into the clone.
Previous transformed cell lines showed high levels of mRNA expression, but undetectable levels of metallohistin protein on western blot analysis. A concatemer sequence has been designed to address concerns that the metallohistin proteins could be inactivated being so tightly bound to the cell wall. The concatemer sequence has four repeats of the metal-binding portion of the coding region fused to the native signal peptide (Fig. 5). The resulting pearl-string of metallohistin proteins could be partly bound to the cell wall and still have free proteins available for metal-binding or form a multimer which can also bind metals. The larger protein will also be easier to localize and capture on western blot analysis.
Figure 5. The AgNt84 concatemer sequence. The first part of the concatemer sequence is the signal peptide coded for by the AgNt84 cDNA that is predicted to be exported out of the cell membrane to the cell wall (82% PSORT). Next, the metal-binding portion of the cDNA is repeated four times followed by a stop codon. The concatemer is flanked by a 5’ SacII and a 3’ ApaI restriction site.
3. Development of herbicide resistant energy and biomass crops
This project has been highly successful. Our prime targeted energy crop, soybean, has been transformed with the DMO gene and shown to be resistant to treatment with dicamba at levels 5 to 10 times the rate normally used for weed control under field conditions. The soybean plants transformed with the modified DMO gene have normal agronomic traits and there is no "yield penalty" associated with the dicamba resistance trait as demonstrated by three years of field testing. The technology has been licensed to Monsanto, Co. and will be in the marketplace within the next three years. The details of this work have been recently published in Science (Behrens MR, Mutlu N, Chakraborty S, Dumitru R, Jiang WZ, Lavallee BJ, Herman PL, Clemente TE, Weeks DP. Dicamba resistance: enlarging and preserving biotechnology-based weed management strategies. Science. 2007 May 25;316(5828):1185-8).
In addition, we have gained a great deal of knowledge concerning the biochemistry, molecular biology and microbiology associated with the enzymes, genes and bacteria associated with dicamba degradation in vivo and in vitro. The kinetics of the DMO reaction with dicamba and other substrates have been determined not only for the wild-type enzyme, but also with a mutant that displays enhanced enzymatic activity. Likewise, the enzymatic properties have been described for the reductase and ferredoxin enzymes that, along with DMO, make up the three component enzyme system called dicamba O-demethylase. All of these studies are reported in a recent publication in Archives of Biochemistry and Biophysics (Chakraborty S, Behrens M, Herman PL, Arendsen AF, Hagen WR, Carlson DL, Wang XZ, Weeks DP. A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: purification and characterization. Arch Biochem Biophys. 2005 May 1;437(1):20-8).
The cloning and characterization of the genes encoding the reductase, ferredoxin and DMO components of dicamba O-demethylase revealed that the former two genes were located on the chromosome of Pseudomonas maltophilia, strain DI-6, while the DMO gene was located on a number of the eight megaplasmids associated with strain DI-6. Conversion of E. coli to a dicamba degrading bacterium was achieved by transferring the megaplasmids isolated from strain DI-6 into E. coli by electroporation. This strongly implies that most, if not all, of the remaining genes needed for dicamba degradation reside on the strain DI-6 megaplasmids. Ongoing studies are aimed at defining the entire pathway for dicamba degradation and the (megaplasmid) genes responsible for each reaction. Details of the cloning and characterization of the DMO, reductase and ferredoxin genes have been published recently in the Journal of Biological Chemistry (Herman PL, Behrens M, Chakraborty S, Chrastil BM, Barycki J, Weeks DP. A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: gene isolation, characterization, and heterologous expression. J Biol Chem. 2005 Jul 1;280(26):24759-67).
4. Environmentally Superior Soybean Genome Development
Newly created BC4F1 hybrid seeds were grown in a lighted nursery in Puerto Rico during winter 2006-2007. Leaf tissue was collected from each BC4F1 individual plant and DNA extracted to confirm true double-hybrids at the low-phytate loci, using molecular markers Satt237 and Satt561 via capillary gel electrophoresis on our Beckman-Coulter CEQ 8800 genetic analysis system. This enabled identification of 15 double heterozyotes in the 5601T genetic background and 2 double heteroygotes in the ‘Allen’ genetic background. (Allen is a Roundup Ready conversion of 5601T, released as a new cultivar in 2006 by the Tennessee Agricultural Experiment Station).
SSR markers (dispersed throughout the genome) which were polymorphic between the recurrent parent and the donor were screened to determine genetic recovery of the 5601T genome (Table 1). Two BC4F1 plants of 5601T background (Plant 005 from BC3F1 pollen donor 91-10 and Plant 029 from BC3F1 pollen donor 91-21) and two BC4F1 plants of Allen background (Plant 217 from BC3F1 pollen donor 82-2 and Plant 230 from BC3F1 pollen donor 82-4) showed perfect genetic identity with the recurrent parent genome.
We chemically analyzed the phosphorous content of seeds of hundred of soybean lines derived from earlier stages of this project (BC1, BC2, BC3) in order to identify those that expressed the low phytate trait, following DNA molecular marker assisted trait introgression. All generation lines were planted in rows at the East Tennessee Research and Education Center in Knoxville, TN for field evaluation and seed production. Field harvests of those materials are currently in progress.
The molecular markers Satt237 and Satt561 proved to be effective for dual marker assisted selection for gene transfer of two recessive genes which collectively confer the low phytate trait, as evidenced by chemical analysis of seed phosphorous in selected progeny. The original donor line (Cx1834-1-2) is poorly agronomic: it suffers from significant losses in seed germination, flowers and sets pods too early in southern USA latitudes, and produces low seed yields. A strategy was needed to rapidly transfer the donor’s low phytate trait to a high yielding genetic background. Molecular markers dispersed across the genome proved to be effective for facilitating genome recovery of the high yielding 5601T recurrent parent every backcross generation (see Figures 1 and 2). By the BC4 stage we have fully captured the recurrent parent genome while simultaneously confirming the presence of both low phytate loci. We have successfully accomplished the specific aim of this research project.
Table 1. DNA selections identified as heterozygous BC4F1 individuals at the Satt237 (linkage group N and Satt561 (linkage group L) loci. SCORE: summation over linkage groups, where 1 = Heterozygote, 0 = 5601T allele. We expect plants with the lowest score sum to have retained more of the 5601T and Allen genome.
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Linkage Groups
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| N | L | F | N | D2 | K | L | A2 | O | O | G | |||||
| CROSS | FEMALE ♀ | MALE ♂ | BC4F1 PLANT | Satt 237 | Satt 561 | Satt 114 | Satt 152 | Satt 226 | Satt 260 | Satt 373 | Satt 429 | Satt 243 | Satt 259 | Satt 517 |
SCORE |
| 06-45 | 5601T | 91-10 | 001 | HET | HET | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| 06-45 | 5601T | 91-10 | 002 | HET | HET | 1 | . | . | 0 | 0 | 0 | 0 | 0 | 1 | 2 |
| 06-45 | 5601T | 91-10 | 005 | HET | HET | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 06-45 | 5601T | 91-10 | 009 | HET | HET | 1 | . | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| 06-46 | 5601T | 91-12 | 014 | HET | HET | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 2 |
| 06-46 | 5601T | 91-12 | 017 | HET | HET | 0 | 0 | 0 | 0 | . | 1 | 0 | 0 | 0 | 1 |
| 06-47 | 5601T | 91-21 | 029 | HET | HET | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 06-48 | 5601T | 91-24 | 030 | HET | HET | 0 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 2 |
| 06-41 | 5601T | 90-19 | 052 | HET | HET | 0 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | . | 2 |
| 06-41 | 5601T | 90-19 | 054 | HET | HET | 0 | 0 | 0 | . | 0 | . | 1 | 0 | 0 | 1 |
| 06-42 | 5601T | 90-22 | 062 | HET | HET | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 2 |
| 06-42 | 5601T | 90-22 | 063 | HET | HET | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 1 |
| 06-42 | 5601T | 90-22 | 064 | HET | HET | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 2 |
| 06-43 | 5601T | 90-23 | 072 | HET | HET | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 1 | . | 3 |
| 06-43 | 5601T | 90-23 | 073 | HET | HET | 0 | 0 | . | . | 0 | 1 | 1 | 0 | 0 | 2 |
| 06-50 | ALLEN | 82-2 | 217 | HET | HET | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 06-51 | ALLEN | 82-4 | 230 | HET | HET | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Figure 1. Molecular markers effectively transferred the low phytate trait from the poorly adapted donor (Cx1834-1-2) and captured much of the high yielding genome of recurrent parent (5601T) in BC1 progeny.
Figure 2. Molecular markers effectively transferred the low phytate trait from the poorly adapted donor (Cx1834-1-2) and captured the full high yielding genome of recurrent parent (5601T) in BC4 progeny.
5. CPBR Fellowship: Optimizing willow transformation for enhanced phytoremediation and biofuel production
First we tried producing callus using Woody Plant Medium (WPM) with only cytokinins added. We used media containing 0.5 mg BAP/L, 1.0mg BAP(benzyl amino purine)/L, or 0.1mg thidiazuron/L, as outlined in a study that produced callus tissue from black willow inflorescences (Lyra et al. 2006). Although there was a strong response to the hormone treatments, no callus developed that was viable for genetic transformation (Figures 1a and 1b.).
Next we tried combining cytokinins and auxins at different ratios. Using BAP with either 0.1 or 0.2mg NAA/L elicited a response from stem sections within a week, however the callus that was formed hardened and turned brown (Figures 2a. and 2b). After exposure to the auxin, NAA, stem sections formed white, fluffy, non-embryogenic callus. We began to experiment with Zeatin, another cytokinin, that produced strong, compact callus that appeared to be embryogenic, but the callus samples died before expanding large enough to be separated from the original stem tissue to be used in transformation experiments. We decided to follow the protocol found in Stoehr et al. 1989. Stoehr was successful in producing callus from willow using a combination of BAP and 2,4-D. We used 1mg BAP/L 0.6mg 2,4-D/L, which also produced some hydrolyzed callus, but some of the tissue appeared more compact. We also started cutting the stems perpendicularly rather than lengthwise, which seemed to result in better callus (Figure 3.).
Figure 4a. S x 67 after 18 days. Figure 4b. S x 61 after 35 days.
Figure 5. S x 67 on 0.6 BAP 0.5 ZEA 0.5 NAA After 12 days.
We also tried 0.974mg zeatin/L 0.5mg NAA/L that initially yielded the best response from S x 61 and S x 67 clones. S x 61 grew new embryogenic callus even after being transferred to a new area of the plate, but some hydrolyzed callus was also present. Next we increased zeatin and decreased NAA since roots began to form, (Figures 4a. and 4b.) Since we observed that willow reacts strongly to zeatin, but that the hormone breaks down quickly, we decided to combine it with another cytokinin, BAP. The media also contained either NAA or 2,4-D. So far the callus from S x 67 looked embryogenic (Figure 5). This was not the case, however, since although the combination did induce some callus growth, it was of a lower quality than that original produced by Zeatin and an auxin alone. This warrants further study. Perhaps by using media containing Zeatin to initiate callus formation, and then switching to a separate media containing BAP we could successfully induce callus expansion. Alternatively - assuming that the Zeatin itself is degrading in the media over time, rather than the plant tissue becoming resistant to the presence of the plant growth regulators – we could test the effects of transferring the samples to freshly prepared Zeatin media more frequently. We need to continue optimizing the parameters until we can get the callus to start forming that will be suitable for gene transformation and further studies.
Figure 6. The LifeRaft tissue culture system.
We also tried the LifeRaft tissue culture system (Figure 6) that consisted of a permeable membrane attached to a plastic frame, which is supported by a plastic float. Both the frame and the float are placed in sterile liquid media, and the float keeps the membrane at the level of the media. Plant tissue samples can then be placed on the wetted membrane and allowed to grow as on traditional agar media. We became interested in using the LifeRaft system because we hypothesized that willow – a moisture-loving tree – would benefit from the use of liquid media, and the LifeRaft system provides this without the decrease in air circulation that would occur in standard liquid tissue culture. Another possible benefit is the increased exposure of the tissue samples to more of the added plant growth regulators.
Our initial results were similar to those found by using solid media, but more experiments are planned. Because of the limited number of LifeRafts available (the item has been discontinued), we chose to standardize our experiments by using solid media in petri dishes until a suitable combination of plant growth regulators had been found. Now that we have found promising combinations, we will begin to test the difference in callus growth between tissue samples cultured on solid media and those grown using the LifeRaft system.
Our work with enhancing phytoremediation in aspen trees by overexpressing CYP2E1 has been very effective. This project, sponsored by the DOE and NIEHS, was published in PNAS this week and has received widespread publicity. We will introduce this gene into willow when the transformation method has been developed.
6. Development of tightly regulated ecdysone receptor-based gene switches for use in agriculture
1. Optimization of reporter gene expression cassette:
- Optimization of minimal promoter sequence: We have conducted experiments to optimize the reporter gene expression cassette by manipulating the minimal promoter sequence used to drive the transgene expression. We cloned the luciferase reporter gene under the control of -39 35S (including TATAA box and 8 bp upstream) and -31 35S (includes only TATAA box) minimal promoter sequences and tested the expression levels of the luciferase by electroporating only the reporter gene constructs into tobacco protoplasts. The performance of these new constructs was compared with our regular -46 35S reporter construct. Compared to -46 35S reporter construct, there was about 20% reduction in the luciferase activity when the luciferase gene was placed under the control of -39 35S minimal promoter and 65 to 70% reduction in the activity when the luciferase gene was placed under -31 35S minimal promoter. These truncations were then tested in a two-hybrid gene switch (CfEcR+LmRXR) format. These experiments showed a reduction in the background levels of luciferase by 25% when -31 35S reporter construct was used in place of -46 35S reporter construct.
- Optimization of translational start site: In order to optimize the EcR gene switch for higher induction levels, we have tested different KOZAK sequences cloned upstream of the luciferase reporter gene translational start site. Among several KOZAK sequences tested, ‘AACAATGG’ KOZAK sequence was found to be the best consensus sequence in enhancing the expression of luciferase reporter gene activity when compared to the luciferase reporter gene without any KOZAK sequence. We have tested the induction levels of KOZAK:luciferase reporter gene when electroporated with a two-hybrid gene switch constructs containing CfEcR and LmRXR. By using KOZAK:luciferase construct in a two-hybrid gene switch, we found that the luciferase induction levels were increased significantly while the background activity does not significantly affected compared to the two-hybrid switch containing regular luciferase construct.
2. Optimization of receptor gene expression cassette:
We have tested the possibility of reducing the background activity of transgene expression in the absence of ligand by optimizing the receptor gene expression cassette using autoregualtion format and by performing RXR mutagenesis.
- Autoregulation format switch: In an autoregulation format the CfEcR and LmRXR receptors were placed under the control of GAL4 response elements and a minimal 35S promoter. This is in contrast to standard format switch where the receptor expression cassettes were under the control of constitutive promoters. We have cloned both CfEcR and LmRXR cassettes under minimal promoters and tested their efficiency in lowering the reporter gene expression in the absence of ligand. We have used -46 truncations of 35S promoter to drive the expression of receptor genes in autoregulation format switch. By placing the receptor genes under -46 35S minimal promoter, we could see a reduction in the background expression levels of luciferase reporter gene in the absence of ligand.
- RXR mutagenesis: We performed site-directed mutagenesis on LmRXR to change the amino acid residues responsible for the heterodimerization with EcR in the absence of ligand. Initial experiments were carried out by generating 5 single LmRXR mutants (Lm1, Lm-2, Lm-3, Lm-4 and Lm-5) and screening in a two-hybrid switch format. The performance of these mutants in inducing the luciferase reporter gene expression in a two-hybrid gene switch format was compared with wild-type LmRXR construct. Of the 5 mutants screened, two of them (Lm-4 and Lm-5) appear to work better than the wild-type LmRXR. Lm-4 showed low background expression in the absence of ligand and Lm-5 showed high induction levels compared to the wild-type LmRXR. In order to combine the desirable properties of these two LmRXR mutants (Lm 4 and Lm 5), we incorporated these two mutations together and generated a double mutant (Lm6). As desired, Lm6 showed low background activity in the absence of ligand compared to either Lm4 or Lm5 and wild-type LmRXR, however the induction levels were low compared to either of the mutants tested. With the aim to increase the induction levels and keeping the background reporter gene expression low, further mutagenesis experiments were carried out using Lm6 as a template. We finally selected three LmRXR mutants (Lm7, Lm8 and Lm9) containing 3 to 4 amino acid changes in the ligand binding domain for further study. These three mutants supported low background activity in the absence of ligand and high induced reporter gene expression levels in the presence of nanomolar concentrations of chemical ligand compared to wild-type LmRXR.
Stable transformation and generation of transgenic plants:
The information obtained from optimization of minimal promoter sequence, translational start site, autoregulation format and RXR mutagenesis were used to make binary vectors for Arabidopsis plant transformation. The binary vectors constructed were mobilized into Agrobacterium strain GV3850. Transgenic Arabidopsis plants were generated and screening of T2 transgenic lines is in progress.
Functional genomics studies:
The information obtained from the above experiments was also used to test the utility of the modified two-hybrid gene switch in functional genomics applications by regulating the expression of a SUPERMAN-like single zinc finger protein 11 (ZFP11) gene isolated from Arabidopsis thaliana (AtZFP11). The expression levels of the AtZFP11 gene in wild-type control Arabidopsis plants are extremely low and no mutant phenotype is presently associated with this gene. This AtZFP11 protein caused mortality and a deformed phenotype when overexpressed under the control of a 35S promoter in both Arabidopsis and tobacco (Tavva et al., 2007). There was difficulty in recovering healthy transgenic plants and the seed collected from the transgenic tobacco expressing AtZFP11 under the CaMV 35S promoter failed to germinate on agar plates supplemented with kanamycin. Therefore, the AtZFP11 gene is an ideal candidate for testing the efficiency of the modified two-hybrid EcR gene switch in functional genomics applications. The binary vectors constructed in regulating the AtZFP11 gene were transferred to Arabidopsis plants. We are in the process of recovering T1 seed from the infiltrated plants.
7. Engineered plant virus proteins for biotechnology
Accomplishments will be listed per objective and repetitions of explanations and statements in last year’s report are avoided to stay within the page limits. In case explanations of concepts are desired we respectfully refer to the original proposal and last year’s progress report.
Objective 1
The previously reported Potato virus X (PVX) gene vector was used to reproducibly express the coat protein of satellite panicum mosaic virus (Scp). Passage experiments were performed on Nicotiana benthamiana plants. As described with last year’s report, leaves of each passage plant were collected and analyzed for expression of Scp with immunoblotting techniques (i.e., western blots) using our Scp-specific rabbit antiserum. Results reported last year showed detectable levels of Scp until passage three, this has now been reproducibly extended to passage 4 and sometimes passage 5. Experiments are currently being conducted to test the stability at different temperatures
As explained previously, TBSV vectors expressing the gene for green fluorescent protein (GFP) only express the foreign gene in inoculated leaves. Due to vector instability, TBSV molecules that infect upper tissues are devoid of a functional insert. However, we now can reproducibly demonstrate that when TBSV-GFP is inoculated onto plants expressing Scp (by PVX-Scp), green fluorescent spots appear on upper non-inoculated leaves. This directly supports the notion that Scp benefits expression of foreign genes from virus vectors.
We have generated a library of Scp mutants and as listed with the plans submitted with last year’s report, these mutants have now been tested for several biochemical properties such as RNA binding and solubility. We have determined that the stabilizing effect by the Scp (i.e., avoidance of deletion mutant accumulation during infection) is conveyed by the N- and C- terminal regions. These will now be incorporated in vector stability tests. We have also constructed a GFP-Scp fusion gene and inserted this into our TBSV vector. Inoculation of this vector onto N. benthamiana yielded intense green florescent infection foci a few days after inoculation. Results of experiments that were listed in last year’s plan show that this fusion does not noticeably stabilize the vector.
We have generated a new TBSV-GFP coat protein gene substitution construct in which most of the remaining coat protein encoding RNA sequences are deleted. Preliminary studies suggested that this had a positive effect on foreign gene expression when compared with the original construct that also expresses GFP but retained a substantial stretch of coat protein RNA sequences downstream of the GFP gene. These results need to be verified.
Objective 2
As mentioned last year we tested several Agrobacterium strains and several suppressorexpressing T-DNA vectors and different agroinfiltration conditions to determine which combinations yield optimal and reliable results. This has been completed and we now routinely use strains C58 and EHA for agroinfiltration of N. benthamiana leaves to express for instance P19, HC-Pro or γb. We can reliably perform these experiments and demonstrate that GFP expression (from a separate T-DNA construct) is maintained for at least ten days whereas in absence of any suppressor the GFP signal has disappeared. Specific combinations of suppressors will be tested using this assay.
Last year we reported on a quick method to screen virus mutants for their ability to suppress RNA silencing. Based on this screen, specific versions have now been selected and these will be incorporated in the agroinfiltration experiments. We will also experiment with infiltrating whole plants. Similarly we will determine if agroinfiltration with suppressors enhances the performance of PVX and TBSV gene vectors. Along those same lines, we are also in the process of creating a TBSV-GFP vector devoid of P19 expression, and compare its performance with the current version hat expresses P19. We have also incorporated agroinfiltration experiments with a newly developed TBSV-GFP vector (in collaboration with J. Lindbo). This vector contains the CaMV 35S promoter, a ribozyme, and poly(A) signal that lead to transcription of infectious RNA upon agroinfiltration of the T-DNA construct containing the expression cassette, into leaves. Based on recent literature, we are testing whether infectivity of this construct is enhanced by co-infiltration with suppressor-expressing constructs into leaves and whole plants.
Objective 3
Thus far we have tested 26 plant species for TBSV-GFP susceptibility by measuring green fluorescence 3-4 days after inoculation with in vitro generated infectious RNA. In parallel we also perform western blotting for detection of specific host proteins that we have identified previously to interact with TBSV movement-related proteins. The idea is to determine if a correlation exists between presence and amount of these host proteins and the ability and robustness of TBSV to invade these species, and to convey vector stability.
As outlined last year, during the course of the project we have developed a new bioassay to test virus vector integrity in different hosts. For this we passage material from TBSV-GFP inoculated leaves to cowpea indicator plants that produce visible lesions upon infection. Upon UV illumination we then determine the fraction of these lesions that is green fluorescent, indicating there is no mutation or deletion in the foreign insert. For N. benthamiana we obtain levels of 40-50%. For a more elaborate description I refer to last year’s report. Conducting these experiments relies on implementing a variety of plant growth facilities and conditions simultaneously to have different plant species ready at the same time for RNA inoculation and have this coincide with the availability of 5-6 old cowpea seedlings. We are testing if freezing of originally inoculated green fluorescent leaf material is still suitable for these experiments. The ultimate aim is to compare the percentage green lesions for different species and to see if that can be correlated with the presence or quantity of host factors.
We are now also incorporating the new TBSV-GFP vector (described above) into these efforts. Preliminary results showed that this vector gives rise to enlarged spots of green fluorescence in N. benthamiana and pepper compared with those obtained with the original construct. This and other observations are in line with the hypothesis that coat protein sequences can rearrange more effectively than other regions, to either restore the native coat protein coding region, or to remove foreign sequences.
Future Activities:
1. Transgenic plants for bioremediation of atrazine and related herbicides
We are currently finishing the evaluation of selected plant lines for degradation of atrazine in planta, and are using TLC analyses to quantify atrazine degraded in plant samples. Also, we are continuing soil studies to determine the ability of selected plant lines to take-up and degrade atrazine in natural soil. Upon completion, selected plant lines will be evaluated for there ability to reduce and degrade atrazine in simulated run-off experiments.
2. Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Biding Proteins.
- Cloning of the metallohistin gene from A. glutinosa in an attempt to produce transgenic plants that stably express higher levels of metallohistin protein. To date no stably transformed plants show high levels of metallohistin protein despite the relatively high levels of metallohistin mRNA.
- Transformation of Nicotiana tabacum and Brassica juncea with a concatemer sequence.
- Assay of metal-uptake in aspen, Nicotiana tabacum, Arabidopsis thaliana, and Brassica juncea plants transgenic for metallohistin as they become available.
- Complete characterization of pBIN-AgNt84, pMDC32-AgNt84, and AgNt84 concatemer cell lines through Southern, northern, and western blot analysis.
3. Development of herbicide resistant energy and biomass crops
Our project has demonstrated that a genetically engineered version of the dicamba monooxygenase gene can be used to produce energy and biomass crops that are resistant to treatment with the herbicide, dicamba. Transgenic soybean plants carrying this gene are well on their way to commercialization by our corporate sponsor, Monsanto, Co.
4. Environmentally Superior Soybean Genome Development
We plan to complete field harvests of selected single plants and progeny rows and initiate chemistry analyses of seeds to confirm low phytate expression. The best selected lines will be planted in our Florida or Puerto Rico winter nursery for generation advancement and seed stock increase for future testing. Funding for this project concludes December 31, 2007.
5. CPBR Fellowship: Optimizing willow transformation for enhanced phytoremediation and biofuel production
We recently obtained more elite lines of willow from Professor Larry Smart of SUNY that we are growing in tissue culture now. The undergraduate researcher, Gabe Verdugo, is continuing to work on this project. A graduate student, Xu Ping, who has a scholarship, will work on the project beginning spring 2008. As the callus develops, we will begin doing the particle bombardment experiments and will not end this project until we have successfully developed efficient transformation protocols for this important genus.
6. Development of tightly regulated ecdysone receptor-based gene switches for use in agriculture
Molecular characterization of T2 transgenic lines: Southern and northern blot analysis will be carried out on Arabidopsis T2 transgenic lines to verify the transgene integration pattern and its expression. The induction characteristics of the new versions of twohybrid EcR gene switch will be carried out in T2 and T3 transgenic Arabidopsis plants through time-course and dose-response studies
7. Engineered plant virus proteins for biotechnology
These are summarized with the progress summaries provided above for each objective separately.
References:
- Curtis, M and U Grossnilaus. 2003. A Gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiology 133: 462-469.
- Lyyra S, Lima A, Merkle SA. 2006. In vitro regeneration of Salix nigra from adventitious shoots. Tree Physiol. Jul;26(7):969-75.
- Stoehr, M.U., M. Cai and L. Zsuffa. 1989. In vitro plant regeneration via callus culture of mature Salix exigua. Can. J. For. Res. 19:1634–1638.
Journal Articles: 45 Displayed | Download in RIS Format
| Other center views: | All 212 publications | 49 publications in selected types | All 45 journal articles |
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Altpeter F, Positano M. Efficient plant regeneration from mature seed derived embryogenic callus of turf-type bahiagrass (Paspalum notatum Flugge). International Turfgrass Society Research Journal 2005;10(Part 1):479-484. |
R829479 (Final) R829479C017 (Final) |
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Altpeter F, James VA. Genetic transformation of turf-type bahiagrass (Paspalum notatum Flugge) by biolistic gene transfer. International Turfgrass Society Research Journal 2005;10(Part 1):485-489. |
R829479 (Final) R829479C017 (Final) |
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Chakraborty S, Behrens M, Herman PL, Arendsen AF, Hagen WR, Carlson DL, Wang XZ, Weeks DP. A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6. Archives of Biochemistry and Biophysics 2005;437(1):20-28. |
R829479 (2007) R829479 (Final) R829479C022 (2005) R829479C022 (2006) |
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Chaufaux J, Seguin M, Swanson JJ, Bourguet D, Siegfried BD. Chronic exposure of the European corn borer (Lepidoptera: Crambidae) to Cry1Ab Bacillus thuringiensis toxin. Journal of Economic Entomology 2001;94(6):1564-1570. |
R829479 (2006) R829479 (Final) R829479C014 (Final) |
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Fan ZL, South C, Lyford K, Munsie J, van Walsum P, Lynd LR. Conversion of paper sludge to ethanol in a semicontinuous solids-fed reactor. Bioprocess and Biosystems Engineering 2003;26(2):93-101. |
R829479 (2006) R829479 (Final) R829479C006 (2004) R829479C006 (Final) |
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Flannagan RD, Yu C-G, Mathis JP, Meyer TE, Shi XM, Siqueira HAA, Siegfried BD. Identification, cloning and expression of a Cry1Ab cadherin receptor from European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae). Insect Biochemistry and Molecular Biology 2005;35(1):33-40. |
R829479 (2006) R829479 (Final) R829479C014 (Final) |
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Gaspar YM, Nam J, Schultz CJ, Lee L-Y, Gilson PR, Gelvin SB, Bacic A. Characterization of the Arabidopsis lysine-rich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of agrobacterium transformation. Plant Physiology 2004;135(4):2162-2171. |
R829479 (2006) R829479 (Final) R829479C001 (Final) |
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Gelvin SB. Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool. Microbiology and Molecular Biology Reviews 2003;67(1):16-37. |
R829479 (2006) R829479 (Final) R829479C001 (2003) R829479C001 (Final) |
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Gelvin SB. Improving plant genetic engineering by manipulating the host. Trends in Biotechnology 2003;21(3):95-98. |
R829479 (2006) R829479 (Final) R829479C001 (2003) R829479C001 (Final) |
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Herman PL, Behrens M, Chakraborty S, Chrastil BM, Barycki J, Weeks DP. A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6:gene isolation, characterization, and heterologous expression. Journal of Biological Chemistry2005;280(26):24759-24767. |
R829479 (2007) R829479 (Final) R829479C022 (2005) R829479C022 (2006) |
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Hogenhout SA, Redinbaugh MG, Ammar E-D. Plant and animal rhabdovirus host range: a bug's view. Trends in Microbiology 2003;11(6):264-271. |
R829479 (2006) R829479 (Final) R829479C004 (2003) |
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Kayihan GC, Huber DA, Morse AM, White TL, Davis JM. Genetic dissection of fusiform rust and pitch canker disease traits in loblolly pine. Theoretical and Applied Genetics 2005;110(5):948-958. |
R829479 (Final) R829479C025 (Final) |
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Ke TY, Sun XZ. Melting behavior and crystallization kinetics of starch and poly(lactic acid) composites. Journal of Applied Polymer Science 2003;89(5):1203-1210. |
R829479 (2006) R829479 (Final) R829479C012 (2003) R829479C012 (Final) |
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Ke TY, Sun XZS. Thermal and mechanical properties of poly(lactic acid)/starch/methylenediphenyl diisocyanate blending with triethyl citrate. Journal of Applied Polymer Science 2003;88(13):2947-2955. |
R829479 (2006) R829479 (Final) R829479C012 (2003) R829479C012 (Final) |
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Ke TY, Sun SXZ, Seib P. Blending of poly(lactic acid) and starches containing varying amylose content. Journal of Applied Polymer Science 2003;89(13):3639-3646. |
R829479 (2006) R829479 (Final) R829479C012 (2003) R829479C012 (Final) |
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Ke TY, Sun XZS. Starch, poly(lactic acid), and poly(vinyl alcohol) blends. Journal of Polymers and the Environment 2003;11(1):7-14. |
R829479 (2006) R829479 (Final) R829479C012 (Final) |
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Liu X, Zhu Y, Yang S-T. Construction and characterization of ack deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid and hydrogen production. Biotechnology Progress 2006;22(5):1265-1275. |
R829479 (2006) R829479 (Final) R829479C016 (Final) R829479C023 (2006) |
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Liu XG, Zhu Y, Yang S-T. Butyric acid and hydrogen production by Clostridium tyrobutyricum ATCC 25755 and mutants. Enzyme and Microbial Technology 2006;38(3-4):521-528. |
R829479 (2006) R829479 (Final) R829479C016 (Final) R829479C023 (2005) R829479C023 (2006) |
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Liu X, Yang S-T. Kinetics of butyric acid fermentation of glucose and xylose by Clostridium tyrobutyricum wild type and mutant. Process Biochemistry 2006;41(4):801-808. |
R829479 (Final) R829479C023 (2005) R829479C023 (2006) |
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Morse AM, Nelson CD, Covert SF, Holliday AG, Smith KE, Davis JM. Pine genes regulated by the necrotrophic pathogen Fusarium circinatum. Theoretical and Applied Genetics 2004;109(5):922-932. |
R829479 (Final) R829479C025 (Final) |
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Noureddini H, Gao X, Joshi S. Immobilization of Candida rugosa lipase by sol-gel entrapment and its application in the hydrolysis of soybean oil. Journal of the American Oil Chemists' Society 2003;80(11):1077-1083. |
R829479 (2006) R829479 (Final) R829479C008 (Final) |
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Noureddini H, Gao X, Philkana RS. Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil. Bioresource Technology 2005;96(7):769-777. |
R829479 (2006) R829479 (Final) R829479C008 (2003) R829479C008 (Final) |
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Redinbaugh MG, Seifers DL, Meulia T, Abt JJ, Anderson RJ, Styer WE, Ackerman J, Salomon R, Houghton W, Creamer R, Gordon DT, Hogenhout SA. Maize fine streak virus, a new leafhopper-transmitted rhabdovirus. Phytopathology 2002;92(11):1167-1174. |
R829479 (2006) R829479 (Final) R829479C004 (2003) |
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Rong JK, Abbey C, Bowers JE, Brubaker CL, Chang C, Chee PW, Delmonte TA, Ding XL, Garza JJ, Marler BS, Park C-H, Pierce GJ, Rainey KM, Rastogi VK, Schulze SR, Trolinder NL, Wendel JF, Wilkins TA, Williams-Coplin TD, Wing RA, Wright RJ, Zhao XP, Zhu LH, Paterson AH. A 3347-locus genetic recombination map of sequence-tagged sites reveals features of genome organization, transmission and evolution of cotton (Gossypium). Genetics 2004;166(1):389-417. |
R829479 (2006) R829479 (Final) R829479C009 (Final) |
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Rugh CL. Genetically engineered phytoremediation: one man's trash is another man's transgene. Trends in Biotechnology 2004;22(10):496-498. |
R829479 (Final) R829479C020 (2005) R829479C020 (Final) |
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Rugh CL, Susilawati E, Kravchenko AN, Thomas JC. Biodegrader metabolic expansion during polyaromatic hydrocarbons rhizoremediation. Zeitschrift fur Naturforschung C 2005;60(3-4):331-339. |
R829479 (Final) R829479C020 (2005) R829479C020 (Final) |
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Siqueira HAA, Nickerson KW, Moellenbeck D, Siegfried BD. Activity of gut proteinases from Cry1Ab-selected colonies of the European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae). Pest Management Science 2004;60(12):1189-1196. |
R829479 (2006) R829479 (Final) R829479C014 (Final) |
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Siqueira HAA, Moellenbeck D, Spencer T, Siegfried BD. Cross-resistance of Cry1Ab-selected Ostrinia nubilalis (Lepidoptera: Crambidae) to Bacillus thuringiensis δ-endotoxins. Journal of Economic Entomology 2004;97(3):1049-1057. |
R829479 (2006) R829479 (Final) R829479C014 (Final) |
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Tao Y, Rao PK, Bhattacharjee S, Gelvin SB. Expression of plant protein phosphatase 2C interferes with nuclear import of the Agrobacterium T-complex protein VirD2. Proceedings of the National Academy of Sciences of the United States of America 2004;101(14):5164-5169. |
R829479 (2006) R829479 (Final) R829479C001 (Final) |
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Tian L, Wang JL, Fong MP, Chen M, Cao HB, Gelvin SB, Chen ZJ. Genetic control of developmental changes induced by disruption of Arabidopsis histone deacetylase 1 (AtHD1) expression. Genetics 2003;165(1):399-409. |
R829479 (2006) R829479 (Final) R829479C001 (2003) R829479C001 (Final) |
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van Attikum H, Bundock P, Overmeer RM, Lee L-Y, Gelvin SB, Hooykaas PJJ. The Arabidopsis AtLIG4 gene is required for the repair of DNA damage, but not for the integration of Agrobacterium T-DNA. Nucleic Acids Research 2003;31(14):4247-4255. |
R829479 (2006) R829479 (Final) R829479C001 (2003) R829479C001 (Final) |
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Wang H, Sun XZ, Seib P. Mechanical properties of poly(lactic acid) and wheat starch blends with methylenediphenyl diisocyanate. Journal of Applied Polymer Science 2002;84(6):1257-1262. |
R829479 (2006) R829479 (Final) R829479C012 (Final) |
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Wang H, Sun XZ, Seib P. Effects of starch moisture on properties of wheat starch/poly(lactic acid) blend containing methylenediphenyl diisocyanate. Journal of Polymers and the Environment 2002;10(4):133-138. |
R829479 (2006) R829479 (Final) R829479C012 (Final) |
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Wang H, Sun XS, Seib P. Properties of poly(lactic acid) blends with various starches as affected by physical aging. Journal of Applied Polymer Science 2003;90(13):3683-3689. |
R829479 (2006) R829479 (Final) R829479C012 (Final) |
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Wang L, Samac DA, Shapir N, Wackett LP, Vance CP, Olszewski NE, Sadowsky MJ. Biodegradation of atrazine in transgenic plants expressing a modified bacterial atrazine chlorohydrolase (atzA) gene. Plant Biotechnology Journal 2005;3(5):475-486. |
R829479 (2007) R829479 (Final) R829479C019 (2005) |
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Yi HC, Mysore KS, Gelvin SB. Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation. The Plant Journal 2002;32(3):285-298. |
R829479 (2006) R829479 (Final) R829479C001 (Final) |
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Zhu Y, Yang S-T. Effect of pH on metabolic pathway shift in fermentation of xylose by Clostridium tyrobutyricum. Journal of Biotechnology 2004;110(2):143-157. |
R829479 (2006) R829479 (Final) R829479C016 (Final) |
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Zhang J-F, Sun XZ. Mechanical properties of poly(lactic acid)/starch composites compatibilized by maleic anhydride. Biomacromolecules 2004;5(4):1446-1451. |
R829479 (2006) R829479 (Final) R829479C012 (Final) |
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Zhang J-F, Sun XZ. Mechanical properties and crystallization behavior of poly(lactic acid) blended with dendritic hyperbranched polymer. Polymer International 2004;53(6):716-722. |
R829479 (2006) R829479 (Final) R829479C012 (2003) R829479C012 (Final) |
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Zhang J-F, Sun XZ. Mechanical and thermal properties of poly(lactic acid)/starch blends with dioctyl maleate. Journal of Applied Polymer Science 2004;94(4):1697-1704. |
R829479 (2006) R829479 (Final) R829479C012 (Final) |
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Zhang J-F, Sun XZ. Physical characterization of coupled poly(lactic acid)/starch/maleic anhydride blends plasticized by acetyl triethyl citrate. Macromolecular Bioscience 2004;4(11):1053-1060. |
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Zhu Y, Nam J, Humara JM, Mysore KS, Lee L-Y, Cao HB, Valentine L, Li JL, Kaiser AD, Kopecky AL, Hwang H-H, Bhattacharjee S, Rao PK, Tzfira T, Rajagopal J, Yi HC, Veena, Yadav BS, Crane YM, Lin K, Larcher Y, Gelvin MJK, Knue M, Ramos C, Zhao X, Davis SJ, Kim S-I, Ranjith-Kumar CT, Choi Y-J, Hallan VK, Chattopadhyay S, Sui X, Ziemienowicz A, Matthysse AG, Citovsky V, Hohn B, Gelvin SB. Identification of Arabidopsis rat mutants. Plant Physiology 2003;132(2):494-505. |
R829479 (2006) R829479 (Final) R829479C001 (2003) R829479C001 (Final) |
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Zhu Y, Yang S-T. Adaptation of Clostridium tyrobutyricum for enhanced tolerance to butyric acid in a fibrous-bed bioreactor. Biotechnology Progress 2003;19(2):365-372. |
R829479 (2006) R829479 (Final) R829479C016 (Final) |
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Zhu Y, Liu XG, Yang ST. Construction and characterization of pta gene-deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentation. Biotechnology and Bioengineering 2005;90(2):154-166. |
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Behrens MR, Mutlu N, Chakraborty S, Dumitru R, Jiang WZ, LaVallee BJ, Herman PL, Clemente TE, Weeks DP. Dicamba resistance:enlarging and preserving biotechnology-based weed management strategies. Science 2007;316(5828):1185-1188. |
R829479 (2007) |
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Supplemental Keywords:
Genetic engineering, heavy metal contamination, phytoremediation, Dicamba resistance, herbicide resistance, agricultural biotechnology, weed control, environmentally-friendly, soybeans, Phosphorous, Phytate, Poultry Nutrition, Swine Nutrition, Confined Animal Feeding Operations (CAFOs), bioenergy, plant biotechnology, RFA, Scientific Discipline, Waste, Sustainable Industry/Business, Environmental Chemistry, Sustainable Environment, Technology for Sustainable Environment, Bioremediation, New/Innovative technologies, Environmental Engineering, Agricultural Engineering, bioengineering, transgenic plants, biodegradation, biotechnology, plant biotechnology, remediation, bioacummulation, phytoremediation
Progress and Final Reports:
Original Abstract Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829479C001 Plant Genes and Agrobacterium T-DNA Integration
R829479C002 Designing Promoters for Precision Targeting of Gene Expression
R829479C003 aka R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
R829479C005 Development of Novel Plastics From Agricultural Oils
R829479C006 Conversion of Paper Sludge to Ethanol
R829479C007 Enhanced Production of Biodegradable Plastics in Plants
R829479C008 Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
R829479C009 Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
R829479C010 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals
R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C014 Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
R829479C015 Coordinated Expression of Multiple Anti-Pest Proteins
R829479C016 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C017 Molecular Improvement of an Environmentally Friendly Turfgrass
R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
R829479C019 Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
R829479C021 Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C024 Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
R829479C025 Chemical Induction of Disease Resistance in Trees
R829479C026 Development of Herbicide-Tolerant Hardwoods
R829479C027 Environmentally Superior Soybean Genome Development
R829479C028 Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
R829479C029 Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
R829479C030 Engineered Plant Virus Proteins for Biotechnology
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.