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 | Lyyra S, Lima A, Merkle SA. 2006. In vitro regeneration of Salix nigra from adventitious shoots. Tree Physiol. Jul;26(7):969-75. Journal Articles: 45 Displayed | Download in RIS Format
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, Sustainable Industry/Business, RFA, Scientific Discipline, Waste, Technology for Sustainable Environment, Sustainable Environment, Agricultural Engineering, Environmental Chemistry, Bioremediation, New/Innovative technologies, Environmental Engineering, biotechnology, phytoremediation, remediation, biodegradation, bioacummulation, bioengineering, bioenergy, transgenic plants 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). 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. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||