Grantee Research Project Results

2012 Progress Report: Environmental Research and Technology Transfer Program of The Consortium for Plant Biotechnology Research, Inc.

Objective:

Iowa State University- Bryony C. Bonning

Objective of Research: The overall goal of this project was determine whether an aphid gut binding peptide such as GBP3.1 can be combined with the Bt-derived cytolytic toxin for production of aphicidal toxins. Our ultimate goal is to produce transgenic plants that are aphid resistant. The objectives of this research project were to (i) determine which peptide characteristics are essential for binding to the aphid gut, and (ii) insert the gut binding residues into a Bt-derived cytolytic toxin, and test the modified toxins for stability in and increased binding to the aphid gut, and for toxicity. We have successfully completed both project objectives and as a consequence have developed an effective novel approach for management of aphid pests.

University of Tennessee - Vincent Pantalone

The long-term objective of this research is to develop a commercially acceptable, environmentally superior, high yielding soybean variety with low seed phytate. We have four specific aims:

1. Production of low phytate soybean seeds to enable multiple environment field research and pilot plant research processing of soy meal.
2. Analytical testing of the improved low phytate soybean seeds.
3. Chicken feeding trials to document enhanced nutrition.
4. Insertion of a novel industry proprietary RFO transgene to soybean that modifies expression as an alternative strategy to improve livestock nutrition and metabolic growth rates.

North Carolina State University - Rongda Qu

Objectives:

1. Test AtAVP1, and maize CBP (calcium binding domain of careticulin) transgenic rice lines, and F1 progeny of the CBP-expressing transgenic rice lines crossed with AVP1-expressing transgenic rice lines for drought tolerance.
2. Test AtAVP1, and maize CBP (calcium binding domain of careticulin) transgenic rice lines, and F1 progeny of the CBP-expressing transgenic rice lines crossed with AVP1-expressing transgenic rice lines for salt stress tolerance.

Virginia State University - Shuxin Ren

Objective 1: Test the null hypothesis that there is no difference in field growth characteristics and seed traits (maturity, height, lodging resistance, seed yield, protein concentration, amino acid composition of the protein, oil concentration, fatty acid composition of the oil, and inorganic phosphorous content) between TN09-239 and USG 5601T.

Objective 2: Test the hypothesis that DNA collected from vegetative leaf tissue can distinguish whether the plant will produce seeds at harvest that will contain normal versus low levels of phyate, by evaluating two genetic loci: SSR markers SATT237 (chromosome 3, linkage group N) and SATT561 (chromosome 19, linkage group L).

Objective 3: Extend the knowledge of SSR molecular markers to develop a test of single nucleotide polymorphisms (SNPs) at two genetic loci controlling soybean seed phytate.

North Carolina A&T State University - Guochen Yang

Objectives:

• To perform salt tolerance assays of transgenic rice plants in collaboration with NCSU's CPBR-funded project.
• Student training through fellowship: actively engaged students in the area of plant tissue culture related to transformation, molecular breeding and environmental research.

University of Tennessee - Zong-Ming Cheng

Objective 1: To characterize the transgenic plants for enhanced tolerance to high and low temperature, drought and flooding.

Objective 2: To determine whether the PtXTH22 promoter can be used as a broad stress-inducible promoter.

Objective 3: To characterize the promoter of the PtXTH22 gene.

Iowa State University - Michael Kessler

The goal of this project is to develop bio-based resins, prepared by co-polymerization of agricultural oils (e.g., corn, linseed, and soybean) and derivatives, with commercially available co-monomers, for the manufacture of fiberglass-reinforced composites used in pultrusion processing. Pultrusion is a continuous manufacturing process for creating composites with a constant cross-section. In the pultrusion process (shown schematically in Figure 1 below), the fiber reinforcement is impregnated with the resin matrix and pulled through a heated die where it is shaped and cured in one continuous step.

Figue 1.Schematic of the pultrusion process

The specific objectives for the 2-year project are the following:

1. develop appropriate resin formulations with the right combination of processing viscosity, cure kinetics, and ultimate thermo-mechanical properties for pultrusion processing.
2. determine the best processing conditions for the pultruded fiberglass/bio-resin systems in order to maximize the thermo-mechanical properties of the composites and economics of the system, and
3. evaluate the long-term strength and durability of the composites compared to existing polyester materials through accelerated aging and environmental exposure testing.

University of Georgia - Andrew Paterson

Objectives:

• Paired-end sequencing of about 120,000 BACs, roughly equally sampling the two libraries described above, to be done by subcontract to a fee-for-service vendor selected on a competitive basis.
• Computational analysis of the resulting sequence assembly by several tests to identify possible errors, conducted in the PI's lab as described (Paterson, et al., 2009), including:
  1. Comparison to the order of sequence-tagged-sites along the (~1 cM density) cotton genetic map (Rong, et al., 2004), to investigate accuracy across regions of several cM or more;
  2. Comparison to the order of overgo and BAC-end sequences in BAC clones comprising the physical map (detailed above), testing contiguity of regions of less than the ~1 cM resolution of the genetic map, down to the ~100 kb average size of a BAC;
  3. Comparison to the Arabidopsis genome, the most complete angiosperm genome and the closest relative of cotton that has been fully sequenced. We have previously shown appreciable microsynteny and even some regions of macrosynteny between Arabidopsis and cotton (Rong, et al., 2005a). A sequence scaffold would not be expected to show long stretches of cotton-Arabidopsis synteny if incorrectly assembled. However, because there will surely be many (hundreds?) small rearrangements between these taxa, synteny information will be used only to support other lines of evidence, and the assembly will never exclusively rely on synteny to support any genome arrangement.

Georgia Institute of Technology - Gerald Pullman

Hypothesis 1: Natural redox agents control redox potential in developing female gametophyte and embryo.
Objective 1: Use LC/MS and LC/MS/MS to confirm and extend preliminary results to identify and profile ascorbic acid (reduced), dehydroascorbate (oxidized), glutathione (reduced) and glutathione disulfide (oxidized) in female gametophyte and embryo tissues.

Hypothesis 2: Redox potential controls somatic embryo development in vitro.
Objective 2: Run early-stage, maturation, and germination somatic embryo growth tests with natural redox chemicals.
Objective 3: Run early-stage, maturation, and germination somatic embryo growth tests with alternative redox chemicals.
Objective 4: Measure ascorbic acid (reduced), dehydroascorbate (oxidized), glutathione (reduced) and glutathione disulfide (oxidized) in somatic embryos to determine if target concentrations found in zygotic embryos are achieved during growth in vitro.

Hypothesis 3: Medium redox potential changes over time due to air exposure, tissue growth and medium components.
Objective 5: Determine medium redox potential change over time due to air exposure and tissue growth.
Objective 6: Develop methods to control medium redox potential to desired levels over time and in presence of growing somatic embryos.

Progress Summary:

Iowa State University- Bryony C. Bonning

Figure 1. CGALn and CGSLn bind to pea aphid gut BBMV
 more strongly than Cyt2aAa. (A) Pull down assays were 
conducted following incubation of activated Cyt2Aa, CGALn
or CGSLn and pea phid gut BBMV. Membrane bound toxin
wsa detected by western blot with anti-Cyt2Aa antiserum. 
Western blot images (below) were scanned and processed
using ImageJ to estimate the relative amount of activated
toxin in associated with pea aphid BBMV. Relative toxin
binding is shown for the mean ± SE for two pull-down
assays. DIfferent letters indicate the statistical significance
at p ≤ 0.05 (one-way ANOVA).

Figure 2. BIAcore surface plasmon analysis
of toxin binding to small unilamellarvesicles (SUV).
Sensorgram showing the real-time interaction 
between 6 µm Cyt2Aa, CGAL1, CGAL3, CGAL4, 
CGSL1, CGSL4 and immobilized pea aphid gut
membrane SUV. L1 chip surfaces were prepared
with 4999 RU of ligands. The sata shown are 
representative of two independent experiments. 

Figure 3. CGAL1 and CGSL4 cause
extensive damate to the pea aphid
gut epithelium. Transmission electron
micrographs show the intact apical surface
of the gut epithelial membrane with 
microvilli (NM) projecting into the gut lumen
(L) in aphids fed on control diet (Control). 
The microvilli of Cyt2As-fed asphids showed
some damage, consistent with the low level of
toxicity seen again A.pisum with this toxin. The
integrity of the gut phithelia of aphids fed on
CGAL1 and CGSL4 was severely compromised.
Mocrographs were taken at the same 
magnification with the 1 µm scale indicated
for the Control. 

Figure 4. Relative binding of wild type 
Cyt2Aa, CGAL134 and CGSL14 to pea aphid
gut BBMV in pull down assays. 

In 2009, through backcrossing and molecular marker technology, we created the low phytate line TN09-239.  We validated gene transfer of two low phytate recessive genes into our high yielding commercial line 5601T by utilizing the simple sequence repeat (SSR) markers Satt 237 and Satt 561. These markers are linked to chromosomal locations on linkage group N (Chromosome 3) and L (Chromosome 19) that govern soybean seed phytate (Scaboo, et al., 2009). In that work, our lab confirmed QTL cq-Pha-001, a major allele affecting seed phytate, and cq-Pha-002, which has a smaller affect. This remains a major accomplishment of this research and has allowed us to move forward.

 

Our success in confirming the two low phytate soybean loci led to increased collaboration with USDA-ARS scientists (Drs. Bilyeu and Gillman) at Columbia, MO, in the discovery of soybean gene sequence with complete association between the low phytate phenotype in soybean and homozygosity for mutant alleles of the two low phytic acid lpa1 homologs (Gillman, et al., 2009).  Molecular marker assays then were designed as perfect markers for the low phytate trait so that molecular breeders can use SNP technology to directly select with absolute accuracy the mutant alleles that control the low phytate phenotype. 

 

Over the duration of this research we secured additional funding and institutional investment to purchase a new Roche Lightcycler 480 for the University of Tennessee Soybean Molecular Marker Laboratory, which upgraded our DNA laboratory and enabled us to utilize the newly developed SNP markers. Based on double helix melting temperatures of PCR products using SNP simple probes in our laboratory, we have increased our efficiency of detecting the two low phytate alleles as demonstrated in Table 1 and Figure 1. We also have been able to measure inorganic phosphorous concentrations using a modified version of a colorimetric assay developed by Raboy, et al. (2000) and measured with a BioTek PowerWave spectrophotometer.

 

We produced seed stocks in the 2010 winter nursery to enter low phytate TN09-239 to the 2010 USDA Southern Uniform Preliminary Test. The experiment was planted at 12 field trial sites in 9 states, with 2 replications, throughout the southern region:  Rohwer, AR, Pine Tree, AR, Ullin, IL, McCune, KS, Pittsburg, KS, Queenstown, MD, Portageville, MO, Stoneville, MS, Plymouth, NC, Kinston, NC, Jackson, TN, and Warsaw, VA.  The multi-environment field test was used to gauge the agronomic and seed quality performance of TN09-239 relative to its recurrent parent commercial cultivar 5601T, in order to gauge commercial opportunity. The low phytate line TN09-239 was equivalent to its recurrent parent 5601T for seed protein concentration (405 g Kg-1 for TN09-239 vs. 403 g Kg-1 for 5601T), seed oil concentration (202 g Kg-1 for TN09-239 vs. 201 g Kg-1 for 5601T), reaction to soybean cyst nematode HG types 1.2.5.7 and 5.7, stem canker resistance score, flower, pubescence, and pod color, and days to plant maturity. However, the seed yield of the low phytate line TN09-239 (2,714 Kg ha-1) was 85% the yield of 5601T (3,185 Kg ha-1) and not at commercial yield levels. It was notable that TN09-239 (at 114 cm plant height) was significantly taller than 5601T (at 74 cm plant height). This contributed to higher plant lodging score and reduced seed yield in TN09-239.

 

Thus, we had to continue to use single nucleotide polymorphism (SNP) technology, specifically our confirmed molecular markers cq-Pha-001and cq-Pha-002, to select individual plant progenies with 100% accuracy for each of the two low phytate genes. We have used SNP markers this year to confirm that our most recent phytate lines are double homozygous recessive for the two alleles that express low phytate concentration in soybean seeds. These lines are BC5 derivatives of the high yielding recurrent parent commercial cultivar 5601T and donor low phytate line TN09-239, both developed by our program.

 

The BC5 derived lines 56CX-1273 and 56CX-1283 have as common parents 5601T and our donor low phytate line TN09-239.  We had speculated in the past that TN09-239 was segregating at the Dt1 locus on chromosome 19 (LGL) where the dominant form of that locus has the indeterminate growth habit. The two genes for low phytate were introgressed originally from the indeterminate line CX 1834-2, and one of the genes (Pha-002) resides on LG L, approximately 18 cM from the Dt1 locus. Selection for that low phytate locus likely brought along the dominant form of the relatively close Dt1 locus. The indeterminate trait (Dt1) in a MG V soybean would lead to excessive plant height, contribute to lodging, and possibly reduce yield. The fifth backcross that we performed to the recurrent parent 5601T was done specifically to harvest BC5F2 single plants that appeared determinate (dt1dt1), and to increase recovery of the parent genome 5601T.  Both 56CX-1273 and 56CX-1283 demonstrated a determinate growth habit in our yield trial, which likely contributed to their increased yields. Breaking the genetic linkage between the indeterminate trait and the second low phytate locus was an important milestone accomplished this year.

 

A major goal of this project was to develop an environmentally and agronomically superior soybean variety with low seed phytate.  Towards that aim, we now have developed two promising BC5 derived lines that are high yielding, determinate, and that continue to display high seed germination.  It also should be noted that both 56CX-1273 and 56CX-1283 were earlier in maturity than 5601T.

 

For 2013, we intend to test these two new lines, 56CX-1273 and 56CX-1283, in the Southern Uniform Quality Trait Test (MG V). The experiment will be planted in Stuttgart, AR, Kaiser, AR, Portageville, MO (clay), Portageville, MO (loam), Knoxville, TN, Blacksburg, VA and Warsaw, VA.  This past year in 2012, we conducted a yield trial in Knoxville, TN, and Milan, TN, for 10 of our most promising BC5 low phytate lines and data analyzed from this test showed that two of our low phytate lines, 56CX-1273 and 56CX-1283, yielded at 97% and 96%, respectively of 5601T, their recurrent parent and high yielding commercial cultivar. This is a noteworthy accomplishment, because experimental lines bred for the low phytate trait typically do not yield as high as lines bred directly for yield. An important goal of this project is to remedy the yield limitations of low phytate soybean, and we were encouraged by the yield performance of these two lines in our test. We now will be able to increase seeds for further tests of seed quality and performance.

 

Genotype	Mat	yld bu/A	tld rank	LG N Chomosome 2 melting temperature	LG L Chromosome 19 melting temperature	µg Pig-1	Growth habit	Protein
5601T	282	69.2	1	57 0C	60 0C	201	dd	40.7
TB09-239	281	62.4	7	62 0C	54 0C	1560	DD	41.8
56CX-1273	280	67.0	2	62 0C	54 0C	1808	dd	41.3
56CX-1283	281	66.8	3	62 0C	54 0C	1555	dd	41.9

Table 1. Effective genotyping helped us select these low phytate lines with improved
agronomic characteristic

Figure 1. SNP Melting Curves

North Carolina State University

 

Drought and salinity are the leading causes for crop loss around the globe. Calcium and other nutrients play important roles in plant response to drought and salinity. It was previously shown that constitutive expression of CBP, the calcium binding domain of the maize calreticulin gene, and AVP1, the Arabidopsis gene encoding H+-pyrophosphatase, each confers drought tolerance in transgenic Arabidopsis plants. We introduced 35S:CBP and AVP1 driven by the CaMV 35S promoter separately into cultivar Taipei 309 of rice, a model cereal crop plant, and then crossed them to see their effects on elemental homeostasis and the abiotic stress response. Confocal microscopy revealed that the CBP is predominantly located in ER. We used inductively coupled plasma (ICP)-emission spectrometry to quantify elemental composition and found that the total amount of calcium and phosphorous was higher in F1 progeny of the CBP x AVP1 cross compared to control plants. F1 progeny from CBP-, AVP1- and the cross of CBP x AVP1-transgenic plants all exhibited better drought tolerance than controls. Transgenic plants and F1 progeny from CBP x AVP1 transgenic rice plants showed higher leaf chlorophyll content and relative water content (RWC), and less wilting after intermittent drought treatment. Panicle seed yield was higher in transgenic plants and their F1. We also have tested our transgenic rice plants for salt tolerance and F1 progeny from CBP-, AVP1- and the cross of CBP x AVP1- transgenic plants all exhibited improved salt tolerance than control rice lines. They lost less chlorophyll after two different salt treatments and survived longer after the salt treatments. OsCIPK5 expression was nearly doubled in CBP transgenic plants, implicating a role of this protein in CBP-mediated drought and salt tolerance. The F1 plants performed similarly to the T1 transgenic plants in both tests and better than the control plants. However, no additive or synergistic effects were observed when both transgenes were in the same plants.

Figure 1. Analysis of FGP and FGP-CCP-expressing rice plants. A. Schematic diagram of the
ER-targed GFP-CBP and its control, fgP. B. Southern blot analysis of six independent transgenic lines.
They are )left to right): FPB-CBP 8, 4, 7. 12, 14 and FGP-Lowl1, a FFP transgenetic line as a control
(DIG-labled probe: 700 bp FGP coding sequence). C. qRT-PCR analysis to determine GFP or FGP-CBP
expression in 20 independent transgenetic rice plants. D. Western blot analysis of five GFP-CBP
transgenic lines. They are FGP-CBP 8, 15, 16,04 and 7. Anitbody against FGP was used for the Western
blot. No breakdown products were apparent

 

Figure 2.  The GFP-CBP fusion protein localization in the endoplasmatic reticulum was verified
using a Zeiss confocal laser scanning microscope. A - C, rice root cells in ER-GFP only (A) and
ER-FGP-CBP (B and C) transgenetic rice seedlings, D, root hairs of a GFP-CBP transgenetic rice
seedling, and E; rice root cells of non-ER-targeted GFP transgenetic plants. Rice seeds were
germinated on half strength MS media and grown for one week. 2 cm rice roots were taken from
the rice seedlings and mounted on a microscope slide. 

Figure 3. TOtal calcium content in control rice plants and AVP1-, GFP-, CGP-CBP- and GFPCBPx AVP1
transgenetic rice plant lines. A. Total calcium content in FGP-CBP transgenetic rice plants is increased. 
Total calcium content in the rice leaves of each transgenetic plant lines was measured three times from three
different biological samples. * means they are significanly different (P<0.05). B. Control and transgenetic
rice plants exhibiting normal phenotypes 6 weeks after germination. From left to right: 1. wild type, 2: GFP,
3. AVP1-1, 4. AVP1-2, 5: GFP-CBP Low, 6: GFP-CPB High, 7: GFP-CBPlow % AVP1-2, 9: FGPCBP High %
AVP1-1, 10: GFP-CBP High % AVP1-2

 

Figure 4. A: 1, WT, 2, GFP,3. AVP1-1, 4. AVP1-2, 5. GFP-CBP-Low, 6. GFP-CBP-High, 7.
GFP-CBO-Low % AVP1-1, 8: GFP-CBP-High % AVP1-2, Plants were not watered for 6 days
and then watered. The picture taken wafer withholding water for another 6 days after watering
them. B: Relative water content of transgenic and control rice plants. They were measured three
times; one time point before intermittent drought treatment and two time pints during intermittent
water stress. C. Chlorophys content of transgenic and control rice plants. The chlorophyl content
was measured before and after drought treatments. D. The longest shoot length of control and 
drought treated rice plants. E. The seed weight of seeds per panide in 10 different transgenic rice
lines. All of the transgenic lines show higher seed weight  compared to the control lnes. 

 

 

Figure 5. AVP1 and GFP-CBP shows increased salt tolerance. A and B, After 12 days of 
100 mM(Fig a) and 200 mM (Fig b) NaCl treatment. 1. Wt, 2. GFP, 3. AVP1-1, 4. AVP1-2, 5. 
GFP-CBP-Low, 6. GFP-CBP-High, 7. GFP-CBP-Low % AVP1-1, 8. GFP-CBP-Low % AVP1-2, 9.
GFP-CBP-High % AVP1-1, 10. FGP-CBP-High % AVP1-2. C, chlorophyl content of transgenic
and control rice plants. The chlorophyl content was measured at two different concentrations of
NaCi before and after a period of 12 days. D, Shoot height of control and salttreated rice plants. 

Figure 6. Rice CIPK5 (Os)1g0206700), a homologue of Arabidopsis AtCIPK6, is
unpregulated in transgenic plants expressing CBP, indicating a role in CBP-mediated
drought/salt tolerance. The left axis shows fold change relateive to wild type rice. 
Error bars represent the standard error of three different experiments. 

 

Virginia State University

This project was implemented at the Virginia State University from mid of 2010 to the end of 2012. Soybean lines, TN09-239 and USG5601T, were provided by Dr. Pantalone at University Tennessee and grown in VSU Randolph Farm in 2011 and 2012 in a randomized complete block design, with four row plots. Agronomic traits were recorded and compared between TN09-239 and USG5601T.

Both lines showed white flower and gray pubescence. In addition, they all have same flowering date and are resistant to shattering (score 1). TN09-239 and USG 5601T are all in maturity group 5 (MG V), however TN09-239 is in early MG V, while USG 5601T is in late MG V. In terms of 100 seeds weight, TN09-239 is little bit higher (22 g) than 5601T (21 g) but no significant difference. Although these two soybean lines were considered as near isogenic line with only difference at seedphytate level, we do find that the plant heights between TN09-239 and 5601T were significantly different with TN09-239 of 131.6 cm and 5601T of 68.0 cm. Because of this height difference, TN09-239 appeared more susceptible to lodging stress. This plant height difference may due to environmental difference. However, under this experimental design, we only include one location. Further evaluation at multi-locations on this issue is required to confirm this result. The yields of these two soybean lines showed no significant difference.

In addition to agronomic traits evaluated in the field, the harvested soybean seeds also were analyzed for protein concentration, oil concentration, fatty acid composition of the oil, and seed phosphorous contents. Total protein content in TN-239 seed is 42.06% on average, and in 5601T seed is 41.36% on average. No significant difference was observed. Total seeds oil content for TN239 is 18.45%, and 5601T is 18.59%. Again, no significant difference was observed.  Oil fatty acid composition results showed that TN-239 contains significant more 18:0 fatty acid (4.0%) than 5601T (3.6%). On the other hand, TN-239 has significantly less (6.95%) 18:3 fatty acids than that of 5601T (7.87%). All other oil fatty acid compositions showed no difference between these lines.

Total P content in seeds also was evaluated in two biologically duplicated samples. Results showed that TN-239 has significantly higher phosphate level (1.31g/kg seed) that 5601T (0.52g/kg seed). However, the phytate contents in the seeds are variable from repeat to repeat with the trend that TN-239 has less phytate than 5601T. More investigation is needed to make a solid conclusion on seed phytate content.

We also collected leaves from all plots and isolated DNA from each collected sample. SSR markers SATT237 and SATT561 were used to determine the phytate levels between TN09-239 and USG 5601T. Although the PCR products were successfully amplified from both varieties, separation on 4% agrose gel could not distinguish these two varieties. SNP markers were used to identify genotypes that control phytate levels. As expected, SNP markers used can distinguish these two lines at loci controlling phytate levels.

On the educational propose of this fellowship, the student, together with PIs were traveled to TN. The student was successfully trained on DNA technology, including different DNA isolation methods, SSR markers and SNP markers. Exposure to these state-of-the-art biotechnology facilities will give the student more understanding of the application of biotechnology to agriculture production and stimulate the student’s passion to pursue a biotechnology-related education and career. In fact, the first student who was recruitied has now graduated and has secured funding to continue her education at the graduate level. In addition, a second undergraduate student who was recruited to the project, will be well trained on soybean breeding related techniques upon completion of this project.

North Carolina A&T State University

University of Tennessee

The work in this year has been focused on exploring the mechanism of stress tolerance in transgenic plants over-expressing PtXTH22 gene and functional characterization of the PtXTH22 gene promoter.

Figure 5. Relative mRNA expression  ratios of stress-related genes in Popuhus plants. Relative
mRNA expression opf the gene was represresented as the value in the transgenic lines relative to 
value in the wild type. Line X was ABA-treated or untreated line 18 or 2-B, WT was represented by
wild type, and the target was the ABI1C, ABI3, CBF1, ERD1, bZIP28 genes. The value in Y-axis is -ΔΔCt.
The whole plants were collected from 4-week subcultured plants. 

In our initial work we investigated and optimized suitable bio-based resins for the pultrusion processing method. Among the variety of bioresins that have been developed at Iowa State overthe past 10 years, ROMP-based copolymer of modified linseed oil (Dilulin) and dicyclopentadiene (DCPD) is the first choice we want to develop in pultrusion processing. Because this resin system cures very fast with Grubbs’ catalyst, and the resulting polymer has good mechanical properties, it seems to be an ideal candidate for pultrusion processing. After evaluating several compositions, the optimized composition for this bio-based resin was selected at 30 wt% Dilulin and 70wt%DCPD (designated as Dil30DCPD70 for short). Another potential bio-based resin investigated for the pultrusion process is a copolymer of soybean oil, styrene and divinylbenzene (DVB), which is prepared from a cationic polymerization method. The optimized formulation is 50 wt% soybean oil, 25 wt% styrene and 23 wt% DVB, initiated with 2 wt% boron trifluoride (BFE). With the optimized composition, relatively good mechanical properties and suitable onset cure temperature were obtained. 

 

We noticed that Dilulin/DCPD bio-based resins were very tough, especially compared with commercially available thermosets, including the benchmark unsaturated polyester resins typically used in pultruded fiberglass composites. We have found that while the strength and stiffness of the bio-based Dil30DCPD70 ROMP polymers are lower than the polyester resin, the failure strain and toughness (area under the stress strain curve) are significantly higher than polyester as shown in Figure 2 below.

 

Figure 2. (left) Representative e stress-strain curves for bio-based resin 
and unsaturated polyester resin. (right) while the strength and stiffless 

(Young's modulus) is higher for the polyester resin, the ultimate strain and 
toughness is significanly higher for hte bio-based resin. 

 

Further studies were carried out with Dilulin/DCPD resin system and glass fiber reinforced composites. Essential work of fracture and J-integral fracture testing was carried out to study the fracture toughness and the toughening mechanism of Dilulin/DCPD bioresins. Among a series of compositions of Dilulin/DCPD resins, Dil30DCPD70 shows the highest toughness. We hypothesize that the toughness property is closely related to crosslink density for these thermosets. On one hand, with high crosslink density, more covalent bonds break on the fracture surface, absorbing more energy. On the other hand, high crosslink density usually results in lower shear yielding via network stretching and lower energy absorption in this step. So 30 wt% Dilulin and 70 wt% DCPD is the optimized composition that shows the highest toughness. The promising toughness property of Dil30DCPD70 may bring some potential applications for this bioresin, such as energy absorbing structures and impact resistance.

 

We also have been working on improving the interfacial adhesion of glass fiber reinforced Dil30DCPD70 bio-based resin with various silane coupling agents. Previous study of this composite system showed that poor interface exists, which usually results in lower mechanical properties. Our purpose is to improve the interfacial adhesion between the glass fiber and the bio-based resin matrix. Two types of silanes were applied, norbornenylethyldimethylchlorosilane (MCS) and norbornenylethyltrichlorosilane (TCS). Interfacial adhesion was evaluated by microbond pull-out technique, which showed improvement in the interfacial shear strength (IFSS) with both silanes. IFSS increased ~150% with MCS and IFSS increased ~50% with MCS, compared with as-received glass fiber (see Figure 3 below). Dynamic mechanical analysis (DMA) studies of silane-treated fiberglass/Dil30DCPD70 laminates showed increased storage modulus and glass transition temperature with both types of composites, especially the one containing MCS-treated fiber. Studies indicate that MCS works better than TCS on improving the interfacial adhesion in composites. So in the future, MCS will be applied to glass fiber in the pultrusion process to improve the mechanical properties of the pultruded composite.

 

FIgure 3. Interfacial shear strength (IFSS) for different types of fibers

 

We also have been evaluating the short and long term performance of Dil30DCPD70 bio-based resin under different conditions. Samples were treated in an accelerated weathering chamber at Pella Corporation with UV radiation (see Figure 4), full-spectrum sunlight and severe hot-wet-cold circular weathering exposure. Tensile test resultsof UV-treated samples show that tensile strength and Young’s modulus didn’t change within 1000 h UV treatment, and then decreased slightly with longer exposure time. However, break strain and tensile toughness decreased significantly due to the formation of surface cracks under even short time exposure. Similar results also show in full-spectrum sunlight exposure samples and hot-wet-cold circular exposure samples. Soxhlet extraction results indicate that crosslink density of the bioresin was enhanced a little after UV treatment. Photoacoustic spectroscopy and SEM both demonstrate that degradation is a surface effect. About a 100 µm thick degradation layer was observed on the sample exposed to the most severe condition in our experiment, 2000 h UV radiation.  Because the ROMP-based polymers contain many residual carbon-carbon double bonds (C=C bonds are not consumed in ROMP), we anticipate better environmental stability in the cationic polymerized bio-based resins (in which the C=C bonds are consumed during the cationic polymerization), which will be evaluated in Year 2.

 

Figure 4. Change is polymer appearnce afgter expsure t

QUV accelerated weathering experiments for times ranging
from 0 (left) to 100h (right)

 

A final area we have been working on is setting up a table-top pultrusion machine in our lab to estimate processing conditions for fiberglass/bioresin system (shown in Figure 5). The table-top pultrusion contains roller guide, resin bath, die, heat plates and puller. The die is 600 mm long with a rectangular profile of 25mm x 2mm. The die also was designed with water circulation system for controlling temperature in different heating zones. During processing, the die was divided into three heating zones, two relatively low temperature heating zones on both ends and one high temperature heating zone in the middle. We have made composites using both commercial polyester and the ROMP-based bioresin (Dil30DCPD70) (see Figure 6). We have overcome one of significant problems, sticking, with coating the inner surface of die with Teflon tape. Another significant problem we dealt with is increasing the pulling force of the nip roller puller by using a more powerful motor and designing a suitable gear train. The resulting pultruded fiberglass/Dil30DCPD70 composite is stiff, but results in composites with scratches on the surface. Release agent is being considered to improve the surface finish and processability for next step. Further optimizing of processing parameters is still under way.

 

Figure 5. Bench topo pultrusion syustem deisgned and built for this
project for continous manufacturing of bio-resin fiberglass composites.

Figure 6. Polyester and bio-resin samples made by the pultrusion process. 

 

Future Activities:

University of Tennessee

Now that we have the determinate linkage resolved, we can increase seed and follow up on testing for yield and nutrition for the trait.

North Carolina State University

The project is completed.

North Carolina A&T State University

Analyzing data, and preparing abstracts and manuscripts for presentations and/or publications.

University of Tennessee

1. Determinethe mechanism for drought and ABA tolerance of transgenic plants.
2. Test the transgenic soybean for drought tolerance.
3. Further test the pXTH22:GUS transgenic Arabidopsis to determine whether the promoter is multi-stress inducible.
4. Test different sections of promoter to determine which sections of promoter is responding different stresses.

Iowa State University

In the upcoming year, we plan to (1) continue to improve the process ability and appearance quality of pultruded fiberglass/Dil30DCPD70 composites with our table-top pultrusion machine; (2) evaluate short and long term strength and durability of the pultruded fiberglass/Dil30DCPD70 composites; (3) apply silane coupling agent in fiberglass/Dil30DCPD70 system to further improve the properties of the pultruded composites; and (4) apply the cationic polymerization resin system in thepultrusion process.

More specifically, we have carried out many trials with the ROMP-based Dil30DCPD70 bioresin system on our table-top pultrusion machine. The resulting pultruded composites have a rough surface, effecting the appearance of the composite material. This surface roughness is believed to be caused by a stick-slip progression during processing. Our plans for the coming year are to continue optimizing the processing parameters and to find the best combination of pulling rate, cure temperature and fiber ratio to produce pultruded composites with perfect profile shape and smooth surface. At the same time, we also will add some release agents into the resin. There are a wide range of commercially available release agents in the market. Finding the most suitable type and additive amount of the release agent, as well as the effect of release agents on final pultruded products also will be a part of our future work.

Once we can get good samples from repeatable trials, we will start to characterize the properties of the pultruded composites and compare them to the existing unsaturated polyester pultruded materials. A series of engineering properties that are necessary for structural materials will be evaluated following ASTM standards, such as tensile strength, Young’s modulus, flexure strength, flexure modulus, coefficient of thermal expansion, impact behavior, and compressive properties. The long-term performance of the bio-based pultruded composites will be evaluated with the help of our industrial partner and their extensive environmental chemistry and product test laboratories. Materials will be subjected to a range of elevated temperature and environmental conditions, including humidity, ultraviolet radiation, and full-spectrum sunlight. The effects of those conditions on discoloration, swelling, warping, delaminating, and change in mechanical properties will be analyzed systematically. Because we already studied the long-term performance of the neat resin in the first year of the study, these results will be helpful to gain an understanding of the effect of reinforcement on the long-term performance of pultruded composites.

In the first year of the project, we have investigated the effect of using silane coupling agents on improving the compatibility and adhesive strength between the glass fibers and the bio-based resin. Our studies indicate that interfacial strength between glass fiber and Dil30DCPD70 bio-matrix is increased significantly with the application of norbornenylethyldimethylchlorosilane coupling agent. So applying this silane in the pultrusion process will further enhance properties of the final pultruded composites. The challenge is developing a process for applying the silane in a continuous pultrurion process. Usually, in industry, silane coupling agent can be applied in a solution or in vapor phase. We will compare these two methods first in batch processed conditions that involve treating the glass fiber with silane solution or silane vapor prior to the pultrusion process. Then we will try to add the silane coupling agent during the pultrusion process. Special equipment will be designed to fit the whole continuous pultrusion process. Taking the silane solution method for example, a silane solution bath and a drying tube furnace will be implemented before resin bath in the pultrusion process line.

Our efforts in the second year also will be focused on the cationic polymerization resin system - soybean oil, styrene and divinylbenzeneapplied to the pultrusion process. Following similar experiments as used for the ROMP-based bioresin, processing parameters and a suitable release agent will be optimized first. One challenge for cationic-based bioresin is stabilization of the resin after mixing with initiator. It is difficult for this resin to remain stable at room temperature after it has been mixed with the cationic initiator, especially in large amounts where cure exothermic conditions can increase the temperature of the resin bath. Lowering the resin storage temperature is one approach to increasing pot life of the resin. Towards this end, a special resin bath will be designed for the cationic resin system. Once we can get good samples from pultrution, short-term and long-term performance will be evaluated and also compared with unsaturated polyester materials and the ROMP resin system. 

University of Georgia

Paired-end sequencing of about 120,000 BACs – this has been completed.  Indeed, while our proposal was under consideration by CPBR, another (public) entity supported end-sequencing of about 60,000 BACs from the G. raimondii HindIII library.  Accordingly, the PI and representatives of the matching company (Bayer) agreed to an accordingly reduced investment in BAC end sequencing (that has been met through a contract with a third party, the Hudson Alpha Institute) … redirecting the remaining resources to reduced-representation resequencing of about 30 cotton genotypes, about 14 of which are selected by Bayer for proprietary reasons. The remainder will be selected from publicly available germplasm by the PI but in consideration of feedback from Bayer, giving preference to genotypes from which there exist other publicly available resources (for example Pima S6 and Acala Maxxa have BAC libraries, and G. barbadense K101 was used in reference genetic mapping), and/or which are parents of populations that are being made toward a cotton nested association mapping (NAM) resource.  Several of the public lines have been tentatively completed, although data validation remains pending.  Bayer is preparing DNA of its lines for shipment to the PI. 

Computational analysis of the resulting sequence assembly by several tests to identify possible errors, conducted in the PI’s lab as described (Paterson, et al., 2009), including:

1. Comparison to the order of sequence-tagged-sites along the (~1 cM density) cotton genetic map (Rong, et al., 2004), to investigate accuracy across regions of several cM or more.
2. Comparison to the order of overgo and BAC-end sequences in BAC clones comprising the physical map (detailed above), testing contiguity of regions of less than the ~1 cM resolution of the genetic map, down to the ~100 kb average size of a BAC,
3. Comparison to the Arabidopsis genome, the most complete angiosperm genome and the closest relative of cotton that has been fully sequenced.  We have previously shown appreciable microsynteny and even some regions of macrosynteny between Arabidopsis and cotton (Rong, et al., 2005a). A sequence scaffold would not be expected to show long stretches of cotton-Arabidopsis synteny if incorrectly assembled. However, because there will surely be many (hundreds?) small rearrangements between these taxa, synteny information will be used only to support other lines of evidence, and the assembly will never exclusively rely on synteny to support any genome arrangement.

Items a and b, above, were completed in advance of public release (under Ft Lauderdale principles) of the genome assembly in January 2012. 

Item c was completed as an element of the primary description of the cotton reference genome sequence (Paterson, et al., 2012), confirming early results supportive of the high quality of assembly, and revealing an unexpected dimension of cotton genome evolution, specifically that it has experienced a paleopolyploidy of complexity previously unprecedented … it remains unclear whether this resulted in an aggregate 5x or 6x increase of cotton chromosome numbers, and whether it was a single event or two very closely spaced events (for example 3x followed by 2x).

References:

Febvay, G., B. Delobel & Y. Rahbe (1988) Influence of the amino-acid balance on the improvement of an artificial diet for a biotype of Acyrthosiphon-pisum (Homoptera, Aphididae). Canadian Journal of Zoology-Revue Canadienne De Zoologie, 66, 2449-2453.

Li, J., Koni, P. A. and Ellar, D. J. (1996). Structure of the mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis sp. kyushuensis and implications for membrane pore formation. J. Mol. Biol. 257, 129-152.

Liu, S., Sivakumar, S., Sparks, W.O., Miller, W.A., Bonning, B.C. (2010). A peptide that binds the pea aphid gut impedes entry of Pea enation mosaic virus into the aphid hemocoel. Virology 401 (1): 107-16.

Pérez C., Fernandez L.E., Sun J., Folch J.L., Gill S.S., Soberón M. (2005). Bacillus thuringiensis subsp. israelensis Cyt1Aa synergizes Cry11Aa toxin by functioning as a membrane-bound receptor. PNAS, vol. 102 no. 51 18303-18308.

Supplemental Keywords:

Relevant Websites:

Cotton Incorporated Exit

Progress and Final Reports: