ENERGYCANE GENETIC IMPROVEMENT FOR SOUTHEAST UNITED STATES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1001784
• Project Start Date: Nov 5, 2013
• Project End Date: Oct 31, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Weslaco-TAMU Agr Res Cntr

Non Technical Summary
The goal of this multidisciplinary initiative is to develop new sugarcane germplasm and a new cropping system based on a collection of dedicated sugarcane feedstock and sustainable production systems for the candidate variety in a range of rainfall intensity, temperatures and soil type environments of Florida. For this, we will genetically improve sugarcane using commercial germplasm and related species for the climatic/edaphic environments of SE U.S., identifying high biomass germplasm and other wild species for future use in sugarcane hybridization development, aiming at drought/cold tolerance to cross with sugarcane, in addition to using DNA markers for marker-assisted breeding.

Animal Health Component

50%

Research Effort Categories

Basic

(N/A)

Applied

50%

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
202	2030	1081	30%
203	2030	1081	40%
204	2030	1081	30%

Knowledge Area
204 - Plant Product Quality and Utility (Preharvest); 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 202 - Plant Genetic Resources;

Subject Of Investigation
2030 - Sweet sorghum;

Field Of Science
1081 - Breeding;

Keywords

enhanced biomass yield and stress tolerance

large-scale multiplication process, using modern plant-tissue culture methods

Goals / Objectives
Screening of existing germplasm collections of high-biomass energy canes and "Miscane,™" in addition to other promising bioenergy grasses, for high yield under low input conditions; Interspecific/Generic crossing and introgression to transfer unique traits from Miscanthus, Erianthus, and S. spontaneum to enhance biomass yield, cell-wall composition, stress tolerances, and adaptation; Selection of cane wide-hybrid elite lines with high biomass and adaptation to low-input environments; Use of Bioreactors, a large-scale multiplication process, using modern plant-tissue culture methods (micro propagation), to accelerate seed cane production of new germplasm; Development of a high throughput method for marker-trait association using next generation DNA sequencing technology. This technology is scalable/adaptable and complementary to other marker discovery methods.

Project Methods
Around 30,000 seedlings, produced from true seed resulting from hybridization crosses, will be planted in the field every year as families in a replicated trial (family assessment), to be harvested during the winter (December/January). Biomass yield of cane will be measured with a weigh wagon. The family assessment made with these results will drive the selection rate between families. The seedlings will then be selected (individual plants within families) on the first ratoon stage, with a selection rate between 5 and 10% to Stage 2. Stage 2 trials, with around 2,000 entries, will consist of plots with one row, 30 feet long. At one year of age, all clones will be assessed for vigor and resistance to pests and diseases with the best 400-600 genotypes being selected for promotion to Stage 3 - Clonal Assessment Trial (CAT) and planted on a nursery of 1 x 7-foot plots (Nursery I). Stage 3 trials will consist of one-row plots of 30 feet, replicated three times, with around 500 entries. Each entry will be evaluated for vigor, plant architecture and resistance to pests and diseases. Stage 3 will be harvested with plots being weighted and sampled for cell wall composition analyses using Near Infra Red. Based on these results the best 30% lines will be expanded on a nursery of one row, thirty feet long (Nursery II). Data collected on the first ratoon crop of Stage 3 will be used to select for advancement to Stage 4, which will be planted with three replications of four rows of 30 feet with seed from Nursery II. Each plot will be scored for vigor and sucrose content, and the average performance across replications will be calculated. The best 35-50 clones will be advanced to Stage 4 - Cooperative Trials (CT). Stage 4 - CT trials will be planted with 3 replications of four rows of 30 feet. Each plot will be harvested with plots being weighted and sampled for juice and cell wall composition analyses, and an Analysis of Variance will be performed for all trials. The results of the plant crop will be used to propagate the best 15% (ten entries) in tissue culture (TC), using Bioreactors (TC Nursery) with seed from Nursery II. Stage 4 - CT will be further harvested on first and second ratoons, and the best 5-10 varieties will be advanced to Stage 5 - Agronomy Assessment Trials (AAT). Stage 5 - AAT will be planted at six different sites, representing the different soil types and growing conditions of the South East US. These trials will have 3 replications of 6-row plots of 60 feet, laid out as a complete blocks design, with around eight entries, having two current commercial varieties as controls. The trials will be harvested at the plant crop, as well as first and second ratoons. At each harvest, cane yields will be recorded, and a samples collected for sucrose content, cane quality and cell wall composition parameters. The data will be analyzed by appropriate statistical methods and used to identify the most promising candidates for commercial release. Candidate varieties identified in the plant cane crop will be cleaned from diseases through meristem tip multiplication and propagated at the Texas A&M AgriLife Annex farm in Weslaco, Texas. Those candidate varieties that perform well on the first ratoon crop will be established at Texas A&M Agrilife Research Centers in Corpus Christi and Beaumont. This will ensure that seedcane will be available for growers, should the variety be released for commercial production. The seed increase plots will also be useful for additional observations on disease and agronomic characteristics. Decision on variety release will be made in conjunction with the private sector staff and management.

Progress 11/05/13 to 10/31/18

Outputs
Target Audience:Rio Grande Valley Sugar Growers Inc. Private sector oil companies Changes/Problems:No funding of grant proposals submitted to sponsors. What opportunities for training and professional development has the project provided?Hiring of local undergraduate student How have the results been disseminated to communities of interest?Presentation to potential sponsors. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Energy cane germplasm with high total cellulosic production in sub-tropical areas created.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Fernandez, C.J., Da Silva, J.A., Correa, J.C., Grichar, W.J. 2018. Biomass Production and Water Economy of Sugarcane and Energy Cane Genotypes Grown in Water-Deficient and Well-watered Regimes. Journal of Experimental Agriculture International 20(1): 1-15.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Elsayed-Farag, S., Silva, M. Solis-Gracia, N. Enciso, J., Da Silva, J. 2018. An agronomic approach to screen sugar and energy cane genotypes for drought tolerance. Afr. J. Agric. Res. V.13(23):1220-26.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Yang, Y, Wilson, L.T., Jifon, J., Landivar, J, Da Silva, J. A., Maeda, M., Wang, J., Christensen, E. 2018. Energycane Growth Characteristics and Yield Potential along the Texas Gulf Coast. Biomass and Bioenergy, 113 :1-14. https://doi.org/10.1016/j.biombioe.2018.03.003.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Da Silva, J. A.; Costa, P.M.A.; Marconi, T.G.; Barreto, E.J. S.; Gracia, N.S.; Park, J.W.; Glynn, N.C. 2018. Agronomic and Molecular Characterization of Wild Germplasm (Saccharum spontaneum) for Sugarcane and Energycane Breeding Purposes. Scientia Agricola. Sci. v.75, n.4: 329-338.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Private and public sector enterprises that produce sugar, bio-energy or biofuels. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Oral presentation to principals of Exxon, Inc. to discuss energy cane for South America; Non-disclosure agreement (NDA-001608) between Bioenergy Crops Ltd and The Texas A&M University System to recommend energy cane germplasm to clients overseas; Received visits and had discussions withVERSA for transfer of germplasm for producing pellets for energy. What do you plan to do during the next reporting period to accomplish the goals?Apply micro-propagation to mass multiply sugarcane varieties for the sugarcane commodity group in South Texas; Continue the research with sugarcane wide hybrids to create germplasm for forage and bioenergy.; Breed cassava (Manihot esculenta) with its wild relative and endengered speciesM. walkerae for stress resistance, aiming at starch production.

Impacts
What was accomplished under these goals? Goals1 - 4 have been accomplished.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Da Silva, J. A.; Costa, P.M.A.; Marconi, T.G.; Barreto, E.J. S.; Gracia, N.S.; Park, J.W.; Glynn, N.C. 2017. Agronomic and Molecular Characterization of Wild Germplasm (Saccharum spontaneum) for Sugarcane and Energycane Breeding Purposes. Scientia Agricola. Accepted. Manuscript ID: SA-2017-0028.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Da Silva, J. A. 2017. The Importance of the Wild Cane Saccharum spontaneum for Bioenergy Genetic Breeding. A Review. Sugar Tech 19: 229. DOI: 10.1007/s12355-017-0510-1

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Private and public sector enterprises that produce sugar, bio-energy or biofuels. Changes/Problems:Texas A&M AgrLife Research and BP Biofuels had started a sponsored resarch progam entitled "Perennial Energy Grass Breeding Program", for the duration of three years, renewable for another three years, pending progress. However, a decision was announced by BP Biofuels to shift the focus of its global biofuels business and no longer fund research in this area, given the substantial reduction in oil prices, resulting in the discontinuation of the work to develop ligno-cellulosic ethanol technology. The referred research program was going very well and I was confident on its renewal and working on securing a renewal for the next three year phase of theresearch. In addition, on February 18, 2015, the Rio Grande Valley Sugar Growers, Inc. (RGVSG) Board of Directors made the decision to rescind all funding for the A&M Sugarcane Variety Improvement Program. The RGVSG Board of Directors has also decided to no longer fund our research programs, given (1) disagreements with the Texas A&M Office of Technology Commercialization to charge Intellectual Property fees on the Texas A&M sugarcane germplasm and (2) its difficult financial situation. What opportunities for training and professional development has the project provided?One college undergraduate student has been trained in tissue culture and molecular biology. How have the results been disseminated to communities of interest?Oral presentation entitled "Can we break the sugar content Plateau and create cold resistant sugarcane by Crossing with S. spontaneum?" was madeat the 2015 American Society of Agronomy, Crop Science Society of America, Soil Science Society of America Annual Meeting, Minneapolis, Minnesota; Oral presentation entitled "Using Next Generation Sequencing for tagging stress resistance genes from S. spontaneum" was made at the International Consortium of Sugarcane Biotechnology Workshop. XXIII Plant & Animal Genome Conference, San Diego, USA. 01/11/2015. What do you plan to do during the next reporting period to accomplish the goals?Continue the research on screening different germplasm for tolerance to abiotic stresses, sucha as drought, salinity and cold; in order to identify material that is resistant to these stress and use in hybridization crosses; Use the photoperiod/crossing facilitites present at the Texas A&M AgriLife Research and Extensioin Centier in Weslcao, Texas to make hybridizatin crosses, using sugarcane and other Saccharum spp. germplasm available at this center; Condut and harvest the yield competiotn trials that are currently planted at the Texas A&M AgriLife Research and Extensioin Centier in Weslcao, Texas and select the cane wide-hybrid elite lines shoeing high biomass and adaptation to low-input environments; Aprove research grants and obtain resources to Develop high throughput methods for marker-trait association using next generation DNA sequencing technology.

Impacts
What was accomplished under these goals? A total of 110 Saccharum spontaneum accessions obtained from the World Collection of Sugarcane and Related Grasses in Miami, FL has been characterized phenotypically - morphological and agronomic traits, including composition analysis, and genotypically - molecular markers (Simple Sequence Repeats - SSR). Using these data, a core collection has been established and is being used for choosing parents for hybridization crosses aiming at sugarcane and energy cane genetic improvement.

Publications

• Type: Book Chapters Status: Published Year Published: 2016 Citation: Park, J-W and da Silva, J. 2016. Hybridization of Sugarcane and other Grasses for Novel Traits. In: Compendium of Bioenergy Plants: Sugarcane. Lam, E., Carrer, E., Da Silva, J.A. and Cole, C. (Eds.) CRC Press 129 pp.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Arro, J., Park, J-W., Wai, C.M., VanBuren, R., Pan, Y-B, Nagai, C., da Silva, J., Ming, R. 2016. Domestication of autopolyploid sugarcane (Saccharum officinarum L.) was attributed to balancing selection. Euphytica. DOI: 10.1007/s10681-016-1672-8.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Dos Santos, F.R.C., Zucchi, M.I., Park, J-W, Benatti, T.R., da Silva, J.A., Souza, G.M., Pinto, L.R. 2016. New Sugarcane Microsatellites and Target Region Amplification Polymorphism Primers Designed from Candidate Genes Related to Disease Resistance. Sugar Tech. DOI 10.1007/s12355-016-0457-7.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Can we break the sugar content Plateau and create cold resistant sugarcane by Crossing with S. spontaneum? at the 2015 American Society of Agronomy, Crop Science Society of America, Soil Science Society of America Annual Meeting, Minneapolis, Minnesota.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Biofuels Industry and growers. Scientific Researchers in the US, Braziland other countries. Changes/Problems:Onecommodity group interrupted funding due to Intelectual Property conflict of interest; One private sector funding agency has discontinued the project, and two potential clients have rejected grant proposals on Biofuels, due to low price of liquid fuels. What opportunities for training and professional development has the project provided?One Pos Doc fellow has been hired and one graduate student from Brazil has spent one year of training at the Texas A&M Agrilife Research and Extension Center in Weslaco, Texas. How have the results been disseminated to communities of interest?Progress report has been written and scientific papers have been submitted at one International Conference and one workshop in Brazil. What do you plan to do during the next reporting period to accomplish the goals?Multiply and advance newly created germplasm; screen germplasm bank for stress resistance/tolerance; apply Genomics to develop molecualr markers to be used in molecular breeding.

Impacts
What was accomplished under these goals? 1. Wild germplasm has been identified with resistance to abiotic stress (cold, drought and salinity); 2.Thousands of S. spontaneum x sugarcane F1 hybrids and hundreds of energycane elite lines using its germplasm bank with 120 accessions of S. spontaneum and its photoperiod/crossing facilities have been created; 3. This research and development program produced a group of new, purpose-built and high-yielding perennial energy grasses, adapted to a range of climatic (rainfall intensity, temperatures) and edaphic (soil types) environments of the South and Southeastern United States. Sustainable production systems for these germplasm were developed. In addition to high yield, these new high-biomass crops have their biomass composition optimized for liquid biofuel production; 4.Propagatioin material of new energy cane and cassava germplasm has beenproduced using Bioreactors;

Publications

• Type: Book Chapters Status: Published Year Published: 2015 Citation: Park, J-W and da Silva, J. 2015. Hybridization of Sugarcane and other Grasses for Novel Traits. In: Compendium of Bioenergy Plants: Sugarcane. Lam, E., Carrer, E., Da Silva, J.A. and Cole, C. (Eds.) CRC Press 129 pp.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Park, J., Benatti, T., Marconi, T., Yu, Q., Gracia, N. S., Mora, V., da Silva, J. 2015. Cold Responsive Gene Expression Profiling of Sugarcane and Saccharum spontaneum with Functional Analysis of a Cold Inducible Saccharum Homolog of NOD26-Like Intrinsic Protein to Salt and Water Stress. PLoS ONE 10(5): e0125810. doi:10.1371/journal.pone.0125810
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Pedrozo, C.A. Jifon, J., Barbosa, M.H., da Silva, J.A., Park, J.-W. and Gracia, N.S. 2015. Differential, Morphological Physiological and Molecular Responses to Water Deficit Stress in Sugarcane. Journal of Plant Breeding and Crop Science. Vol. 7 (7): 226-233. DOI: 10.5897/JPBCS2015.0500.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Dos Santos, F.R.C., Zucchi, M.I., Park, J-W, Benatti, T.R., da Silva, J.A., Souza, G.M., Pinto, L.R. 2015. New Sugarcane Microsatellites and Target Region Amplification Polymorphism Primers Designed from Candidate Genes Related to Disease Resistance. Molecular Breeding.

Progress 11/05/13 to 09/30/14

Outputs
Target Audience: Private sector companies in the Biofuels and Cellulosic Ethanol business, such as Chevron Technology Ventures, BP Biofuels and CANERGY. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? It served to train a Post Doctoral student on Next Generation Sequencing. How have the results been disseminated to communities of interest? Through quarterly repoorts. What do you plan to do during the next reporting period to accomplish the goals? I will conduct the project giving priority to: (1) biomass production, aiming at increasing fiber quantity/quality for energy production; (2) adaptability to the South and Southeastern U.S. and mechanical harvest; (3) stress resistance, including cold tolerance, aiming at tailoring the crop for less than optimal conditions.

Impacts
What was accomplished under these goals? A collection of 122 Saccharum spontaneum accessions maintained at the Texas A&M Agrilife Research in Weslaco, TX were characterized and a core collection representing the variability in the available germplasm was established based on agronomic traits and molecular markers fingerprinting. The agronomic traits comprised number of tillers, dry weight, brix, lignin, cellulose and hemicellulose content, which were measured in plants maintained in 10-gallons pots on a concrete slab. The molecular markers data consisted of 12 microsatellite markers (SSR) genotyping using a Licor 4300 DNA analyzer. The individuals showed considerable variation in these traits. Number of tillers ranged from 6 to 69, with an average of 22.0 ± 13.1. Dry weight ranged from 22 to 762 g (per unit area evaluated), with an average of 180.62 ± 133.23 g. Brix ranged from 3.7 to 18.0, with an average of 11.2 ± 3.0. Lignin, cellulose and hemicellulose content ranged from 5.5 to 21.7, 28.9 to 43.5 and 14.9 to 24.9%, with averages of 11.6 ± 3.1, 37.7 ± 3.0 and 21.8 ± 1.7%, respectively. Hybridization crosses were made in Weslaco, TX and Hawaii, involving sugarcane and different accessions of S. spontaneum. Seed was sent to Weslaco, TX, wherre seedlings resulting from these crosses were produced and plnated in the field. A total of thirteen energy cane wide-hybrid breeding lines from the Texas A&M Agrilife Research breeding program"Second Line" stage, have been visually selected and advanced to a competiution field trial in Weslaco, TX. Tissue culture seedlings were produced of three energycane (Type 1) genotypes in addition to one energycane wide hybrid line and one Miscane line in a total of 16,448 seedlings. Five Single Nucleotide Polymorphism (SNP)markers were used to test the Illumina Next Generation Sequence platform. The DNA sequence flanking these SNPs were used to design primers for the amplification by Polymerase Chain Reaction (PCR). The size of the PCR products (amplicons) were between 150-200 base pairs, having the SNP site within 50 bp from either ends, facilitating the use of 76-single end sequencing read length derived from the Illumina GAIIx instrument. The forward primer were comprised of 6 bases upstream of a SbfI (restriction enzyme) site, the corresponding SbfI recognition restriction site and 18-25 bases of the target-specific sequence. The reverse primer was only comprised of the target specific sequence.Transcriptome analysis of sugarcane hybrid CP72-1210 (cold susceptible) and Saccharum spontaneum TUS05-05 (cold tolerant) using Sugarcane Assembled Sequences (SAS) from the SUCEST-FUN Database showed that a total of 35,340 and 34,698 SAS genes, respectively, were expressed before and after chilling stress. The analysis revealed that more than 600 genes are differentially expressed in each genotype after chilling stress. Blast2Go annotation revealed that the major differences in gene expression profiles between CP72-1210 and TUS05-05 after chilling stress are present in the genes related to the transmembrane transporter activity. The SUCEST-FUN Database was utilized as a reference for Saccharum species RNA-seq data analysis and demonstrated a streamlined procedure from transcriptome comparison between cold susceptible and tolerant genotypes of Saccharum species, leading to the identification and validation of the functionality of one gene (named SspNIP2) on the improvement of abiotic stress tolerance. The RNA-Seq approach is a powerful tool to further investigate the genes that contribute to abiotic stress tolerance in many economically important crop species with lack of reference genome, as is the case with sugarcane.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Monge, J.J., Ribera, L.A., Jifon, J.L., da Silva, J.A. and Richardson, J.W. 2014. Economics and Uncertainty of Lignocellulosic Biofuel Production from Energy Cane and Sweet Sorghum in South Texas. Journal of Agricultural and Applied Economics, 46, 4 (November 2014): 457485.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Silva, M. A., Jifon, J.L., Santos, C.M, Jadoski, C.J. and da Silva, J.A. 2013. Photosynthetic Capacity and Water Use Efficiency in Sugarcane Genotypes Subject to Water Deficit During Early Growth Phase. Braz. Arch. Biol. Technol. v.56 n.5: pp. 735-748.