Source: UNIVERSITY OF FLORIDA submitted to
CHARACTERIZATION OF TRAIT COMPONENTS AND GENETIC IMPROVEMENT OF GRASSES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1003932
Grant No.
(N/A)
Project No.
FLA-AGR-005373
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 1, 2014
Project End Date
Aug 31, 2019
Grant Year
(N/A)
Project Director
Altpeter, FR.
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Agronomy
Non Technical Summary
The grass family includes many crops of great commercial importance such as sugarcane, cereals, turf and forage grasses and bioenergy feedstocks. Breeding and biotechnology for genetic improvement of grasses will continue to make significant contributions for a sustainable production of food feed and biofuel. This research program integrates traditional plant breeding, translational genomics and biotechnology to identify critical heredity units and develop genetically improved grasses. New biotechnology tools will allow genome editing and targeted mutagenesis and will increase the precision of plant breeding. Therefore, these new biotechnology tools will reduce costs associated with deregulation and commercialization of genetically improved grasses. Improvement of stress tolerance will enhance the productivity and persistence of commercially important grasses and will result in a more efficient use of natural resources. Alternatively, improving the turf or biomass quality of grasses which are well adapted to stress will significantly increase their value. Lignocellulosic materials produced by biomass grasses are a promising feedstock to produce low-cost transportation fuel, their improvement using breeding or biotechnological approaches will reduce costs associated with biomass conversion to fuel. Risk assessment and development of risk management strategies are essential components of this research program.
Animal Health Component
0%
Research Effort Categories
Basic
30%
Applied
30%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2012020104060%
2032020102020%
2011629108110%
2011550104010%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
2020 - Sugar cane; 1550 - Barley; 1629 - Perennial grasses, other;

Field Of Science
1040 - Molecular biology; 1081 - Breeding; 1020 - Physiology;
Goals / Objectives
1. Develop advanced biotechnology protocols for grasses including intragenic, targeted mutagenesis, genome editing and gene stacking.2. Isolation and characterization of gene families and pathways relevant for increasing the value of grasses as biofuel feedstocks, turf, forage or cereal.3. Genetic improvement of grasses for improved biomass quality and stress tolerance.4. Risk assessment and risk management of genetically improved grasses.
Project Methods
1. Develop advanced biotechnology protocols for grasses A. Development of plastid transformation protocols: Optimization of gene transfer-, selection- and tissue culture parameters will also include different genotypes, explant- types and environmental conditions of donor plants. Vectors will be introduced in regenerable grass tissue cultures, regenerated plants and progenies will be analyzed by Southern and Western blotting for transgene expression and homo- or heterotransplastomic nature.B. Targeted genome engineering/site directed gene stacking: Targeted insertion of transgenes into sugarcane will avoid significant position effects, limiting the predictability and stability of transgene expression and complicating the co-expression of multiple genes via gene stacking. Site-specific transgene integration can occur in plants with the Cre-lox site-specific recombination system. This system relies upon previously engineered loxP genomic sites for the integration of transgenes, and a single enzyme, a recombinase 'Cre', to trigger gene insertions at the loxP sites. LoxP sites consist of inverted repeats which serve as Cre recombinase binding sites.TALE proteins can be designed to bind to any specific DNA sequence and can be fused with a nuclease (TALEN) to create double strand breaks in specific DNA sequences. These double strand breaks will stimulate repair using the plant cell's DNA repair system. Non-homologous end joining (NHEJ) and homologous recombination (HR) are two highly conserved DNA repair pathways in eukaryotic cells for the repair of double strand breaks in the DNA duplex. Repair through NHEJ results in minor deletions and insertions, whereas repair through HR, in the presence of donor template DNA, stimulates recombination or gene replacement.2. Isolation and characterization of gene families relevant for increasing the value of grasses as biofuel feedstocks, turf, forage or cereal.A. Herbicide resistance in grasses:While this process will hopefully be streamlined in the near future we plan to explore also intragenic, targeted mutagenesis and genome editing as alternative genetic improvement strategy which may not be subject of this deregulation requirement or may require a reduced deregulation requirement for cultivar development. Therefore, we need to isolate candidate genes for herbicide resistance by either PCR based approaches or transcriptome sequencing from the target grass species.B. Biomass quality/Lignin biosynthesis: The biosynthesis of lignin proceeds from phenylalanine through the phenylpropanoid pathway to the cinnamoyl CoAs which are the general precursors of a wide range of phenolic compounds. We plan to isolate full length sequences of the caffeic acid 3-O-methyltransferase (COMT) and 4-coumarate:CoA ligase (4CL) enzymes from bioenergy and forage grasses. For this we will use a PCR based approach using sequence information from related species for design of degenerate primers, followed by 3' and 5' RACE-PCR. Following sequence confirmation we will study tissue and developmental expression of different 4CL and COMT orthologs by realtime RT-PCR and design RNAi vectors for suppression of selected enzymes.C. Development of molecular markers for control of flowering time in elephantgrass: Late flowering elephantgrass will result in no or reduced seed production which will enhance the biosafety of this bioenergy and forage crop and may also enhance the biomass yield. To develop molecular markers linked to the flowering time, F1 progeny of elephantgrass segregating for flowering time was recently developed and phenotyped in the PI's program and will be treated as a pseudo F2 segregating mapping population. For genotyping, about 2000 SSR markers and genotyping by sequence approach will be developed and used for genotyping of each individual in the population. Comparative mapping between the closely related pearl millet and elephant grass will also be carried out.D. Plant-Pathogen Interaction: Effector proteins secreted by pathogens suppress host defenses to promote nutrient acquisition and colonization. However, the molecular mechanisms by which these effectors manipulate these processes are poorly understood. This project aims to fill this knowledge gap by identifying the networks of interacting host and pathogen proteins in the well-characterized barley-powdery mildew, host-microbe system. Host targets, as well as key effectors, will be evaluated for their roles in host immunity by silencing via RNA interference (RNAi) and by overexpression.3. Genetic improvement of grasses for improved biomass quality and stress tolerance.Different methodologies will be used to accomplish this part. Generation and analysis of transgenic grasses is discussed under 1., development and use of molecular markers for development of late flowering elephantgrass is discussed under 2.A. Reduce lignin content and alter lignin composition in biofuel crops or forage grasses: The lines generated with targeted mutations in lignin biosynthetic genes will be compared relative to lignin reduction, biomass production and saccharification efficiency with our earlier results in RNAi suppression of lignin biosynthetic genes using transgenic or intragenic approaches with analytic methods described by Jung et al. 2013.B. Metabolic engineering of sugarcane to produce high energy dense triacylglycerols: We will explore the prospects of diverting the carbon flux in sugarcane from sucrose to triacylglycerol (TAG) for increased energy density and development of an advanced biofuel. This strategy involves the analysis of combinations of gene expression cassettes for metabolic engineering of TAG in vegetative sugarcane tissues. Genetic improvement of photosynthetic efficiency will allow improving the TAG yield per acre with a photosynthetic more effective canopy. We will also field test transgenic sugarcane lines expressing single or multiple gene constructs for a. TAG biosynthesis and b. photosynthesis at the UF-PSREC unit near Citra FL under USDA-Aphis permit. Biomass yield will be determined at the same time leaf and stem samples will be collected and sent to collaborators at Brookhaven National lab for TAG analysis. Photosynthesis measurements of field grown plants will be carried out.4. Risk assessment and risk management of genetically improved grasses.Herbicide resistance introduced through intragenic or genome editing technologies allows for rapid monitoring of pollen mediated gene transfer. Environmental impacts on gene flow will be studied by comparison of data obtained from divergent locations. Generation of late flowering or triploid grasses by intra or interspecific hybridization will also be explored relative to generation of novel germplasm with good field performance and enhanced biosafety. Our hypothesis is that suppression of flowering and/or ploidy-induced sterility will significantly reduce the risk of elephantgrass's invasiveness while not compromising feedstock production (e.g., biomass yield) suggesting that risk assessment should be carried out at the genotype level rather than the species level. A direct approach to evaluate invasiveness is to estimate individual components that contribute to fitness in the three life-stages:, i.e. plant establishment, growth and reproduction.The success of the 4 different project components will be measured by collecting and analyzing data describing identification of target genes/markers, vector construction, gene delivery, gene integration/targeted mutation, altered gene expression, plant performance and composition under controlled environment and field conditions. Molecular, biochemical, physiological and phenotypic data will be subjected to statistical analysis and will be compared to non-altered genotypes of the same species.

Progress 09/01/14 to 08/31/19

Outputs
Target Audience:Students, postdocs and scientists attending the following conferences and workshops: International Plant and Animal Genome (PAG) Conference, San Diego, California, January 2019; 2019 Genomic Sciences Program Annual Principal Investigator (GSP PI) Meeting. Tysons Corner, Virginia. February 2019; International Forage and Turf Breeding Conference, Lake Buena Vista, Florida, March 2019; Society for In Vitro Biology (SIVB) Meeting. Tampa, Florida, June 2019; Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) Annual Retreat. Champaign, Illinois, June 2019; International Conference on Plant Synthetic Biology, Bioengineering and Biotechnology, Clearwater, Florida, November 2018. Students attending formal courses at University of Florida AGR 4320/5321C - Plant Breeding / Genetic Improvement of Plants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training opportunities for 2 undergraduate students, 2 graduate students, 2 postdoctoral research associates and 3 visiting scientists How have the results been disseminated to communities of interest?By various conference presentations, formal lectures and during an in service training event What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Optimization of recombinant DNA constructs supported the generation of transgenic plants from recalcitrant energycane and sugarcane cultivars. Recombinant DNA vectors for mulltiallelic genome editing by CRISPR/Cas9 and other RNA guided nucleases were constructed and introduced into sugarcane. A rapid readout system for genome editing of sugarcane by non-homologous end joining was developed and allows now the in-vivo evaluation of alternative genome editing reagents or delivery methods. Golden Gate cloning provided additional constructs to support metabolic engineering and genome editing of sugarcane and energycane. Target genes for the enhancement of biomass and lipid production in sugarcane and energycane, grain production in wheat as well as modification of lignin biosynthesis in bahiagrass and sorghum were introduced into these crops by biolistic gene transfer. Transgenic plants were either regenerated and confirmed by PCR of genomic DNA or are currently regenerating.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhao, Y. , Kim, Y. J., Karan, R., Jung, J. H., Pathak, B., Wang, D., Williamson, B., Fan, C., Yu, W., Dong, S., Srivastava, V. and Altpeter, F. 2019. Generation of a safe harbor locus for transgene stacking in sugarcane. Plant Molecular Biology. 100: 247-263.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Parajuli, S., Kannan, B., Karan, R., Liu, H., Garcia-Ruiz, E., Deepak, K., Singh, V., Zhao, H., Long, S., Shanklin, J. and Altpeter, F. Oilcane: Metabolic engineering for production of lipids in vegetative sugarcane biomass. International Conference on Plant Synthetic Biology, Bioengineering and Biotechnology. Clearwater, Florida. November 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Kannan, B., Grennan, A., Zhang, X., Kim, J. Y., Ort, D. and Altpeter, F. Altering Chloroplast size in sugarcane by RNAi suppression of FtsZ, is bigger better? International Conference on Plant Synthetic Biology, Bioengineering and Biotechnology. Clearwater, Florida. November 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Kannan, B., Jung, J. H., Ko, J. K., Kim, H. E., Kim, K. H., Alper, H. S., Um, Y., Lee, S. M. and Altpeter, F. Processing genome edited, lignin-modified sugarcane biomass with a xylose fermenting yeast strain drastically elevates biofuel production. International Conference on Plant Synthetic Biology, Bioengineering and Biotechnology. Clearwater, Florida. November 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Thakku, R., Zhao, Y., Kannan, B., Liu, C. J. and Altpeter, F. Altering lignin polymerization in sugarcane biomass by expression of an engineered Monolignol 4-O-Methyltransferase. Society for In Vitro Biology (SIVB) Meeting. Tampa, Florida. June 2019. P-3021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Schneider, M., Luo, G., Kannan, B., Liu, H., Shanklin, J. and Altpeter, F. Developing a genetic transformation protocol for elite sugarcane cultivars to enhance energy content. Society for In vitro Biology (SIVB) Meeting. Tampa, Florida. June 2019. P-3020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Paudel, D., Kannan, B., Yang, X., Harris-Shultz, K., Thudi, M., Varshney, R., Altpeter, F. and Wang, J. Napiergrass (Cenchrus purpureus Schumach.) genome survey and high-density genetic map construction. International Forage and Turf Breeding Conference. Lake Buena Vista, Florida. March 2019. P-72.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Parajuli, S., Karan, R., Kannan, B., Liu, H., Garcia-Ruiz, E., Deepak, K., Singh, V., Zhao, H., Long, S., Shanklin, J. and Altpeter, F. Oilcane: Metabolic engineering for elevating lipid content in vegetative tissues of sugarcane. International Forage and Turf Breeding Conference. Lake Buena Vista, Florida. March 2019. P-73.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Mohan, C., Wang, D. and Altpeter, F. Establishing a rapid readout system for genome editing of sugarcane. Society for In vitro Biology (SIVB) Meeting. Tampa, Florida. June 2019. P-3018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Mohan, C., Wang, D. and Altpeter, F. Targeted mutagenesis of the complex sugarcane genome with CRISPR/Cas9. International Forage and Turf Breeding Conference. Lake Buena Vista, Florida. March 2019. P-87.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Luo, G., Shanklin, J. and Altpeter, F. Modular assembly of gene constructs for engineering lipid accumulation into energycane. Society for In Vitro Biology (SIVB) Meeting. Tampa, Floria. June 2019. P-3005.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kannan, B., Altpeter, A., Nguyen, T., South, P., Long, S. and Altpeter, F. Over-expression of miscanthus pyruvate orthophosphate dikinase (PPDK) in energycane for improving cold tolerance and biomass yield. Society for In Vitro Biology (SIVB) Meeting. Tampa, Florida. June 2019. P-3015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Cano, S., Parajuli, S., Kannan, B., Liu, H., Garcia-Ruiz, E., Zhao, H., Shanklin, J. and Altpeter, F. Environmental impacts on triacyl glycerol accumulation in vegetative tissues of oilcane. Society for In Vitro Biology (SIVB) Meeting. Tampa, Florida. June 2019. P-3037.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Altpeter, F., Oz, T., Altpeter, A., Karan, R. and Merotto, A.. Precision nucleotide editing in sugarcane following homology directed repair of targeted DNA breaks. Society for In Vitro Biology (SIVB) Meeting. Tampa, Florida. June 2019. P-3012.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Oz, T., Karan, R., Merotto, A. and Altpeter, F. Homology directed repair of CRISPR/Cas9 induced DNA breaks in sugarcane. International Plant and Animal Genome (PAG) Conference. San Diego, California. January 2019. PE0010.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Oz, T., Karan, R., Merotto, A. and Altpeter, F. Homology directed repair of CRISPR/Cas9 induced DNA breaks in sugarcane. International Plant and Animal Genome (PAG) Conference. San Diego, California. January 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Altpeter, F. Precision breeding of bahiagrass. 2019 Forage Workers Tour & In-Service Training. Ft. Pierce, Florida. August 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kannan, B., Schneider, M., Nguyen, T., South, P., Ort, D., Long, S. and Altpeter, F. Overcoming recalcitrance of genetic transformation in energycane for improving cold tolerance and biomass yield. 2019 Genomic Sciences Program (GSP) Annual PI Meeting. Tysons Corner, Virginia. February 2019. P-377.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Altpeter, F., Kannan, B., Luo, G., Oz, T., Wang, D., Parajuli, S., Karan, R., Liu, H., Zhao, Y., Liu, C. J. and Shanklin, J. Oilcane: Metabolic Engineering and Genome Editing to Improve Energy Density, Agronomic and Conversion Performance of Sugarcane. 2019 Genomic Sciences Program (GSP) Annual PI Meeting. Tysons Corner, Virginia. February 2019. P-169.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Altpeter, F., Oz, T., Altpeter, A., Karan, R., Merotto, A.. Editing the complex sugarcane genome. National Association of Plant Breeders. Pine Mountain, Georgia. August 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Altpeter, F., (Keynote lecture) Precision editing of the complex sugarcane genome by homology directed repair of CRISPR/Cas9 induced DNA breaks. Lake Buena Vista, Florida. March 2019.


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Target Audience:Students, postdocs and scientists attending the following conferences and workshops: ASA-CSSA-SSSA International Annual Meetings, Tampa Florida, October 2017; Biotechnology Havana 2017, Havana Cuba, December 2017; DOE Genomic Sciences Program Annual Principal Investigator Meeting, Tysons Corner VA, February 2018; 40th Symposium on Biotechnology for Fuels and Chemicals, Clearwater Beach FL, May 2018; Inter-BRC Lignin Workshop National Renewable Energy Laboratory Golden, CO. May 2018; Center for Advanced Bioenergy and Bioproduct Innovation (CABBI) Retreat, Urbana Champaign IL, July 2018. New Frontiers in Genetic Evaluation, Corteva Agriscience, Johnston, Iowa. July 2018. Students attending formal courses at University of Florida AGR 4320/5321C - Plant Breeding / Genetic Improvement of Plants; AGR 5307 Molecular Genetics for Crop Improvement. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training opportunities for 2 undergraduate students, 3 graduate students, 2 postdoctoral research associates, 4 visiting scientists How have the results been disseminated to communities of interest?By various conference presentations and formal lectures What do you plan to do during the next reporting period to accomplish the goals?We plan to expand our effort in genome ediing and metabolic engineering in a range of crops

Impacts
What was accomplished under these goals? Culture media optimizations supported the development of embryogenic tissue cultures for genetic transformation of recalcitrant energycane and sugarcane cultivars. Mulltiallelic precision genome editing by CRISPR/Cas9 was confirmed in the complex sugarcane genome by Sanger and Pacbio sequencing. Genome edited plants transmitted the multiallelic nucleotide substitutions, which conferred herbicide resistance, faithfully to vegetative progenies without compromising biomass accumulation. Golden Gate cloning was established and a library of L0 constructs was generated for modular assembly of multi-gene constructs to support metabolic engineering of sugarcane and energycane. Target genes for the enhancement of biomass production in sugarcane and energycane, grain production in wheat as well as modification of lignin biosynthesis in bahiagrass and sorghum were identified and sequenced to inform construct assembly. traits

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oz, T. M.. Carter, M., Xu, W., Chapman, A., Helm, M., Bogdanove, A., Innes, R., Wise, R. and Altpeter, F. Decoding host-pathogen interactions with transgenic and genome edited barley. ASA-CSSA-SSSA Meeting. 2017. 152-810.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Paudel, D., Kannan, B., Altpeter, F. and Wang, J. Construction of high-density genetic linkage map of napiergrass (Pennisetum purpureum). ASA-CSSA-SSSA Meeting. 2017. 38-25.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Kim, J. Y., Kim, C. H., Kim, K. H., Gallo, M., Preston, J. and Altpeter, F. In Planta produced hyperthermostable GH10 xylanase Xyl10B improves hydrolysis of sugarcane xylan to fermentable sugars for biofuel production. ASA-CSSA-SSSA Meeting. 2017. 410-1242.
  • Type: Book Chapters Status: Published Year Published: 2018 Citation: Altpeter, F. and Karan R. In: Achieving sustainable cultivation of sugarcane. Rott P. (ed.), Genetic improvement of sugarcane by transgenic, intragenic and genome editing technologies. Burleigh Dodds Science Publishing, Cambridge UK. Volume 2. 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Kannan B., Jung J.H., Moxley G.W., Lee S.-M., Altpeter F. 2018. TALEN mediated targeted mutagenesis of more than 100 COMT copies/alleles in highly polyploid sugarcane improves saccharification efficiency without compromising biomass yield. Plant Biotechnology Journal 16: 856-866.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Ko, J. K., Jung, J. H., Altpeter, F., Kannan, B., Kim, H. E., Kim, K. H., Alper, H. S., Um, Y. and Lee, S. M. 2018. Synergistic improvement of ethanol production by targeted mutagenesis of lignin biosynthesis in sugarcane and utilizing an engineered Saccharomyces cerevisiae. Bioresource Technology 256: 312-320.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Paudel, D., Kannan, B., Yang X., Harris-Shultz K., Thudi M., Varshney R.K., Altpeter, F. and Wang, J. Surveying the genome and constructing a high-density genetic map of napiergrass (Cenchrus purpureus Schumach.). 2018. Scientific Reports 8, 14419
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Lopez, Y., Kurashev, A., Chase, C., Gallo, M., Sollenberger, L., Altpeter, F. and Wang, J. Developing and validating microsatellite markers in elephantgrass (Pennisetum purpureum S.). 2018. Euphytica 214: 185
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Altpeter F., Kannan B., Jung J.H., Parajuli S., Karan R., Oz T, Liu H., Garcia-Ruiz E., Zhao H., Shanklin J., Long S. Metabolic engineering and genome editing to improve sugarcane as biofuel feedstock. 40th Symposium on Biotechnology for Fuels and Chemicals. Clearwater Beach, Fl. April 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Kannan, B., and Altpeter, F. Multiplex editing of flowering genes in sugarcane with CRISPR/Cas9 to enhance biomass and lipid yield. Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) Annual Retreat, Champaign, IL. July 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Luo G.B., Altpeter, F. Elevating oil yield from sugarcane with targeted mutagenesis and synthetic transcription activators. Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) Annual Retreat, Champaign, IL. July 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Oz, T., Karan, R., Merotto, A., Altpeter, F. Targeted precision nucleotide substitutions in sugarcane following CRISPR/Cas9 and template mediated genome editing confer ⿿gain of function⿝. DOE Genomic Sciences Program Principal Investigator meeting, Tysons Corner, VA, February 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Oz, T., Karan, R., Merotto, A., Altpeter, F. Editing the sugarcane genome with targeted nucleotide substitutions confers gain of function. Center for Advanced Bioenergy and Bioproduct Innovation (CABBI) Retreat, Champaign, IL. July 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Parajuli, S., Karan, R., Kannan, B., Liu, H., Garcia-Ruiz, E., Deepak, K., Singh, V., Zhao, H., Long, S. Shanklin, J. and Altpeter, F. Converting sugarcane into an oil producing crop by metabolic engineering. Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) Annual Retreat, Champaign, IL. July 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Paudel, D., Kannan, B., Yang, X., Altpeter, F., and Wang, J. Constructing a high-density genetic map of napiergrass (Cenchrus purpureus Schumach.). New Frontiers in Genetic Evaluation, Corteva Agriscience, Johnston, Iowa. July 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wang D. and Altpeter, F. Comparing the efficiency and specificity of different transgene-free genome editing reagent delivery systems in sugarcane. Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) Annual Retreat, Champaign, IL. July 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Altpeter, F., Parajuli, S., Karan, R., Kannan, B., Liu, H., Garcia-Ruiz, E., Deepak, K., Singh, V., Zhao, H., Long, S. and Shanklin, J. Metabolic engineering of lipid biosynthesis to convert sugarcane into an oil producing crop. Havanna Biotechnology. 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Altpeter, F., Kannan, B., Oz. T. M., Jung, J. H., Karan, R. and Merotto, A.. Genetic improvement of sugarcane by targeted mutagenesis of precision nucleotide substitutions. Havanna Biotechnology. 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Altpeter, F., Kannan, B., Oz, T. M., Karan, R., Jung, J. H. and Merotto, A. Editing the complex sugarcane genome with TALEN or CRISPR-Cas9. ASA-CSSA-SSSA Meeting. 2017. 225-2.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Kannan, B., Jung, J. H., Moxley, G. and Altpeter, F. Field performance of genome edited sugarcane. ASA-CSSA-SSSA Meeting. 2017. 152-814.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Kannan, B., Sinche, M., Corsato, C. and Altpeter, F. Developing high biomass napiergrass (Pennisetum purpureum) hybrids with enhanced biosafety. ASA-CSSA-SSSA Meeting. 2017. 410-1240.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oz, T. M., Karan, R., Merotto, A. and Altpeter, F. Precision nucleotide substitutions in sugarcane following CRISPR/Cas9 and template mediated genome editing. ASA-CSSA-SSSA Meeting. 2017. 152-809.


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

Outputs
Target Audience:Target Audience:Students, postdocs and scientists attending the following conferences and workshops: SIVB Raleigh, NC June 2017,University of Sao Paulo, BIOEN Seminar (broadcasted live to satellite sites in Brazil). Sao Paulo, Brazil. May 2017, Plant and Animal Genome XXV Conference. San Diego, California. January 2017, Keystone Symposia on Precision Genome Engineering. Breckenridge, Colorado. January 2017, International Conference on Plant Synthetic Biology and Bioengineering. Miami, Florida. December 2016. Students attending formal courses at University of Florida AGR 4320/5321C - Plant Breeding / Genetic Improvement of Plants; Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Mentoring 1 postdoctoral research associate, 3 graduate students and 1 undergraduate student. How have the results been disseminated to communities of interest?In form of peer reviewed publications, oral and poster presentations at conferences and workshops What do you plan to do during the next reporting period to accomplish the goals?Apply for additional grant funding, hire and train additional graduate students or postdoctoral research associates, form additional collaborations

Impacts
What was accomplished under these goals? A reproducible protocol was developed for precision nucleotide substitutions via homology directed repair of targeted DNA double strand breaks in planta and will allow to accomplish crop improvement with greater precision than conventional plant breeding.Targeted co-mutagenesis of more than 100 copies/alleles of the lignin biosynthetic COMT gene was accomplished which did not compromise field performance of the target sugarcane plants but elevated biofuel yields from lignocellulosic sugarcane biomass.The first molecular map for napiergrass was developed following genotyping by sequencing of a mapping population. This will accelerate crop improvement by marker assisted selection for biomass related and biosafety related traits.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Kim, J. Y., Nong, G., Rice, J. D., Gallo, M., Preston, J. F. and Altpeter, F. 2017. In planta production and characterization of a hyperthermostable GH10 xylanase in transgenic sugarcane. Plant Molecular Biology. 93: 465-478.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Huang, H., Moreau, R. A., Powell, M. J., Wang, Z., Kannan, B., Altpeter, F., Grennan, A. K., Long, S. and Singh, V. 2017. Evaluation of the quantity and composition of sugars and lipid in the juice and bagasse of lipid producing sugarcane. Biocatalysis and Agricultural Biotechnology. 10:148-155.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Rios, E., Kenworthy, K., Blount, A. R., Quesenberry, K., Unruh, B., Erickson, J., Altpeter, F. and Munoz, P. 2017. Breeding apomictic bahiagrass (Paspalum notatum Flugge) with improved turf traits. Plant Breeding. 136: 253-260.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Altpeter, F. Genetic improvement of sugarcane by targeted loss- or gain of function mutations using TALEN or CRISPR-Cas9. Plant and Animal Genome XXV Conference. San Diego, CA W699. 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Karan, R., Zhao, Y., Sanahuja, G., Kim, J. Y., Jung, J. H. and Altpeter, F. Gene stacking into an engineered safe-harbor locus of sugarcane. In Vitro Biology Meeting (SIVB). Raleigh NC. 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oz, T. M., Kannan, B., Jung, J. H., Merotto, A., Moxley, G. and Altpeter, F. TALEN or CRISPR-Cas9 mediated genome editing in highly polypoid sugarcane. Keystone Symposia on Precision Genome Engineering. Breckenridge CO. 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oz, T. M., Karan, R., Merotto, A. and Altpeter, F. Targeted nucleotide substitutions in the higly polyploid sugarcane genome using CRISPR/Cas9 and template mediated homology directed repair. Genome Engineering 5.0 Conference. Cambridge MA. 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Roa, C. C., Oz, T. M. and Altpeter, F. Development of herbicide resistant bahiagrass by chemical mutagenesis. University of Florida Undergraduate Research Symposium of the 2016 University Scholars Program. Gainesville, FL. 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Altpeter, F., Oz, T. M., Kannan, B., Karan, R., Jung, J. H. and Merotto, A. Crispr-Cas9 or Talen mediated genome editing in sugarcane. International Conference on Plant Synthetic Biology and Bioengineering. Miami, FL, 2016.


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

Outputs
Target Audience:Students, postdocs and scientists attending the following conferences and workshops: Plant and Animal Genome Conference, San Diego CA, January 2016; World Congress for In Vitro Biology, San Diego, CA, June 2016; Redbio Conference, Lima Peru, June 2016. Students attending formal courses at University of Florida AGR 4320/5321C - Plant Breeding / Genetic Improvement of Plants; AGR 5307 Molecular Genetics for Crop Improvement. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training and professional development of 3 postdoctoral research associates, 3 graduate students, 4 undergraduate students and 2 visiting scientists in plant molecular biology, plant breeding, grant proposal development, manuscript preparation, instructional activities, presentation of results, lab management. How have the results been disseminated to communities of interest?In form of peer reviewed publications, oral and poster presentations at the following conferences and workshops: Plant and Animal Genome Conference, San Diego CA, January 2016; World Congress for In Vitro Biology June 2016, San Diego CA, Redbio Congress Lima Peru June 2016; What do you plan to do during the next reporting period to accomplish the goals?Apply for additional grant funding, hire and train additional graduate students or postdoctoral research associates, form additional collaborations Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Mentoring 3 postdoctoral research associates, 3 graduate students and 4 undergraduate students. How have the results been disseminated to communities of interest?In form of peer reviewed publications, oral and poster presentations at the following conferences and workshops: Plant and Animal Genome Conference, San Diego CA, January 2016; World Congress for In Vitro Biology, San Diego CA, June 2016; Redbio Congress Lima Peru June 2016 What do you plan to do during the next reporting period to accomplish the goals?Apply for additional grant funding, hire and train additional graduate students or postdoctoral research associates, form additional collaborations

Impacts
What was accomplished under these goals? CRISPR/Cas9 mediated targeted gene editing was established for sugarcane and bahiagrass and will allow to accomplish crop improvement with greater precision than conventional plant breeding. TALEN mediated targeted mutagenesis of the lignin biosynthetic gene COMT and intragenic sugarcane with suppression of the lignin biosynthetic 4CL gene resulted in field grown sugarcane with improved saccharification efficiency of cell wall bound sugars which translates into drastically elevated biofuel yields from lignocellulosic biomass. Metabolic engineering for production of energy dense triacyl glycerol in transgenic sugarcane leaves and stems was further refined and resulted in biosynthesis and accumulation of lipids at levels that can easily be converted to biodiesel. Homozygous transgenic barley plants were generated which stably express an evolutionary conserved effector of the barley pathogen powdery mildew Blumeria graminis f. sp. hordei (Bgh). Transgenic plants will be crossed with resistance signaling mutants for biochemical (e.g. pull-down assay) and genetic assays to determine cellular localization and confirm target interactions to unravel novel strategies for disease resistance. Bahiagrass with expression of a mutated acetolactase synthase (ALS) expression cassette from sorghum displayed high level of herbicide resistance (including herbicides Imazapyr and Nicosulfuron) which will facilitate weed control. Sterile, triploid interspecific hybrids of napiergrass and pearl millet were generated and lines accumulating superior amounts of biomass were identified.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Altpeter, F., Parajuli, S., Karan, R., Sanahuja, G., Liu, H. and Shanklin, J. Converting sugarcane into an oil producing crop with metabolic engineering. IX REDBIO Conference. 2016. Altpeter, F., Karan, R., Kannan, B., Jaikumar, N., Xie, L., Swaminathan, K., James, B., Moose, S. and Long, S. Engineering elevated photosynthetic efficiency to boost biomass accumulation in sugarcane. IX REDBIO Conference. 2016. Altpeter, F., Jung, J. H., Oz, T. M., Karan, R. and Merotto, A. Genome editing in sugarcane to support the sustainable production of biofuels and other carbon based products. IX REDBIO Conference. 2016. Altpeter, F., Jung, J. H., Oz, T. M., Karan, R. and Merotto, A. Targeted genome modifications in sugarcane with CRISPR/Cas9 and TALEN. World Congress on In Vitro Biology (SIVB). 2016. P-6. Kannan, B., Jung, J. H. and Altpeter, F. Talen mediated multi-allelic mutagenesis of COMT improves saccharification efficiency from lignocellulosic biomass of field grown sugarcane. International Conference on Plant Synthetic Biology and Bioengineering. 2016. Kannan, B., Parajuli, S., Karan, R., Sanahuja, G., Liu, H., Shanklin, J. and Altpeter, F. Metabolic engineering converts sugarcane into an oil producing crop. International Conference on Plant Synthetic Biology and Bioengineering. 2016. Kannan, B., Karan, R., Grennan, A., Jaikumar, N., Vilarinho, A., Zhang, X., Kim, J. Y., Chen, X., Slattery, R., Moose, S., Long, S., Ort, D. and Altpeter, F. Field performance of transgenic sugarcane with reduced chlorophyll content, altered chloroplast size and elevated levels of PPDK. International Plant and Animal Genome (PAG) Conference. 2016. P0022. Kannan, B., Jung, J. H. and Altpeter, F. Field performance of sugarcane with TALEN or RNAi mediated lignin reduction. International Plant and Animal Genome (PAG) Conference. 2016. W090. Kannan, B., Jung, J. H. and Altpeter, F. Field performance of sugarcane with TALEN mediated targeted mutagenesis of COMT. International Plant and Animal Genome (PAG) Conference. 2016. W917. Karan, R., Kannan, B., Jaikumar, N., Xie, L., Swaminathan, K., James, B., Moose, S., Long, S. and Altpeter, F. Engineering elevated photosynthetic efficiency into sugarcane. International Conference on Plant Synthetic Biology and Bioengineering. 2016. Karan, R., Kannan, B., Jaikumar, N., Swaminathan, K., Xie, L., James, B., Moose S., Altpeter, F. and Long, S. Improving photosynthetic efficiency in sugarcane by expression of pyruvate orthophosphate dikinase from miscanthus x giganteus. World Congress on In Vitro Biology (SIVB). 2016. P-1022. Oz, T. M., Xu, W., Wise, R. and Altpeter, F. Stable expression of the fungal effector BEC1019 in transgenic barley to evaluate host-pathogen interactions. International Conference on Plant Synthetic Biology and Bioengineering. 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Altpeter F., Springer N.M., Bartley L.E., Blechl A.E., Brutnell T.P., Citovsky V., Conrad L.J., Gelvin S.B., Jackson D.P., Kausch A.P., Lemaux P.G., Medford J.I., Orozco-C�rdenas M.L., Tricoli D.M., Van Eck J., Voytas D.F., Walbot V., Wang K., Zhang Z.J., Stewart C.N. 2016. Advancing Crop Transformation in the Era of Genome Editing. Plant Cell. 28: 1510-1520. Jung, J. H. and F. Altpeter. 2016. TALEN mediated targeted mutagenesis of the caffeic acid O-methyltransferase in highly polyploid sugarcane improves cell wall composition for production of bioethanol. Plant Molecular Biology 92: 131-142. Zale, J. , J.H. Jung, J.Y. Kim, B. B. Pathak, R. Karan, H. Liu, X. Chen, H. Wu, J. Candreva, Z. Zhai, J. Shanklin, F. Altpeter. 2016. Metabolic engineering of sugarcane to accumulate energy-dense triacylglycerols in vegetative biomass. Plant Biotechnology Journal 14: 661-669. Dermawan H., R. Karan, J.H. Jung, Y. Zhao, S. Parajuli, G. Sanahuja, F. Altpeter. 2016. Development of an intragenic gene transfer and selection protocol for sugarcane resulting in resistance to acetolactate synthase-inhibiting herbicide. Plant Cell Tissue and Organ Culture 126: 459-468. Faleiro, F.G., B. Kannan, F. Altpeter. 2016. Regeneration of fertile, hexaploid, interspecific hybrids of elephantgrass and pearl millet following treatment of embryogenic calli with antimitotic agents. Plant Cell Tissue and Organ Culture 124: 57-67. Jung J. H., B. Kannan, H. Dermawan, G. W. Moxley and F. Altpeter. 2016. Precision breeding for RNAi suppression of a major 4-coumarate: coenzyme A ligase gene improves cell wall saccharification from field grown sugarcane. Plant Molecular Biology 92: 505-517.


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

Outputs
Target Audience:Students, postdocs and scientists attending the following conferences and workshops: Plant and Animal Genome Conference, San Diego CA, January 2015; Consortium for Plant Biotechnology Research, Washington DC, March 2015; Society for In Vitro Biology Conference, Tucson AZ, June 2015; International Plant Molecular Biology Conference Iguacu, Brazil, October 2015; NSF workshop for transformation enabled genomic research in crop plants, November 2015; Florida Genetics Symposium, Gainesville Fl Nov. 2015; USDA-ARS Miami; UF-IFAS EREC, Belle Galde, Fl December 2015, students attending formal courses at University of Florida AGR 4320/5321C - Plant Breeding / Genetic Improvement of Plants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training and professional development of 3 postdoctoral research associates, 4 graduate students and 4 undergraduate students in plant molecu;lar biology, plant breeding, grant proposal development, manuscript preparation, instructional activities, presentation of results, lab management. How have the results been disseminated to communities of interest?In form of peer reviewed publications, book chapters, oral and poster presentations at the following conferences and workshops: Plant and Animal Genome Conference, San Diego CA, January 2015; Consortium for Plant Biotechnology Research, Washington DC, March 2015; Society for In Vitro Biology Conference, Tucson AZ, June 2015; International Plant Molecular Biology Conference Iguacu, Brazil, October 2015; NSF workshop for transformation enabled genomic research in crop plants, November 2015; Florida Genetics Symposium, Gainesville Fl Nov. 2015; What do you plan to do during the next reporting period to accomplish the goals?Apply for additional grant funding, hire and train additional graduate students or postdoctoral research associates, form additional collaborations

Impacts
What was accomplished under these goals? TALEN mediated targeted mutagenesis of the lignin biosynthetic gene COMT resulted in sugarcane with reduced lignin content, brown midrib and brown stem phenotype. Field testing of these events revealed no significant difference in agronomic performance compared to the original cultivar. Intragenic sugarcane with suppression of the lignin biosynthetic 4CL gene was generated by biolistic co-transfer of an intragenic RNAi expression cassette targeting a conserved region of the 4CL gene and a selectable marker gene conferring herbicide resistance. Intragenic events displayed both herbicide resistance and reduced lignin content as well as field performance similar to the original cultivar. Transgenic sugarcane plants were generated with co-expression and co-suppression of several genes associated with lipid accumulation. Drastic elevation of energy dense triacyl glycerol in transgenic sugarcane leaves and stems confirmed the feasibility of engineering sugarcane for biosynthesis and accumulation of lipids.Transgenic barley plants were generated expressing an evolutionary conserved effector of the barley pathogen powdery mildew Blumeria graminis f. sp. hordei (Bgh). Transgenic plants will be crossed with resistance signaling mutants for biochemical (e.g. pull-down assay) and genetic assays to determine cellular localization and confirm target interactions. Bahiagrass with expression of a mutated acetolactase synthase (ALS) expression cassette from sorghum was generated and is currently evaluated for response to various ALS inhibiting herbicides. Sterile, triploid interspecific hybrids of napiergrass and pearl millet were generated and lines accumulating superior amounts of biomass were identified. Chromosome doiubling of the superior interspecific hybrids was acomplished with antimititic agents and restored fertility in the hexaploid germplasm.

Publications

  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Wu, H. and Altpeter, F. Sugarcane (Saccharum spp. hybrids). In: Agrobacterium Protocols (3rd edition, Volume 2). Wang, K. (ed.), Springer Science and Business Media, LLC. 2015. pp 307-316.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Fouad, W. M., Wu, H., Xiong, Y., Steeves, C., Sandhu, S. and Altpeter, F. 2015. Generation of transgenic energy cane plants with integration of minimal transgene expression cassette. Current Pharmaceutical Biotechnology. 16: 407-413. Kannan, B., Davila-Olivas, N. H., Lomba, P. N. and Altpeter, F. 2015. In vitro chemical mutagenesis improves the turf quality of bahiagrass. Journal of Plant Biotechnology. 120: 551-561. Wu, H., Awan, F., Vilarinho, A., Zeng, Q., Kannan, B., Phipps, T., McCuiston, J., Wang, W., Caffall, K. and Altpeter, F. 2015. Transgene integration complexity and expression stability following biolistic or agrobacterium-mediated transformation of sugarcane. In Vitro Cellular and Developmental Biology - Plant. 51: 603-611.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Clemente, T. E., Altpeter, F., Long, S., Shanklin, J., Grennan, A., Ort, D. and Moose, S. P. Towards the development of sustainable feedstocks for renewable biobased products: the case for sugarcane and sweet sorghum. Plant and Animal Genome (PAG) Conference. 2015. W084. p. 147. Jung, J. H. and Altpeter, F. Targeted genome editing in sugarcane. 11th International Congress of Plant Molecular Biology (IPMB). 2015. Jung, J. H., Dermawan, H. and Altpeter, F. TALEN-mediated precision mutagenesis and intragenic RNAi for lignin reduction in sugarcane. Plant and Animal Genome (PAG) Conference. 2015. W813. p. 198. Jung, J. H., Zale, J., Kim, J. Y., Pathak, B.,Vilarinho, A., Grennan, A., Liu, H., Karan, R., Kannan, B., Zhang, X., Chen, X., Candreva, J., Slattery, R. A., Zhai, Z., Long, S., Ort, D., Shanklin, J. and Altpeter, F. Metabolic engineering of sugarcane for production of oil and increased biomass yield. Plant and Animal Genome (PAG) Conference. 2015. W814. p. 198. Jung, J. H., Zale, J., Kim, J. Y., Pathak, B., Kannan, B., Karan, R., Liu, H., Candreva, J., Zhai, Z., Shanklin, J. and Altpeter, F. Metabolic engineering of sugarcane for production of oil. Plant and Animal Genome (PAG) Conference. 2015. P0546. p. 247. Jung, J. H., Zale, J., Kim, J. Y., Pathak, B., Liu, H., Kannan, B., Karan, R., Candreva, J., Zhai, Z., Shanklin, J. and Altpeter, F. Precision genome and metabolic engineering for genetic improvement of the biofuel feedstock sugarcane. Plant and Animal Genome (PAG) Conference. 2015. W833. p. 199. Jung, J. H. and Altpeter, F. TALEN-Mediated editing of the caffeic acid 3-O-methyltransferase for genetic improvement of the biofuel feedstock sugarcane. Plant and Animal Genome (PAG) Conference. 2015. P1092. p. 294.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kannan, B., Vilarinho, A., Grennan, A., Zhang, X., Karan, R., Chen, X., Slattery, R. A., Long, S., Ort, D. and Altpeter, F. Improving canopy level photosynthesis in sugarcane. Plant and Animal Genome (PAG) Conference. 2015. P0010. p. 205. Karan, R., Kannan, B., Jaikumar, N., Long, S., Moose, S. and Altpeter, F. Expression of miscanthus pyruvate orthophosphate dikinase (M-PPDK) improves photosynthesis and biomass accumulation in sugarcane. Florida Genetics Symposium. 2015. #46. Karan, R., Zhang, X., Vilarinho, A., Grennan, A. K., Kannan, B., Jaikumar, N. S., Slattery, R., Cahill, C., Ort, D., Long, S. and Altpeter, F. Sugarcane biotechnology for improved photosynthesis and biomass production. Society for In Vitro Biology (SIVB) Conference. 2015. P-2024. Oz, M. T., Xu, W., Wise, R. P. and Altpeter, F. Towards decoding host signaling pathways with transgenic barley stably expressing Blumeria effector candidate (BEC)1019. Florida Genetics Symposium. 2015. #72. Parajuli, S., Sanahuja, G., Karan, R., Zale, J., Jung, J. H., Pathak, B., Kim, J. Y., Liu, H., Shanklin, J. and Altpeter, F. Metabolic engineering for production of oil in vegetative tissues of sugarcane. 11th International Congress of Plant Molecular Biology (IPMB). 2015. Parajuli, S., Sanahuja, G., Karan, R., Liu, H., Shanklin, J. and Altpeter, F. Towards lipid producing sugarcane for biodiesel production. Florida Genetics Symposium. 2015. #75. Parajuli, S., Sanahuja, G., Karan, R., Liu, H., Shanklin, J. and Altpeter, F. Elevating the lipid content in vegetative sugarcane biomass by metabolic engineering. Society for In Vitro Biology (SIVB) Conference. 2015. P-2034. Paudel, D., Kannan, B., Wang, J. and Altpeter, F. Interspecific hybridization between pearl millet and napiergrass for high biomass production and biosafety. Florida Genetics Symposium. 2015. #77. Ravanfar, S. A., Karan, R. and Altpeter, F. Characterization of tonoplast monosaccharide transporter (TMT) genes of sugarcane. Florida Genetics Symposium. 2015. #82. Silva, T. N., Oz, M. T. and Altpeter, F. Stacking of intragenic herbicide resistant genes in sugarcane. Society for In Vitro Biology (SIVB) Conference. 2015. P-2006. Silva, T. N., Oz, M. T. and Altpeter, F. Generation of intragenic sugarcane with stacked herbicide resistant genes. Florida Genetics Symposium. 2015. #97. Strauss, T. and Altpeter, F. CRISPR/Cas9-mediated genome editing for conferring ALS-type herbicide resistance in bahiagrass. Florida Genetics Symposium. 2015. #103. Wu, H., Vilarinho, A., Awan, F., Zeng, Q., Kannan, B., Phipps, T., McCuiston, J., Wang, W., Caffall, K. and Altpeter, F. Transformation efficiency, transgene integration complexity and expression stability following biolistic or agrobacterium-mediated transformation of sugarcane. Plant and Animal Genome (PAG) Conference. 2015. P1093. p. 294. Wu, H., Vilarinho, A., Awan, F., Zeng, Q., Kannan, B., Phipps, T., McCuiston, J., Wang, W., Caffall, K. and Altpeter, F. Transformation efficiency, transgene integration complexity and expression stability following biolistic or agrobacterium-mediated transformation of sugarcane. Plant and Animal Genome (PAG) Conference. 2015. W625. p. 186. Zhao, Y., Kim, J. Y., Jung, J. H., Pathak, B., Williamson, B., Fan, C., Yu, W., Dong, S., Srivastava, V. and Altpeter, F. Towards targeting multiple expression cassettes into a precharacterized genomic locus of sugarcane for predictable transgene performance. Society for In Vitro Biology (SIVB) Conference. 2015. P-2005.


Progress 09/01/14 to 09/30/14

Outputs
Target Audience: Nothing Reported 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? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? 1. Develop advanced biotechnology protocols for grasses including intragenic, targeted mutagenesis, genome editing and gene stacking. Evaluate a mutated ALS gene as selectable marker to create intragenic events with herbicide resistance. Evaluate TALEN for targeted mutagenesis of a lignin biosynthetic gene, generate transgenic lines with high level expression of a pre-characterized site and a unique target sequence for gene stacking by re-transformation. 2. Isolation and characterization of gene families and pathways relevant for increasing the value of grasses as biofuel feedstocks, turf, forage or cereal. Evaluate RNAi and/or overexpression strategies for target genes to enhance canopy level photosynthesis. Evaluate the effect of transgene combinations on accumulation of oil in vegetative tissues. 3. Genetic improvement of grasses for improved biomass quality and stress tolerance. Evaluate herbicide resistance of sugarcane carrying an intragenic, mutated ALS gene. Evaluate lignin content of bahiagrass and sugarcane with mutated lignin biosynthetic genes. 4. Risk assessment and risk management of genetically improved grasses. Evaluate flowering time, seed viability and seedling establishment from F1 hybrids of contrasting napiergrass genotypes for flowering time.

Impacts
What was accomplished under these goals? This new project was initiated September 1st 2014. The accomplishments since October 2013 were reported in the previous CRIS project which ended in summer 2014. During the short reporting period of this new project (September 1st to September 30th 2014) progress was made towards all goals but no significant additional accomplishments are to be reported.

Publications