Source: UNIVERSITY OF FLORIDA submitted to
DISSECTION OF TRAIT COMPONENTS AND MOLECULAR IMPROVEMENT OF GRASSES THROUGH GENETIC ENGINEERING
 
PROJECT DIRECTOR: Altpeter, F.
 
PERFORMING ORGANIZATION
AGRONOMY
UNIVERSITY OF FLORIDA
GAINESVILLE,FL 32610
 
NON TECHNICAL SUMMARY: Environmental limitations of forage, turfgrass and cereral production systems require advanced germplasm, that will allow the efficient use of natural resources. This project develops and applies advanced genetic transformation technology to develop grasses with improved stress response that will allow for more efficient use of natural resources. The research will also address and minimize enviromental risks associated with transgenic grasses.
 
OBJECTIVES: 1.Develop advanced transformation protocols for grasses. 2.Molecular dissection of physiological traits defining the environmental limitations of sub-tropical forage grass production. 3.Modulate bioactive gibberellic acid content in transgenic grasses for improved turf characteristics and reduced mowing requirements. 4.Risk assessment and risk management in transgenic grasses.
 
APPROACH: This project focuses on the molecular improvement of grasses through genetic engineering. The grass family (Gramineae) includes many commercially important species including turf, forage grasses and cereals. Genetic engineering has a great potential for trait dissection and germplasm improvement of grasses and requires efficient genetic transformation protocols. Efficient genetic transformation protocols support the reproducible generation of transgenic plants, idealy with defined transgene integration patterns, stable and defined expression of transgenes with minimum tissue culture derived somaclonal variation. We will develop or establish advanced biolistic or Agrobacterium-mediated transformation protocols for bahiagrass, ryegrass, seashore paspalum, wheat, barley and rye. The significant problem with forage production in the Southern Costal Plain is the gap between the decline of warm season grass production (e.g. bahiagrass) in fall and availability of winter forage, mainly small grain cereals and ryegrass in December. The interaction of photoperiodic response, induction of dormancy and cold acclimation in grasses has not been studied yet and its dissection should provide clues for conventional and molecular germplasm improvement to provide an optimal distribution of forage production over the course of the year. Stress inducible transcription activators will be isolated and overexpressed under control of stress specific promoters. Targets for turfgrass germplasm improvement by genetic engineering include improved turf characteristics, reduced mowing requirements, stress tolerance and resistance to biodegradable and environmentally friendly herbicides. Initially we will focus on the molecular modulation of the bioactive gibberellic acid content for a more efficient use of natural resources. Several of the grasses are not only valuable turf and forages but are at the same time serious weeds in cereal production systems. Some of the targets for molecular improvement of grasses have the potential to promote weediness. These include resistance against abiotic- and biotic stresses and herbicides. Before introducing stress resistant transgenic grasses into the environment we propose to investigate the likelihood and consequences of the transfer of transgenes through pollination to other fields, in which they would be undesirable, or to wild, weedy relatives of the crops. Strategies that allow risk mamagement (e.g. inducible cell death) will also be implemented.
 
CRIS NUMBER: 0194585 SUBFILE: CRIS
PROJECT NUMBER: FLA-AGR-04076 SPONSOR AGENCY: CSREES
PROJECT TYPE: HATCH PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Oct 1, 2002 TERMINATION DATE: Sep 30, 2008

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
111162910406.110%
201154910402.210%
201155010402.210%
201163110402.210%
201213010402.210%
203162910202.210%
203163110202.210%
203163110402.210%
213162910404.210%
213162910804.210%

CLASSIFICATION HEADINGS
KA203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants
KA213 - Weeds Affecting Plants
KA201 - Plant Genome, Genetics, and Genetic Mechanisms
KA111 - Conservation and Efficient Use of Water
S1549 - Wheat, general/other
S2130 - Turf
S1550 - Barley
S1629 - Perennial grasses, other
S1631 - Winter annual grasses
F1020 - Physiology
F1080 - Genetics
F1040 - Molecular biology
G2.2 - Increase Efficiency of Production and Marketing Systems
G4.2 - Reduce Number and Severity of Pest and Disease Outbreaks
G6.1 - Ensure Clean Water and Air


RESEARCH EFFORT CATEGORIES
BASIC 20%
APPLIED 40%
DEVELOPMENTAL 40%

KEYWORDS: cereals; stress tolerance; risk assessment; risk management; transgenic plants; turf grasses; forage grasses; resource utilization; genetic engineering; plant genetics; traits; plant improvement; water use efficiency; plant physiology; gibberellic acid; mowing; germplasm; somaclonal variation; gene expression; biolistics; wheat; barley; rye; paspalum notatum; lolium; paspalum

PROGRESS: Oct 1, 2006 TO Sep 30, 2007
OUTPUTS: 1. Advanced transformation protocols for grasses: A genetic transformation protocol for seashore paspalum was developed for the first time. Relevant regulatory elements, selectable marker genes and sugarcane chloroplast sequences have been isolated for the development of integrative chloroplast transformation vectors for sugracane. 2. Generation and evaluation of transgenic grasses: Integration and expression of a synthetic, insecticidal BT cry gene was confirmed in seashore paspalum by Southern blot analysis and RT-PCR. An RNA interference construct was generated with a partial cDNA sequence of the 4-coumarate:coenzyme A ligase (4-CL)and stably introduced into bahiagrass. Transgenic bahiagrass plants will be analyzed for suppression of 4-CL and reduction of lignin. 3. Molecular dissection and engineering of environmental stress tolerance: Transgenic bahiagrass over-expressing WRKY 38 transcription activators displayed enhanced biomass production along with elevated relative water content and photosynthetic activity following dehydration stress. 4. Molecular improvement of turf characteristics: A protocol which combines chemical mutagenesis with somatic embryogenesis was developed to induce mutations in bahiagrass. Regenerated mutants from more than 20,000 treated seeds include phenotypes with shorter stems and leaves and more vegetative tillers. Seedhead production and seedset will be evaluated. 5. Risk assessment and risk management in transgenic grasses: A gibberellin catabolizing enzyme under transcriptional control of a tapetum specific promoter was stably introduced into bahiagrass by biolistic gene transfer to eliminate pleiotrophic effects of constitutive expression and evaluate the potential suppression of pollen development. Seeds from wild type bahiagrasses surrounding the transgenic, apomictic glufosinate herbicide resistant bahiagrass were harvested under field (USDA permit # 05-365-01r) and greenhouse conditions to study short distance gene transfer from apomictic bahiagrass by pollen. These seeds were germinated in the greenhouse and more than 28,000 seedlings were sprayed with glufosinate to determine the gene transfer frequency. Sexual diploid bahiagrass used as pollen receptor under greenhouse conditions with constant air movement resulted in 13 glufosinate resistant hybrids out of a total of 8,300 seedlings (0.15% gene flow frequency). Apomictic tetraploid genotypes used as pollen receptor under greenhouse conditions resulted in 4 glufosinate resistant hybrids out of 7,078 seedlings (0.06% gene flow frequency). Seed progeny analysis from 2006 field grown, sexual, diploid pollen receptors identified 3 glufosinate resistant hybrids out of a total of 8,330 seedlings(0.036% gene flow frequency). While none of 4,330 seedlings derived from 2005 field grown diploid pollen receptors displayed glufosinate resistance. Results of these studies were presented at the Society for In Vitro Biology Conference (awarded first place in the graduate student poster competition), IFAS Research Symposium, University of Florida Graduate Student Council Forum, American Society of Agronomy Conference and Florida Genetics Symposium. PARTICIPANTS: J. Leeds (undergraduate student), J. Thomas (undergraduate student), N. Davila (student OPS), L. Martin (student OPS), J. Yactayo-Chang (student OPS), S. Sandhu (graduate student), M. Agharkar (graduate student), P. Lomba (graduate student), G. Luciani (graduate student), J. Celedon (graduate student), W. Fouad (postdoctoral research associate), X. Xiong (postdoctoral research associate), V. James (postdoctoral research associate), Nameirakpam Singh (postdoctoral research associate), H.Zhang (senior biologist), A. Blount (Co-PI, University of Florida - IFAS, North Florida Research and Education Center Marianna), M. Gallo (Co-PI, University of Florida - IFAS), R. Meagher (Co-PI, USDA ARS), D. Wofford (Co-PI, University of Florida - IFAS), K. Kenworthy (Co-PI, University of Florida - IFAS), T. Sinclair (Co-PI, University of Florida - IFAS), W. Vermerris (Co-PI, University of Florida - IFAS), J. Preston (Co-PI, University of Florida - IFAS), B. Moudgil (Co-PI, University of Florida - PERC), Elide Valencia (Co-PI, University of Puerto Rico),Scott Morris (student OPS), Henry Daniell (Co-PI, University of Central Florida), F. Altpeter (PI, University of Florida - IFAS) TARGET AUDIENCES: Sugarcane, forage and turf industry

IMPACT: 2006-10-01 TO 2007-09-30 Chloroplast transformation of sugarcane is expected to support high level expression of transgenic proteins along with transgene containment. For the first time a transgene was stably inserted into the nuclear genome of seashore paspalum. The developed protocol will allow molecular improvement of abiotic or biotic stress tolerance in this high quality turfgrass. The expression of the insecticidal BT gene in seashore paspalum is a first step in this direction. Molecular improvement of drought tolerance and cold tolerance will enhance the productivity and persistence of commercially important grasses and will result in a more efficient use of natural ressources. Over-expression of the WRKY 38 transcription factor in bahiagrass supported better recovery from severe dehydration stress. This might enhance persistence of perennial turf and forage grasses following an extended period of drought without irrigation. Quality improvement in those grasses, that are well adapted to environmental stress, can significantly increase their value. Transgenic bahiagrass plants with integration of an RNAi suppression vector of 4-coumarate:coenzyme A ligase (4-CL) a key enzyme in lignin biosynthesis were generated. We will investigate if RNAi suppression of 4-CL in bahiagrass results in reduced lignin content. Reduced lignin content is expected to enhance digestibility and forage quality of bahiagrass. The developed mutagenesis protocol might contribute to enhancing the turf or forage quality of bahiagrass by increasing leaf biomass and turf density on expense of reproductive tillers and stem biomass. Risk assessment and risk management are essential components of a molecular crop improvement program. The described risk management and risk assessment research provides the first data on a cross pollinating, perennial warm season turf and forage grass and involves co-existing apomictic and sexual genotypes. The described research evaluates promising strategies for transgene containment in bahiagrass in subsequent greenhouse and field experiments. Gene flow studies showed low hybridization frequencies under field conditions (< 0.04%) between the apomictic transgenic and wild-type bahiagrass even if pollen receptor and donor are in close distance(1m). All hybrids derived from sexual diploid pollen receptors have been confirmed for bar gene integration by Southern blot analysis and bar gene expression by immuno-chromatographic lateral membrane flow stix assay (Liberty link PAT/bar). All six of the so far analyzed hybrids have been confirmed as triploids by both flow cytometry and cytogenetic root-tip chromosome counting. Embryo sac analysis showed these hybrids as facultative apomicts with both apomictic and sexual embryo sacs. Fertility of hybrids will be evaluated. Molecular markers will be used to determine apomictic or sexual nature of triploid hybrid plants. All triploid hybrids showed reduced vigor compared to diploid or tetraploid bahiagrass. This suggests that using apomictic bahiagrass cultivar Argentine as target for bahiagrass transformation provides a high level of transgene containment compared to open-pollinating sexual diploid turf and forage grasses.

PUBLICATION INFORMATION: 2006-10-01 TO 2007-09-30
Agharkar, M., Lomba, P. N., Altpeter, F., Zhang, H., Kenworthy, K. E. and Lange, T. Stable expression of AtGA2ox1 in a low-input turfgrass (Paspalum notatum Flugge) reduces bioactive gibberellin levels and improves turf quality under field conditions. Plant Biotechn. J. 2007. 5: 791-801.

PROJECT CONTACT INFORMATION
NAME: Altpeter, F.
PHONE: 352-392-1823
FAX: 352-392-7248