Progress 09/01/05 to 08/31/08

Outputs
OUTPUTS: A primary output of this project was the training of a M.S. graduate student in all aspects of sub-tropical and tropical legume plant cell and tissue culture. In this program we developed methods for the culture of several different species with these protocols contained in the student's thesis and in preparation for publication in manuscripts. An additional outcome was the discovery to high levels of root-knot nematode resistance to near immunity in several of the native tropical forage legumes evaluated in this project. This finding has been disseminated at national professional meetings and in journal publications. An additional output was the release of two cultivars (UF Tito and UF Peace) of rhizoma perennial peanut in summer 2008. These cultivars will be available to producers in 2009. PARTICIPANTS: The principal investigators of this project were Drs. K. H.Quesenberry and M. J. Williams. During the period of the grant we have both participated in the research project and gained valuable knowledge related to native and introduced tropical and subtropical forage legumes. The Master of Science student trained in this project, Ms. Laura Vidoz is a native of Argentina and obtained new and varied experience related to the responses of these leguminous species to plant cell and tissue culture. She produced an outstanding MS Thesis and publications documenting the reseearch are in preparation. Subcontract coopeartive research in Puerto Rico and in Uruguay with Dr. Elide Valencia and Dr. Daniel Real, respectively, led to the development and strengthening of research ties between these institutions. The findings of their research have been published in refered publications as documented in previous annual reports of this grant. TARGET AUDIENCES: The traget audience of this project was a combination of scientists worldwide who work with tropical and subtropical forage legumes and livestock producers who might incorporate some of the legumes identified in this project into their production system. Our findings regarding root-knot nematode resistance in native legumes were shared with the international research community at the International Grasslands Congress in Hohhot, China in summer 2008. We officially released two rhizoma perennial peanut cultivars in summer 2008 that resulted from research conducted under this and a previous TSTAR grant project. These new cultivars offer genetic diversity, improved competitive ability in mixed grass swards and possible tolerance to virus diseases that seem to be attacking current released cultivars. Producers will be notified of these releases for the spring 2009 planting season. PROJECT MODIFICATIONS: A one year non-funded extension was approved to complete all objectives originall outlined in this project. All objectives as outlined in the original project were completed.

Impacts
Two changes in knowledge have resulted from this project. One of these was the knowledge developed related to methods (tissues and media protocols) appropriate for the regeneration of various tropical and subtropical legumes evalauted in this project. Prior to this research little to no plant cell and tissue culture research had been conducted with these species and the research knowledge developed in this project is a significant additional to the scientific knowledge base. Additionally, prior to this research, the response of these legume species to root-knot nematodes (Meloidogyne spp.) was unknown. Our reseearch has shown that several of these species have high levels of resistance and could possibly be a source of genes for resistance that could be transferred to crop species by genetic transformation.

Publications

• Quesenberry, Kenneth H., Judith M. Dampier, Billy Crow, and Donald W. Dickson. 2008. Response of native southeastern USA legumes to root-knot nematodes. Crop Sci. 48:in press.
• Quesenberrry, K.H., J.M. Dampier, and E. Valencia. 2008. Forage potential of legumes native to subtropical southeastern USA. p. 466. In Organizing Committee of ICG/IRC 2008 (ed.). Multifunctional Grasslands in a Changing World. Vol. II. Proc. XXI International Grasslands Congress and VII International Rangelands Congress. Hohhot, China. 29 June to 4 July 2008. Guangdong Peoples Publishing House, Beijing.
• Williams, M.J., T.R. Sinclair, P. Mislevy, K.H. Quesenberry, A.S. Blount, and S.W. Coleman. 2008. Photoperiod sensitivity of rhizoma peanut germplasm. Agronomy J. 100:1366-1370.

Progress 09/01/06 to 08/31/07

Outputs
OUTPUTS: A field experiment of selected accessions of Desmodium incanum, a species native to Uruguay and the southeastern USA established as a second stage monoculture evaluation experiment, showed infection by plant virus in late fall 2006 and severe infection in spring 2007. Initial pathological testing with a general potyvirus antisera was positive for the presence of potyvirus. Subsequent tests with available antisera showed negative results for bean yellow mosaic virus (BYMV), soybean mosaic virus (SMV) and watermelon mosaic virus (WMV). Antisera were not available for blackeye cowpea mosaic virus (BCMV) or desmodium mosaic virus (DMV), two other potyviruses that have been identified on Desmodium species. By mid summer 2007 all plants in the monoculture experiment had died, although the earlier experiment interplanted with bahiagrass showed good persistence and negligible virus symptoms. Similar to the results reported for the Gainesville experiments in 2006, data on tannin content of Desmodium accessions grown in Puerto Rico also showed variability and levels above and below those previously identified in the more tropical species Desmodium heterocarpon. These results suggest that this species may have levels of tannin that could be of an antiquality concern in livestock diets, but that also could have positive antihelminthic effects for animals grazing these plants. Data from the ongoing field evaluation experiment with Arachis glabrata advanced selections was summarized in 2007 along with data from two other Florida locations and a decision was made to propose three accessions from this experiment as new cultivar releases. The documentation for this release will be presented to the UF/IFAS cultivar release committee in 2008 and source nursery plantings of these lines will be established at Gainesville and at Marianna. The new germplasm of perennial Arachis species that was established in field plots in summer 2006 at Gainesville was maintained and allowed to fill in as full plots through summer 2007. On the flatwoods soil of this experimental site, weed control during establishment continues to be a production constraint for this species. We have not identified any 4x Trifolium polymorphum accessions from either our stem tip or in vitro chromosome doubling treatments. Additional experiments with tissue culture protocols have show that the growth regulator thiadazuron (TDZ) that had positive effects with some of these subtropical legume species also had a positive effect on red clover regeneration from tissue culture. PARTICIPANTS: Dr. Elide Valencia, University of Puerto Rico, Mayaguez, PR Dr. Daniel Real, INIA, Uruguay Dr. Marco Dalla Rizza, INIA Uruguay TARGET AUDIENCES: The target audience for this project is forage livestock producers in the Southeastern USA subtropics and other regions of the subtropics and tropics. PROJECT MODIFICATIONS: A special additional year extension was approved to complete some objectives of this project.

Impacts
Development of new and improved cultivars of sub-tropical legumes for use in grazed pastures in the southeastern USA and other subtropical regions will improve efficiency of livestock production. This is an environmentally friendly approach in that it reduces the chemical nitrogen fertilizer required for grass production through biological nitrogen fixation by the forage legumes. The goal of this project is to release three new cultivars of perennial Arachis species in 2008.

Publications

• Dalla Rizza, Marco, Daniel Real, Rafael Renyo,Valentina Porro, Eugenia Errico, and Kenneth H. Quesenberry. 2007. Genetic diversity and DNA content of three South American and three Eurasiatic Trifolium species. Genetics and Molecular Biology 30:1118-1124.
• Real, Daniel, Marco Dalla Rizza, Rafael Renyo, and Kenneth H. Quesenberry. 2007. Breeding system of the areial flowers in an amphicarpic clover species: Trifolium polymorphum. Crop Sci. 47 1401-1406.
• Shoaf, Samantha, and Kenneth Quesenberry. 2007. Thiadiazuron effects on tissue culture of red clover. In ASA-CSSA-SSSA-CSSS Abstracts 2006 [CD-ROM]. ASA, CSSA, and SSSA, Madison, WI.

Progress 09/01/05 to 09/01/06

Outputs
Selected accessions of Desmodium incanum, a species native to Uruguay and the southeastern USA well adapted to Florida pasture conditions and associates well with bahiagrass (Paspalum notatum), were established in a second stage evaluation experiment and were evaluated in 2006 for yield and tannin content. The protein precipitation tannin analysis procedure evaluated in 2005 showed repeatable results in 2006. Spread of transplanted plants appeared to lower in a weed free monoculture than when grown in association with bahiagrass as in the stage one experiments. We have also shown that Lotononis bainesii has potential for use as a forage legume in Florida, but that it is sensitive to self-shading of lover canopy buds leading to poor regrowth when dense stands are defoliated by mowing. This plant should be frequently defoliated to an intermediate growth stage in order to maintain vigorous stands. Additional evaluations are in progress with species in Adesmia. An ongoing field evaluation experiment with Arachis glabrata advanced selections was harvested for the fourth year in 2006 and a decision will be made this winter regarding release of one or more cultivars from this evaluation. New germplasm of perennial Arachis species were established in field plots in 2006 at Gainesville and preliminary data has been collected on rate of spread in the establishment year. Laboratory studies have resulted in the development of in vitro plant regeneration protocols for L. bainesii, Adesmia latifolia and A. bicolor. Studies have also resulted in preliminary protocols for T. carolinianum, but additional research is need for T. polymorphum. This research has shown that the growth regulator thidiazuron (TDZ) can be particularly effective in inducing shoot organogenesis in these subtropical forage legumes. An additional finding of this research is that leaf rachis explants in the pinnately compound leaf Adesmia species may be superior to either leaf petioles or leaflets. Our research under objective two has shown that T. polymorphum may have variable pollination biology depending on origin of accessions. Under field and greenhouse conditions in Uruguay the aerial flowers on native accessions were primarily allogamous, whereas the subterranean flowers were autogamous. In the greenhouse in Florida, an accession from Paraguay showed moderate seed production on aerial flowers when self pollinated suggesting lack of a strong self-incompatibility system. Using molecular techniques we have definitively shown that L. bainesii primarily reproduces by allogamy under field conditions. To accomplish objective three, we currently have small plants growing from both vegetative cutting treatment attempts at chromosome doubling in T. polymorphum and from nitrous oxide treatment of pollinated flowers. Success of ploidy manipulation will be verified in the coming year. We have not completed objective four of our project, but research in progress with D. incanum and perennial Arachis could result in germplasm or cultivar releases.

Impacts
Incorporation of forage legumes into perennial grass pastures will improve overall system animal gains due to increased protein content of the animal's diet.

Publications

• Sullivan, M.L., Quesenberry, K.H. 2006. Red Clover (Trifolium pratense). In: Wang, K. Methods in Molecular Biology, vol. 343: Agrobacterium Protocols. 2nd edition. Totowa, NJ: Humana Press. p369-384.
• Quesenberry, K.H., J.M. Dampier, and M.L.Vidoz. 2006. Evaluation of Desmodium incanum germplasm for tannin concentration. In ASA-CSSA-SSSA-CSSS Abstracts 2006 [CD-ROM]. ASA, CSSA, and SSSA, Madison, WI.
• Vidoz, Maria and Kenneth Quesenberry2006. Shoot Organogenesis and Plant Regeneration of Adesmia bicolor and A. latifolia (Leguminosae).In ASA-CSSA-SSSA-CSSS Abstracts 2006 [CD-ROM]. ASA, CSSA, and SSSA, Madison, WI.