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DEVELOPMENT OF COLD-TOLERANT NAPIERGRASS AS A BIOENERGY FEEDSTOCK *Charlie Dowling, Texas A&M University Department of Soil & Crop Sciences; Russell Jessup, Texas A&M University Department of Soil & Crop Sciences; Byron Burson, USDA-ARS Crop Germplasm Research Unit, Texas A&M University ABSTRACT Perennial C 4 grasses, such as napier grass (Pennisetum purpureum Schumach.; 2n=4x=28), are vital in the recurrent rise in the demand for more output from renewable energy sources. Advancements occurring in the area of biomass feedstock processing and conversion technology makes the lignocellulosic energy platform provided by biomass feedstocks like napiergrass increasingly viable and also most beneficial for carbon sequestration. Similar to other high yielding dedicated energy crops, napiergrass’ production region is limited to areas of the Southern United States. The tertiary gene pool found only in the wild species within Pennisetum harbor enhanced cold tolerance in comparison to the cultivated Pennisetum species, pearl millet and napiergrass, that contains the primary and secondary gene pools of Pennisetum. Interspecific hybridization of napiergrass with the wild species, P. orientale Rich. and P. flaccidum Griseb., allows for the opportunity to incorporate cold tolerance and possibly apomixis into the napiergrass genome. Wide interspecific crosses utilizing oriental grass [P. orientale (2n=4x=36)] and flaccid grass [P. flaccidum (2n=4x=36)] as pollinators with napiergrass as the female parent are being analyzed for viable hybrid production. Embryo rescue techniques in the occurrence of zygote termination in the napiergrass ovule are being investigated while the production of natural hybrids may be a possibility. The characterization of genetic diversity for cold tolerance within napiergrass is also being developed with genotypes that exhibit superior adaptation for winter hardiness and cold tolerance in higher latitude USDA Hardiness Zones. This napiergrass germplasm originated from and is being developed in the Perennial Grass Breeding Program at Texas A&M University. Species- and cultivar-specific microsatellite markers are under development in the genus Pennisetum (napiergrass, pearl millet, oriental grass, and flaccid grass) as a rapid hybrid verification procedure. OBJECTIVES Select individual napiergrass plants and genotypes that display superiority to overwinter in increasingly higher latitudes, ie. USDA Hardiness Zones. Create novel interspecific hybrids between napiergrass and Pennisetum wild species (flaccid grass & oriental grass) naturally and/or through embryo rescue techniques to incorporate cold tolerance into napiergrass’ genome Develop genotype- and species-specific markers in Pennisetum oriental, P. flaccidum, and P. glaucum for high-throughput hybrid verification MATERIALS AND METHODS Evaluate the cold tolerant adaptation of 19 napiergrass selections with improved rhizomes. The selections represent six novel genotypes that were planted in replicates of two at the AgriLife Research Center in Commerce, TX and assessed during the winter of 2012-2013. Plant IDNo. overwinteredNo. tillers, Plant 1No. tillers, Plant 2 PEPU12TX09 247 PEPU12TX10 286 PEPU12TX13 11-- PEPU12TX14 11-- PEPU12TX15 225 PEPU12TX17 118-- PEPU12TX18 212 PEPU12TX19 241 PEPU12TX20 17-- PEPU12TX21 254 PEPU12TX22 110-- Figure 2. (A) Napiergrass surviving the winter of 2012-2013 in Commerce, TX (B) Pollen tube growth in P. glaucum x P. orientale cross [Kaushal and Sidhu, 2000];. RESULTS AND DISCUSSION CONCLUSIONS Conclusion 1 Conclusion 2 Conclusion 3 Fluorescent in-situ hybridization (FISH) of napiergrass gynoecium to determine pollen tube growth of P. orientale and P. flaccidum to the micropyle and ovary in napiergrass Embryo rescue tecniques of napiergrass ovules pollinated with P. orientale and P. flaccidum in progress. Bulked Segregant Analysis (BSA) of P. purpureum x P. orientale and P. purpureum x P. flaccidum utilizing expressed sequence tag (EST)-derived simple-sequence repeats (SSRs) A B Representative plants from each of the six genotypes planted in Commerce, TX exhibited adaptation to colder environments (Fig. 2A). The number of plants that overwintered along with the number of tillers per plant are shown in Table 1. Woodard and Sollenberger (UFL, 2012) defined 8b as the northern zone limit for cold-tolerant selections of napiergrass, however, overwintering of TAMU napiergrass reached zone 8a (See Figure 1.). This demonstrates suitable variability allowing for further selection and introgression of cold-tolerance into other elite, high-biomass genotypes. Table 1. Overwintering of napiergrass genotypes selected for increased cold-tolerance and rhizomatousness Figure 1. TAMU napiergrass adaptation (red line) related to the expected northern limit (blue line). *The red star represents Commerce, TX. No hybrids were recovered via embryo rescue of 400+ napiergrass pistils during winter of 2012-2013. Optimizing this procedure in progress including the consideration of protoplast fusion. Pearl millet stigmas have demonstrated compatibility with pollen from diverse Pennisetum species (Fig. 2B) indicating the feasibility of napiergrass hybrids with Pennisetum wild species. P. orientale- and P. flaccidum-specific markers are currently being developed, and two oriental grass and seven flaccid grass putative hybrids have been produced. Delineation of these to soon follow. EST-SSRs specific to male parent, napiergrass, have recently been reported to delineate hybrids between pearl millet and napiergrass (Dowling, et al., 2013)(Figure 3.). Progenies 1-10 Figure 3. Napiergrass-specific EST-SSR segregation in PMN hybrids Female Male