1. Reactive Oxygen Species Generation and Impact of Allopurinol in Hybrid Necrosis in Wheat D. R. Kandel 1 *, K. D. Glover 1, W. A. Berzonsky 2, Y. Wu 1 and M.R. Anower 3 Several studies in wheat hybrid necrosis documented the reactive oxygen species (ROS) mediated cell death (Khanna-Chopra et al., 1998; Sharma et al., 2003). Oxidative stress due to higher generation of superoxide anion and hydrogen peroxide (H 2 O 2 ) are reported both before and during the progression of necrosis n hybrid plants. Plants cell possesses several enzymes considered to have involved in the synthesis of H 2 O 2 (Apel and Hirt 2004). Xanthine oxidase is one of them. Allopurinol is a xanthine oxidase inhibitor. In several host rust interactions, use of allopurinol altered the susceptible reaction to the hypersensitive type (Montalbini, 1992; Adam et al., 2000). Introduction  To evaluate the change in ROS generation before and during progression of necrosis in necrotic hybrid and in parents  To determine the impact of allopurinol in ROS production and in overall in hybrid necrosis Materials and Methods Study was conducted in spring wheat crossing greenhouse located at South Dakota State University in factorial RCB design with 9 – 11 replications. Three wheat genotypes were included. 1. TA 4152-37 i.e., synthetic hexaploid wheat as a female parent 2. Alsen as a male parent and, 3. F 1 hybrid (TA 4152-37/Alsen). ROS measurement: Amplex® red hydrogen peroxide assay kit was used to detect the released H 2 O 2 as the reactive oxygen species. H 2 O 2 was measured from both without (0 µM) and with allopurinol (30 µM) treated plants. Three different concentrations of allopurinol was applied through soil drenching i.e., 0 µM, 15 µM, and 30 µM. Canon DSLR was used to take images of 5 th leaves and percent necrosis was analyzed using Assess 2 software. ResultsConclusions  Higher generation of ROS or oxidative burst is not always the case that cause programmed cell death in hybrid necrosis. Cell death in hybrid necrosis can occur even in the normal or low ROS production condition.  Allopurinol does not have impact on ROS production, and overall performance in hybrid necrosis. This suggests that the xanthine oxidase is not likely the main source of ROS generation in tested wheat genotypes. Acknowledgements Hybrid necrosis in wheat (Triticum aestivum L.) is the gradual premature death of leaves, shealth tissues or plants in hybrids (Hermsen, 1963). Hybrid necrosis in wheat is caused by the complementary interaction of two dominant genes Ne1 and Ne2 which are brought together due to hybrid combination (Tsunewaki, 1960; Chu et al., 2006). Fig. 4. ROS production from Ist leaf in three wheat genotypes in both with and without allopurinol treated plants when there was not necrosis and when 25% leaf area covered by necrosis in F 1 (TA 4152- 37/Alsen). References Adam, A. L., A. A. Galal, K. Manninger, and B. Barna. 2000. Plant Pathol. 49: 317-323. Apel, K., and H. Hirt. 2004. Annu. Rev. Plant Biol. 55:373–399. Chu, C. G., J. D.Faris JD, T. L. Friesen, and S. S. Xu. 2006. Theor. Appl. Genet. 112: 1374–1381. Dalal M, and R. Khanna-Chopra. 2001. Physiol. Plant. 111:297–304. Hermsen, J.G. Th. 1963. Euphytica 12:1–16. Montalbini, P. 1992. J. Phytopath. 134:218–228. Sharma, G., B. Srivalli, and R. Khanna-Chopra. 2003. Indian J. Biotechnol. 2: 17 – 25. Tsunewaki, K. 1960. Japan J. Genet. 35: 71–75.  South Dakota Wheat Commission and USDA/NIFA for financial support  Spring Wheat Breeding Program  Plant Science Department  Steven S. Xu for providing female parent seeds  Siddhi Bhusal, Mkandawire Thumbiko, Krishna Ghimire Fig. 1. F 1, hybrid pants and leaves showing necrosis Parent 1 F 1 Parent 2 Fig. 2. Experimental plots in Green house 1 South Dakota State University, Brookings, SD; 2 Bayer CropScience, Lincoln, NE; 3 Noble Foundation, OK *Presenting author email (devi.kandel@jacks.sdstate.edu)  H 2 O 2 productions in F 1 leaves (TA 4152-37/Alsen) were not higher than the parent leaves in both before and during the progression of necrosis (Figs. 4 and 5). Furthermore, as the necrosis progressed, H 2 O 2 production decreased in F1. Allopurinol application does not have any effect on H 2 O 2 production (Figs 4 and 5).  Allopurinol use also does not have effect on necrosis development in F 1 (Fig. 6). Fig. 5. ROS production from 5 th leaves in three wheat genotypes in both with and without allopurinol treated plants when necrosis was just started and when necrosis covered >35% of leaf area in F 1 (TA 4152- 37/Alsen). Fig. 6. Necrosis progression in 5 th leaves in F 1 plants (TA 4152- 37/Alsen) supplied with different allopurinol concentration through soil drenching. Objectives 3.1 3.3 3.2 3.3 Parent 1 F1F1 Parent 2 Status of F 1 leaves (TA 4152-37/Alsen) during ROS measurement