1. Performance of Four Mannitol-Accumulating Transgenic Wheat Lines Under Moisture Stress and Non-Stress Conditions MATERIALS AND METHODS  The four mannitol-accumulating transgenic lines, the non- transformed parent line ‘Bobwhite’, and five conventional check varieties (Reeder, RT01-12, Butte 86, Kauz, and MTRWA116) were planted on April 29, 2005 at the ARDEC research station north of Ft. Collins, CO.  In two of the lines (pTA5-1 and pTA5-2), the transgene construct contained the chloroplast transite peptide sequence to transport the mtlD product to the chloroplast. In lines pTA2-1 and pTA2-2 (without the transit peptide sequence), mtlD is expected to accumulate in the cytosol.  In order to establish the stands, both the irrigated and rain-fed trials were furrow irrigated until June 17, when a diversion ditch was dug between the trials, cutting off the irrigation to the rain-fed plots.  To compensate for stand variability between plots, grain weight, 200-kernel weight, harvest index, head number, and number of kernels was calculated from a one-meter sample taken from one row in each plot  Soil moisture was determined with a Delta T Instruments HH2 Moisture Meter used with a six-sensor, one meter long soil probe.  Statistical analysis was conducted with SAS/STAT v. 9.1 (Cary, NC). Analysis of variance was followed by mean separation with Duncan`s New Multiple Range Test (p<0.05), and simple phenotypic (Pearson) correlation.  Experimental Design: Complete Randomized Block Three Replications Plot Measurement: 5’ X 5’ INTRODUCTION Moisture stress is one of the greatest factors affecting yield in agronomic crops worldwide. Traditional breeding methods have met with only limited success at increasing the drought-tolerance of field crops. Some of the most current attempts at improving the performance of crops in water-limited environments have used biotechnology to transfer novel genes conveying drought-tolerance into crop species. A research team from Oklahoma State University has transformed the spring wheat variety ‘Bobwhite’ with the mannitol-dehydrogenase gene (mtlD) from E. coli that causes the wheat to produce the sugar alcohol mannitol. Mannitol is found in several plant species, and is thought to aid in stress tolerance by scavenging reactive oxygen species (ROS) produced during stress. OBJECTIVES  To evaluate the performance of four transgenic lines containing the mtlD gene in the ‘Bobwhite’ background under well watered and moisture stress conditions  To compare the performance of two lines with transgenes containing the chloroplast transite peptide sequence to two lines without the sequence.  To gain insights on the drought tolerance mechanism of the mtlD gene. CONCLUSIONS Grain Weight – 1 Meter 200-Kernel Wt. Harvest Index # of Heads – 1 meter Head Length # of Kernels – 1 meter Grain Weight– 1 Meter 0.80219 0.0052* 0.90398 0.0003* 0.76587 0.0098* -0.46810 0.1724 0.98267 <.0001* 200-Kernel Wt. -0.09084 0.8029 0.64834 0.0426* 0.46094 0.1800 -0.02359 0.9484 0.68429 0.0291* Harvest Index 0.93223 <.0001 * -0.03536 0.9228 0.78168 0.0076* -0.42381 0.2223 0.92664 0.0001* # of Heads – 1 meter 0.88486 0.0007 * -0.15224 0.6746 0.89660 0.0004 * -0.71118 0.0211* 0.80995 0.0045* Head Length -0.53369 0.1121 0.59741 0.0682 -0.41240 0.2363 -0.66271 0.0368 * -0.56572 0.0883 # of Kernels – 1 meter 0.97122 <.0001 * -0.31150 0.3809 0.91586 0.0002 * 0.87850 0.0008 * -0.63766 0.0473 * Pearson Correlation Coefficients Correlations in Fully Irrigated Trial Shown Above Diagonal, Correlations in Partially Irrigated Trial Shown Below (Correlations significant at the 0.05 level are denoted by an asterisk *) Source of Variance Grain Weight – 1 Meter 200-Kernel Wt. Harvest Index # of Heads – 1 meter Head Length # of Kernels – 1 meter Treatment**** ***ns **** Rep (Treatment) ns* Entry**** Treatment* Entry ***nsNsns CV (%)26.36.815.717.24.921.5 Summarized ANOVA Table (all treatments) *, ****: Significant at the 0.05 and 0.0001 levels of probability, respectively. ns: not significant at the 0.05 probability level. Means of Selected Traits a ab a a c c c c c c c c bc c ab abc a cd d bcd a ab cd ab abc a ab a cd d b bc cd a d bcd ab d a bcd f de cde e f bc de a b d cd bcd bc cd e bcd ab cd ab abc f bcd a d ab cd abc a a c bc b a a b b b b b (Means within treatments followed by same letter are not statistically significant at the 0.05 level) Chart 1aChart 1bChart 1c Chart 1dChart 1eChart 1f Chart 3aChart 3b RESULTS AND DISCUSSION  Due to heavy rains early in the season (refer to the raınfall chart), both trials were well watered until anthesis (refer to soil moisture chart). Hot and dry weather during grain filling resulted in a divergence in soil moisture profiles (refer to other soil moisture chart) and a severe stress on the partially irrigated trial. Mean grain weight per plot in the stressed trial was reduced 43% from the well watered trial.  In the analysis of variance, the effect of entries was highly significant for all variables, the treatment effect was significant for all variables except number and length of heads, and the treatment x entry effect was siginificant only for grain weight and 200- kernel weight (refer to anova table).  Several significant correlations were found both in the fully irrigated and partially irrigated trials (refer to correlation table). Correlations found in the fully irrigated trial, but not in the partially irrigated trial include grain weight and 200-kernel weight, harvest index and 200-kernel weight, and 200-kernel weight and number of kernels. One correlation between head length and number of kernels was found in the partially irrigated trial, but not the fully irrigated trial.  Poor emergence in the transgenic lines and the `Bobwhite` control resulted in low plant stands relative to the cultivars. Partially for this reason, the cultivars outperformed the transgenic lines for most variables (refer to mean graphs).  For most traits in both treatments, the transgenic lines did not differ from the `Bobwhite`control (refer to mean graphs). One or more transgenic lines exceeded the control for harvest index, kernel number, and head number.  There was no clear pattern of difference between lines with and without the chloroplast trasit peptide. Partially Irrigated Trial Diversion Ditch Fully Irrigated Trial  Data this year showed no clear differences among the transgenic lines or between the transgenics and the parent line ‘Bobwhite’  This year’s evaluation may have been complicated due to late planting, small plot size, and/or poor stand establishment  Late season rain may have also lessened the amount of moisture stress  Trials will be repeated next year REFERENCES Abebe, Tilahun, Arron C. Guenzi, Bjorn Martin and John C. Cushman. “Tolerance of Mannitol- Accumulating Transgenic Wheat to Water Stress and Salinity.” Plant Physiology 131: 1748-1755. William Nobles 1, Patrick Byrne 1, Arron Guenzi 2, Bjorn Martin 2, Sathya Elavarthi 2, Brett Carver 2 and Mohamed Mergoum 3, (1)Colorado State University, Soil and Crop Sciences Dept., Campus Delivery 1170, Fort Collins, CO 80523-1170, (2)Oklahoma State University, Oklahoma State University, 368 Ag Hall, PASS, Stillwater, OK 74078, (3)Plant Sciences Department, NDSU, Loftsgard Hall, P.O. Box 5051, Fargo, ND 58105