Annual Progress Report on Malting Barley Research March, 2002

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96 ATTEMPTS TO TRANSFORM NEWER CULTIVARS WITH GENES AFFECTING TRICHOTHECENE TOXIN AND FHB LEVELS Lynn S. Dahleen and M. Manoharan Cereal Crops Research Unit, USDA-ARS and Dept. of Plant Sciences, NDSU Objective To produce transgenic commercial barley cultivars that express anti-toxin genes which reduce DON levels and Fusarium infection. Introduction Fusarium head blight (FHB), caused primarily by Fusarium graminearum, has been one of the most destructive diseases of barley since the early 1990s, resulting in huge economic losses for growers (McMullen et al.1997). Of particular concern is the production of deoxynivalenol (DON), a potential pathogen virulence factor (Desjardins et al. 1996, Proctor et al. 1995, 1997) that is harmful to humans and livestock (Busby and Wogan 1981). Chemically modifying DON or transporting it out of cells could improve resistance to FHB while at the same time reducing DON accumulation in grain. FsTri101 (TriR), isolated from F. sporotrichioides, encodes a 3-OH trichothecene acetyltransferase that converts DON to a less toxic acetylated form. PDR5, an ATP-binding cassette transporter isolated from Saccharomyces cerevisiae, acts as an efflux transporter, shunting DON across the plasma membrane from the interior of the cell. We have transformed the commercial malting barley cultivar Conlon with the Tri101 and PDR5 genes with the aim of reducing DON level in infected grain. Methodology Development of the transgenic lines was described in last year’s report. Experiments have resulted in seven transgenic events for Tri101 and six events for PDR5. A transformation event consists of all the plants regenerated from one callus clump that was bombarded with the genes. Four of the events for each gene resulted in tetraploid plants. Lines from the three diploid Tri101 events and the two diploid PDR5 events were advanced two generations by self-pollination and seed used for field trials in 2001. Ten lines each of the five diploid transformation events were planted in short rows in two replicates. One replicate was planted in the misted, inoculated nursery at Langdon, ND and one replicate was planted just outside the inoculated nursery, so each would have different levels of FHB. Non-transgenic Conlon was planted every fifth row. Traits measured included height, % FHB, seed weight per 100 spikes, and DON concentration. Greenhouse tests are underway. Plants are spray inoculated, held in a mist chamber for 24 hours, and then scored for % FHB 14 days later. The two lines from each transformation event that had the lowest FHB in the field have been tested and additional lines are being grown for further testing. After harvest, seed will be sent to the NDSU Barley Malt Quality Lab for DON measurements.
97 Results Results from field and greenhouse tests of the diploid transgenics are presented in Table 1. Acetylase activity tests indicated that Tri101 was expressed in all 30 lines. FHB infections in lines with Tri101 were similar to or somewhat less than the Conlon nontransgenic controls in both the field and greenhouse trials. However, DON levels in the Tri101 lines from the field were equal to or higher than Conlon, sometimes substantially higher. DON tests on the greenhouse-grown plants will be conducted when seed is mature. Lines with PDR5 showed more promising results. Northern analysis showed that PDR5 was transcribed in the transgenic lines. In the field and greenhouse, all PDR5 lines had FHB levels equal or less than the Conlon control rows. Unlike lines with Tri101, only five PDR5 lines showed increased DON. Eleven of the lines had a DON reduction of at least 1 ppm, with DON in three of the lines reduced more than 5 ppm. DON tests on the greenhouse-grown plants will be conducted when seed is mature. Most transgenic lines were shorter than Conlon, with the shortest lines almost 30% shorter than the nontransgenic control. Transgenic lines also had reduced seed weights per 100 spikes, with the poorest lines showing a 60% reduction in seed weight. The reduction in seed weight appears to be related to DON levels. PDR5 lines with the lowest DON generally had the smallest reduction in seed weight. Greenhouse screening of the transgenic lines will continue until spring planting. Lines will be planted in replicated field plots this spring for further FHB evaluation and additional controls will be included. Plants will be scored for height, heading date, % FHB, and yield. After harvest, cleaned seed will be used for DON analysis. Additional transgenic line development is underway. Various combinations of the genes Tri101, PDR5, a rice chitinase and a rice thaumatin-like protein are being inserted into Conlon and Drummond. Conlon lines carrying Tri101 and PDR5 are being crossed to put both genes into the same plant. We also plan to cross the Conlon transgenics with other elite 2- and 6-rowed cultivars to transfer the anti-toxin genes into other backgrounds. As these various lines are developed, they will be tested in the lab for the presence and expression of the transgenes and in the field for the effects of the genes on FHB and DON levels. Personnel Mrs. Luming Brewer – part-time laboratory technician Mrs. Mary Wentz - part-time laboratory technician Recent Publications Dahleen, L.S., Okubara, P.A. and Blechl, A.E. Transgenic approaches to combat Fusarium head blight in wheat and barley. Crop Sci. 40:628-637. 2001. Dahleen, L.S. and McCormick, S.E. Trichothecene toxin effects on barley callus and seedling growth. Cer. Res. Comm. 29:115-120. 2001
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Annual Pro
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-i- INTRODUCTION The March 2002 <st
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Barley Transformation G.J. Muehlbau
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1 2000/2001 WINTER NURSERY: BARLEY
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3 2001 Barley Stripe Rust Screening
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5 Triumph/Tyra//Arupo, a selection
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7 Washington (Fossum Cereals and Wa
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9 This project is supported by the
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11 ‘Triumph’, ‘Valier’, ‘
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13 Table 2. Agronomic data for sele
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15 Table 4. Agronomic data for sele
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17 Table 8. Malting quality data* f
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19 Six-rowed barley selections of c
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21 Table 14. Agronomic data for sel
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23 Table 16. Agronomic data for win
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25 Table 19. Malting quality data*
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Garnet/98Ab11865 Garnet/98Ab12895 9
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29 rust resistant NSGC accessions o
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31 DEVELOPMENT OF SIX-ROWED MALTING
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33 backcrossing. The selfed lines w
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35 MINNESOTA BARLEY IMPROVEMENT PRO
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37 ADVANCED LINE EVALUATION Varieti
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39 OTHER BARLEY DISEASES In 2001, r
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41 Wingbermuehle, W. J., K.M. Belin
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43 In 2001 winter/spring, over 300
Page 53 and 54: 45 assessments were made by countin
Page 55 and 56: 47 IDENTIFICATION OF NOVEL DISEASE
Page 57 and 58: 49 Unfortunately, disease levels in
Page 59 and 60: 51 BARLEY TRANSFORMATION G.J. Muehl
Page 61 and 62: 53 We have available the sugarcane
Page 63 and 64: 55 Secondly, we tried to grow F. gr
Page 65 and 66: 57 References: Issac, S. and Jennin
Page 67 and 68: 59 Huntley-dryland) with two replic
Page 69 and 70: 61 Table 2. 1992 thru 2001 Overall
Page 71 and 72: 63 performance for Montana farmers.
Page 73 and 74: 65 Blake, T,. Hensleigh P., Boss D.
Page 75 and 76: 67 settling tower cannot accommodat
Page 77 and 78: Table 3. Seedling reaction to strip
Page 79 and 80: 71 TWO-ROWED BARLEY IMPROVEMENT PRO
Page 81 and 82: Table 1. Agronomic trait comparison
Page 83 and 84: 75 mutants reduce plant vigor and g
Page 85 and 86: 77 SIX-ROWED BARLEY IMPROVEMENT PRO
Page 87 and 88: 79 this variety has decreased over
Page 89 and 90: Table 7. Malt quality comparisons o
Page 91 and 92: 83 • Management studies for malt
Page 93 and 94: 85 STUDIES ON BARLEY DISEASES AND T
Page 95 and 96: 87 With funding from the US Wheat a
Page 97 and 98: 89 MALTING AND BREWING QUALITY OF B
Page 99 and 100: 91 β-glucans, it might be most ins
Page 101 and 102: 10 9 8 7 6 5 4 3 2 1 0 93 Figure 1.
Page 103: 95 Bolin, P., Schwarz, P., Jones, B
Page 107 and 108: 99 DEVELOPING BARLEY TISSUE CULTURE
Page 109 and 110: 101 trials of plants regenerated fr
Page 111 and 112: 103 Table 2. Agronomic performance
Page 113 and 114: 105 BSMV spread between entries. Ho
Page 115 and 116: 107 involving two susceptible backg
Page 117 and 118: 109 THE OREGON BARLEY IMPROVEMENT P
Page 119 and 120: 111 for traits that are difficult/e
Page 121 and 122: Germplasm Development: 113 Crosses:
Page 123 and 124: 115 • All lines in spring yield t
Page 125 and 126: 117 Table 2. Agronomic data for Ore
Page 127 and 128: 119 Table 6. Malting quality of win
Page 129 and 130: 121 Table 7. Across location summar
Page 131 and 132: 123 MOLECULAR MARKER ASSISTED MODIF
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Page 135 and 136: 127 exchanges. Spring and winter ba
Page 137 and 138: 129 Table 2. 2000-2001 Agronomic Pe
Page 139 and 140: 131 Table 7. Spring 2- and 6-Row #
Page 141 and 142: 133 2. Direct Seeding Cropping Syst
Page 143 and 144: 135 D.M. Wesenberg - spring and win
Page 145 and 146: 137 A STUDY OF THE MALTING QUALITY
Page 147 and 148: 139 Methods. Our malting schedule.
Page 149 and 150: 141 modification measures indicated
Page 151 and 152: 143 CHARACTERIZATION OF THE PROTEIN
Page 153 and 154: 145 Purification of a serine endope
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147 STUDIES ON THE PROTEINASES THAT
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149 with the proteinase T will be s
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151 10°C increase in thermostabili
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153 Sissons MJ and MacGregor AW (19
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µM Carbohydrate / Unit of α-Gluco
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157 Tissue-specific gene products f
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159 The promoters were subcloned fr
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161 Fig. 3. Expression of Trx-Hth f
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Annual Progress Report on Malting Barley Research March, 2002

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