Annual Progress Report on Malting Barley Research March, 2002

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152 adjusted RDF value obtained for mashes with added thermostable α-glucosidase were 69% greater than those obtained for mashes with wild type α-glucosidase added. This study shows that the yield of fermentable sugars from the degradation of adjunct and malt starches can be increased by the presence of thermostable α-glucosidase. Additionally, this work shows that the thermostable α-glucosidase produced significantly more glucose when nongelatinized starch from the malt was present and when the starches from the malt and adjunct were gelatinized. In other words, the thermostable αglucosidase was a significant contributor to the production of fermentable sugars and maltodextrins with DP 4-7 throughout mashing as long as starch and/or large polysaccharides were present. Additional research funded by AMBA supports the production of transgenic barley containing the mutated α-glucosidase gene imparting thermostability. To meet this goal we are subcloning the mutated barley α-glucosidase DNA into the pAHC25 plasmid (Ubi:Gus:Nos/Ubi:Bar:Nos) which will later be used to transform barley calli using particle bombardment. Site directed mutagenesis was used to add a start codon and a Sma1 restriction site to the 5' end of the T340P/PTZ18U construct. During this stage of the research the vendor that supplied our preferred mutagenesis kit and reagents discontinued their production. Replacement sources of helper phage and the enzymes required for mutagenesis have now been found and the research is again progressing. The T340P was cut out of PTZ18U at both the 5' and 3' Sma I sites and purified using GENECLEAN II. pAHC25 was digested with Sma I and Sac I, thus removing the GUS gene, and purified using GENECLEAN II. The sticky end of the pAHC25 plasmid caused by cutting with the SacI will be made blunt-ended with T4 DNA polymerase. The T340P DNA will then be ligated into the pAHC25 vector using T4 DNA ligase. Upon complete generation of this construct it will be sent to Dr. Lynn Dahleen who has agreed to collaboratively produce barley (cv. Conlon) containing this gene for thermostable αglucosidase. Publications Clark SE, Hayes PM, Henson CA (2002) A comparison of β-amylase alleles from two North American barley cultivars. Manuscript in preparation. Im H and Henson CA (1995) Characterization of high pI α-glucosidase from germinated barley seeds. Carbohydrate Research 277:145-159 Muslin EH, Clark SE, Henson CA (2002) The effect of proline insertion on the thermostability of a barley α-glucosidase. Protein Engineering 15:29-33 Sun Z and Henson CA (1990) Degradation of native starch granules by barley αglucosidases. Plant Physiology 94:320-327 Sun A and Henson CA (1991) A quantitative assessment of the importance of α-amylase, β-amylase, debranching enzyme and α-glucosidase in starch degradation. Archives of Biochemistry and Biophysics 28:298-305
153 Sissons MJ and MacGregor AW (1994) Hydrolysis of barely starch granules by αglucosidases from malt. Journal of Cereal Science 19:161-169 Yoshigi N, Okada Y, Maeba H, Sahara H, Tamaki T (1995) Construction of a plasmid used for expression of a sevenfold mutant barley β-amylase with increased thermostability in E. coli and properties of the sevenfold mutant β-amylase. Journal of Biochemistry 118:562-569 Table 1. The effects of α-glucosidase additions to mashing on the calculated RDF values of worts. RDF RDF/unit α-glucosidase added wild type α-glucosidase 63.4 13.5 thermostable α-glucosdiase 70.2* 27.9** * P = 0.03, ** P = 0.0002
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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
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45 assessments were made by countin
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47 IDENTIFICATION OF NOVEL DISEASE
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49 Unfortunately, disease levels in
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51 BARLEY TRANSFORMATION G.J. Muehl
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53 We have available the sugarcane
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55 Secondly, we tried to grow F. gr
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57 References: Issac, S. and Jennin
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59 Huntley-dryland) with two replic
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61 Table 2. 1992 thru 2001 Overall
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63 performance for Montana farmers.
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65 Blake, T,. Hensleigh P., Boss D.
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67 settling tower cannot accommodat
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Table 3. Seedling reaction to strip
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71 TWO-ROWED BARLEY IMPROVEMENT PRO
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Table 1. Agronomic trait comparison
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75 mutants reduce plant vigor and g
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77 SIX-ROWED BARLEY IMPROVEMENT PRO
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79 this variety has decreased over
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Table 7. Malt quality comparisons o
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83 • Management studies for malt
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85 STUDIES ON BARLEY DISEASES AND T
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87 With funding from the US Wheat a
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89 MALTING AND BREWING QUALITY OF B
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91 β-glucans, it might be most ins
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10 9 8 7 6 5 4 3 2 1 0 93 Figure 1.
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95 Bolin, P., Schwarz, P., Jones, B
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97 Results Results from field and g
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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
Page 155 and 156: 147 STUDIES ON THE PROTEINASES THAT
Page 157 and 158: 149 with the proteinase T will be s
Page 159: 151 10°C increase in thermostabili
Page 163 and 164: µM Carbohydrate / Unit of α-Gluco
Page 165 and 166: 157 Tissue-specific gene products f
Page 167 and 168: 159 The promoters were subcloned fr
Page 169: 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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