|
|
char = D;code = 68
char = -;code = 45
char = g;code = 103
char = l;code = 108
char = u;code = 117
char = f;code = 102
char = o;code = 111
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = ;code = 32
char = a;code = 97
char = ;code = 32
char = m;code = 109
char = a;code = 97
char = l;code = 108
char = e;code = 101
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = l;code = 108
char = i;code = 105
char = t;code = 116
char = y;code = 121
char = ;code = 32
char = a;code = 97
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = ;code = 32
char = h;code = 104
char = y;code = 121
char = b;code = 98
char = r;code = 114
char = i;code = 105
char = d;code = 100
char = ;code = 32
char = s;code = 115
char = e;code = 101
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = o;code = 111
char = d;code = 100
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = .;code = 46
result sql = D-glufosinate as a male sterility agent for hybrid seed production.
D-glufosinate as a male sterility agent for hybrid seed production.char = A;code = 65
char = ;code = 32
char = c;code = 99
char = h;code = 104
char = e;code = 101
char = m;code = 109
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = m;code = 109
char = a;code = 97
char = l;code = 108
char = e;code = 101
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = l;code = 108
char = i;code = 105
char = t;code = 116
char = y;code = 121
char = ;code = 32
char = s;code = 115
char = y;code = 121
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = m;code = 109
char = ;code = 32
char = b;code = 98
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = r;code = 114
char = -;code = 45
char = l;code = 108
char = o;code = 111
char = c;code = 99
char = a;code = 97
char = l;code = 108
char = i;code = 105
char = z;code = 122
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = n;code = 110
char = v;code = 118
char = e;code = 101
char = r;code = 114
char = s;code = 115
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = v;code = 118
char = e;code = 101
char = ;code = 32
char = D;code = 68
char = -;code = 45
char = e;code = 101
char = n;code = 110
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = m;code = 109
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = h;code = 104
char = e;code = 101
char = r;code = 114
char = b;code = 98
char = i;code = 105
char = c;code = 99
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = ,;code = 44
char = ;code = 32
char = g;code = 103
char = l;code = 108
char = u;code = 117
char = f;code = 102
char = o;code = 111
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = (;code = 40
char = 2;code = 50
char = -;code = 45
char = a;code = 97
char = m;code = 109
char = i;code = 105
char = n;code = 110
char = o;code = 111
char = -;code = 45
char = 4;code = 52
char = -;code = 45
char = (;code = 40
char = m;code = 109
char = e;code = 101
char = t;code = 116
char = h;code = 104
char = y;code = 121
char = l;code = 108
char = p;code = 112
char = h;code = 104
char = o;code = 111
char = s;code = 115
char = p;code = 112
char = h;code = 104
char = i;code = 105
char = n;code = 110
char = y;code = 121
char = l;code = 108
char = );code = 41
char = -;code = 45
char = b;code = 98
char = u;code = 117
char = t;code = 116
char = a;code = 97
char = n;code = 110
char = o;code = 111
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = );code = 41
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = p;code = 112
char = h;code = 104
char = y;code = 121
char = t;code = 116
char = o;code = 111
char = t;code = 116
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = L;code = 76
char = ;code = 32
char = i;code = 105
char = s;code = 115
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = s;code = 115
char = c;code = 99
char = r;code = 114
char = i;code = 105
char = b;code = 98
char = e;code = 101
char = d;code = 100
char = .;code = 46
char = ;code = 32
char = H;code = 72
char = i;code = 105
char = g;code = 103
char = h;code = 104
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = p;code = 112
char = u;code = 117
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = D;code = 68
char = -;code = 45
char = g;code = 103
char = l;code = 108
char = u;code = 117
char = f;code = 102
char = o;code = 111
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = w;code = 119
char = a;code = 97
char = s;code = 115
char = ;code = 32
char = i;code = 105
char = s;code = 115
char = o;code = 111
char = l;code = 108
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = >;code = 62
char = 9;code = 57
char = 8;code = 56
char = %;code = 37
char = ;code = 32
char = e;code = 101
char = n;code = 110
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = m;code = 109
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = c;code = 99
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = ;code = 32
char = f;code = 102
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = r;code = 114
char = a;code = 97
char = c;code = 99
char = e;code = 101
char = m;code = 109
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = v;code = 118
char = i;code = 105
char = a;code = 97
char = ;code = 32
char = f;code = 102
char = e;code = 101
char = r;code = 114
char = m;code = 109
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = w;code = 119
char = i;code = 105
char = t;code = 116
char = h;code = 104
char = ;code = 32
char = a;code = 97
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = r;code = 114
char = a;code = 97
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = E;code = 69
char = s;code = 115
char = c;code = 99
char = h;code = 104
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = c;code = 99
char = h;code = 104
char = i;code = 105
char = a;code = 97
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = l;code = 108
char = i;code = 105
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = p;code = 112
char = r;code = 114
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = g;code = 103
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = P;code = 80
char = A;code = 65
char = T;code = 84
char = ;code = 32
char = (;code = 40
char = L;code = 76
char = -;code = 45
char = g;code = 103
char = l;code = 108
char = u;code = 117
char = f;code = 102
char = o;code = 111
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = N;code = 78
char = -;code = 45
char = a;code = 97
char = c;code = 99
char = e;code = 101
char = t;code = 116
char = y;code = 121
char = l;code = 108
char = ;code = 32
char = t;code = 116
char = r;code = 114
char = a;code = 97
char = n;code = 110
char = s;code = 115
char = f;code = 102
char = e;code = 101
char = r;code = 114
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = );code = 41
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = p;code = 112
char = u;code = 117
char = r;code = 114
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = u;code = 117
char = n;code = 110
char = r;code = 114
char = e;code = 101
char = a;code = 97
char = c;code = 99
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = D;code = 68
char = -;code = 45
char = e;code = 101
char = n;code = 110
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = m;code = 109
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = f;code = 102
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = b;code = 98
char = r;code = 114
char = o;code = 111
char = t;code = 116
char = h;code = 104
char = ;code = 32
char = b;code = 98
char = y;code = 121
char = ;code = 32
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = c;code = 99
char = h;code = 104
char = a;code = 97
char = n;code = 110
char = g;code = 103
char = e;code = 101
char = .;code = 46
char = ;code = 32
char = A;code = 65
char = ;code = 32
char = m;code = 109
char = o;code = 111
char = d;code = 100
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = (;code = 40
char = F;code = 70
char = 5;code = 53
char = 8;code = 56
char = K;code = 75
char = ,;code = 44
char = ;code = 32
char = M;code = 77
char = 2;code = 50
char = 1;code = 49
char = 3;code = 51
char = S;code = 83
char = );code = 41
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = m;code = 109
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = D;code = 68
char = -;code = 45
char = a;code = 97
char = m;code = 109
char = i;code = 105
char = n;code = 110
char = o;code = 111
char = ;code = 32
char = a;code = 97
char = c;code = 99
char = i;code = 105
char = d;code = 100
char = ;code = 32
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = (;code = 40
char = D;code = 68
char = A;code = 65
char = A;code = 65
char = O;code = 79
char = );code = 41
char = ;code = 32
char = (;code = 40
char = E;code = 69
char = C;code = 67
char = ;code = 32
char = 1;code = 49
char = .;code = 46
char = 4;code = 52
char = .;code = 46
char = 3;code = 51
char = .;code = 46
char = 3;code = 51
char = );code = 41
char = ;code = 32
char = f;code = 102
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = ;code = 32
char = R;code = 82
char = h;code = 104
char = o;code = 111
char = d;code = 100
char = o;code = 111
char = s;code = 115
char = p;code = 112
char = o;code = 111
char = r;code = 114
char = i;code = 105
char = d;code = 100
char = i;code = 105
char = u;code = 117
char = m;code = 109
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = r;code = 114
char = u;code = 117
char = l;code = 108
char = o;code = 111
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = w;code = 119
char = a;code = 97
char = s;code = 115
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = s;code = 115
char = i;code = 105
char = g;code = 103
char = n;code = 110
char = e;code = 101
char = d;code = 100
char = ,;code = 44
char = ;code = 32
char = t;code = 116
char = e;code = 101
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = v;code = 118
char = i;code = 105
char = t;code = 116
char = r;code = 114
char = o;code = 111
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = u;code = 117
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = e;code = 101
char = f;code = 102
char = f;code = 102
char = i;code = 105
char = c;code = 99
char = i;code = 105
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = i;code = 105
char = z;code = 122
char = e;code = 101
char = ;code = 32
char = D;code = 68
char = -;code = 45
char = g;code = 103
char = l;code = 108
char = u;code = 117
char = f;code = 102
char = o;code = 111
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = i;code = 105
char = t;code = 116
char = s;code = 115
char = ;code = 32
char = 2;code = 50
char = -;code = 45
char = o;code = 111
char = x;code = 120
char = o;code = 111
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = v;code = 118
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = v;code = 118
char = e;code = 101
char = ;code = 32
char = [;code = 91
char = 2;code = 50
char = -;code = 45
char = o;code = 111
char = x;code = 120
char = o;code = 111
char = -;code = 45
char = 4;code = 52
char = -;code = 45
char = (;code = 40
char = m;code = 109
char = e;code = 101
char = t;code = 116
char = h;code = 104
char = y;code = 121
char = l;code = 108
char = p;code = 112
char = h;code = 104
char = o;code = 111
char = s;code = 115
char = p;code = 112
char = h;code = 104
char = i;code = 105
char = n;code = 110
char = y;code = 121
char = l;code = 108
char = );code = 41
char = -;code = 45
char = b;code = 98
char = u;code = 117
char = t;code = 116
char = a;code = 97
char = n;code = 110
char = o;code = 111
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = a;code = 97
char = c;code = 99
char = i;code = 105
char = d;code = 100
char = ];code = 93
char = .;code = 46
char = ;code = 32
char = T;code = 84
char = o;code = 111
char = b;code = 98
char = a;code = 97
char = c;code = 99
char = c;code = 99
char = o;code = 111
char = ;code = 32
char = (;code = 40
char = N;code = 78
char = i;code = 105
char = c;code = 99
char = o;code = 111
char = t;code = 116
char = i;code = 105
char = a;code = 97
char = n;code = 110
char = a;code = 97
char = ;code = 32
char = t;code = 116
char = a;code = 97
char = b;code = 98
char = a;code = 97
char = c;code = 99
char = u;code = 117
char = m;code = 109
char = );code = 41
char = ;code = 32
char = p;code = 112
char = l;code = 108
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = s;code = 115
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = t;code = 116
char = r;code = 114
char = a;code = 97
char = n;code = 110
char = s;code = 115
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = m;code = 109
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = p;code = 112
char = r;code = 114
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = i;code = 105
char = s;code = 115
char = ;code = 32
char = m;code = 109
char = o;code = 111
char = d;code = 100
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = u;code = 117
char = n;code = 110
char = d;code = 100
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = n;code = 110
char = t;code = 116
char = r;code = 114
char = o;code = 111
char = l;code = 108
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = T;code = 84
char = A;code = 65
char = P;code = 80
char = 1;code = 49
char = ;code = 32
char = t;code = 116
char = a;code = 97
char = p;code = 112
char = e;code = 101
char = t;code = 116
char = u;code = 117
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result sql = A chemical male sterility system based on anther-localized conversion of the inactive D-enantiomer of the herbicide, glufosinate (2-amino-4-(methylphosphinyl)-butanoate) to the phytotoxic L is described. Highly pure D-glufosinate was isolated in >98% enantiomeric excess from the racemate via fermentation with a strain of Escherichia coli expressing the PAT (L-glufosinate N-acetyl transferase) gene and purification of the unreacted D-enantiomer from the broth by ion exchange. A modified (F58K, M213S) form of the D-amino acid oxidase (DAAO) (EC 1.4.3.3) from Rhodosporidium toruloides was designed, tested in vitro and found to efficiently oxidize D-glufosinate to its 2-oxo derivative [2-oxo-4-(methylphosphinyl)-butanoic acid]. Tobacco (Nicotiana tabacum) plants were transformed to express this modified oxidase under control of the TAP1 tapetum-specific promoter. A number of the resultant transgenic lines exhibited complete male sterility that persisted for two or more weeks immediately following foliar treatment with 75 or 200 g/ha of D-glufosinate without exhibiting obvious phytotoxic symptoms or any measurable decline in female fertility. Similarly, plants containing the same construct and, additionally, a PAT gene expressed from a plastocyanin promoter exhibited significantly reduced male fertility and no reduction in female fertility following foliar application of racemic glufosinate. Thus, foliar application of d-glufosinate either purified or as the commercial herbicide, combined with anther expression of a modified DAAO promises to provide a cost-effective conditional chemical male sterility system with the characteristics necessary for practical F₁ hybrid seed production.
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char = e;code = 101
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = ;code = 32
char = a;code = 97
char = ;code = 32
char = m;code = 109
char = a;code = 97
char = l;code = 108
char = e;code = 101
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = l;code = 108
char = i;code = 105
char = t;code = 116
char = y;code = 121
char = ;code = 32
char = a;code = 97
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = ;code = 32
char = h;code = 104
char = y;code = 121
char = b;code = 98
char = r;code = 114
char = i;code = 105
char = d;code = 100
char = ;code = 32
char = s;code = 115
char = e;code = 101
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = o;code = 111
char = d;code = 100
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = .;code = 46
result sql = D-glufosinate as a male sterility agent for hybrid seed production.
char = ;code = 32
char = c;code = 99
char = h;code = 104
char = e;code = 101
char = m;code = 109
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = m;code = 109
char = a;code = 97
char = l;code = 108
char = e;code = 101
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = l;code = 108
char = i;code = 105
char = t;code = 116
char = y;code = 121
char = ;code = 32
char = s;code = 115
char = y;code = 121
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = m;code = 109
char = ;code = 32
char = b;code = 98
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = r;code = 114
char = -;code = 45
char = l;code = 108
char = o;code = 111
char = c;code = 99
char = a;code = 97
char = l;code = 108
char = i;code = 105
char = z;code = 122
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = n;code = 110
char = v;code = 118
char = e;code = 101
char = r;code = 114
char = s;code = 115
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = v;code = 118
char = e;code = 101
char = ;code = 32
char = D;code = 68
char = -;code = 45
char = e;code = 101
char = n;code = 110
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = m;code = 109
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = h;code = 104
char = e;code = 101
char = r;code = 114
char = b;code = 98
char = i;code = 105
char = c;code = 99
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = ,;code = 44
char = ;code = 32
char = g;code = 103
char = l;code = 108
char = u;code = 117
char = f;code = 102
char = o;code = 111
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = (;code = 40
char = 2;code = 50
char = -;code = 45
char = a;code = 97
char = m;code = 109
char = i;code = 105
char = n;code = 110
char = o;code = 111
char = -;code = 45
char = 4;code = 52
char = -;code = 45
char = (;code = 40
char = m;code = 109
char = e;code = 101
char = t;code = 116
char = h;code = 104
char = y;code = 121
char = l;code = 108
char = p;code = 112
char = h;code = 104
char = o;code = 111
char = s;code = 115
char = p;code = 112
char = h;code = 104
char = i;code = 105
char = n;code = 110
char = y;code = 121
char = l;code = 108
char = );code = 41
char = -;code = 45
char = b;code = 98
char = u;code = 117
char = t;code = 116
char = a;code = 97
char = n;code = 110
char = o;code = 111
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = );code = 41
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = p;code = 112
char = h;code = 104
char = y;code = 121
char = t;code = 116
char = o;code = 111
char = t;code = 116
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = L;code = 76
char = ;code = 32
char = i;code = 105
char = s;code = 115
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = s;code = 115
char = c;code = 99
char = r;code = 114
char = i;code = 105
char = b;code = 98
char = e;code = 101
char = d;code = 100
char = .;code = 46
char = ;code = 32
char = H;code = 72
char = i;code = 105
char = g;code = 103
char = h;code = 104
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = p;code = 112
char = u;code = 117
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = D;code = 68
char = -;code = 45
char = g;code = 103
char = l;code = 108
char = u;code = 117
char = f;code = 102
char = o;code = 111
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = w;code = 119
char = a;code = 97
char = s;code = 115
char = ;code = 32
char = i;code = 105
char = s;code = 115
char = o;code = 111
char = l;code = 108
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = >;code = 62
char = 9;code = 57
char = 8;code = 56
char = %;code = 37
char = ;code = 32
char = e;code = 101
char = n;code = 110
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = m;code = 109
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = c;code = 99
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = ;code = 32
char = f;code = 102
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = r;code = 114
char = a;code = 97
char = c;code = 99
char = e;code = 101
char = m;code = 109
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = v;code = 118
char = i;code = 105
char = a;code = 97
char = ;code = 32
char = f;code = 102
char = e;code = 101
char = r;code = 114
char = m;code = 109
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = w;code = 119
char = i;code = 105
char = t;code = 116
char = h;code = 104
char = ;code = 32
char = a;code = 97
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = r;code = 114
char = a;code = 97
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = E;code = 69
char = s;code = 115
char = c;code = 99
char = h;code = 104
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = c;code = 99
char = h;code = 104
char = i;code = 105
char = a;code = 97
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = l;code = 108
char = i;code = 105
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = p;code = 112
char = r;code = 114
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = g;code = 103
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = P;code = 80
char = A;code = 65
char = T;code = 84
char = ;code = 32
char = (;code = 40
char = L;code = 76
char = -;code = 45
char = g;code = 103
char = l;code = 108
char = u;code = 117
char = f;code = 102
char = o;code = 111
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = N;code = 78
char = -;code = 45
char = a;code = 97
char = c;code = 99
char = e;code = 101
char = t;code = 116
char = y;code = 121
char = l;code = 108
char = ;code = 32
char = t;code = 116
char = r;code = 114
char = a;code = 97
char = n;code = 110
char = s;code = 115
char = f;code = 102
char = e;code = 101
char = r;code = 114
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = );code = 41
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = p;code = 112
char = u;code = 117
char = r;code = 114
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = u;code = 117
char = n;code = 110
char = r;code = 114
char = e;code = 101
char = a;code = 97
char = c;code = 99
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = D;code = 68
char = -;code = 45
char = e;code = 101
char = n;code = 110
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = m;code = 109
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = f;code = 102
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = b;code = 98
char = r;code = 114
char = o;code = 111
char = t;code = 116
char = h;code = 104
char = ;code = 32
char = b;code = 98
char = y;code = 121
char = ;code = 32
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = c;code = 99
char = h;code = 104
char = a;code = 97
char = n;code = 110
char = g;code = 103
char = e;code = 101
char = .;code = 46
char = ;code = 32
char = A;code = 65
char = ;code = 32
char = m;code = 109
char = o;code = 111
char = d;code = 100
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = (;code = 40
char = F;code = 70
char = 5;code = 53
char = 8;code = 56
char = K;code = 75
char = ,;code = 44
char = ;code = 32
char = M;code = 77
char = 2;code = 50
char = 1;code = 49
char = 3;code = 51
char = S;code = 83
char = );code = 41
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = m;code = 109
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = D;code = 68
char = -;code = 45
char = a;code = 97
char = m;code = 109
char = i;code = 105
char = n;code = 110
char = o;code = 111
char = ;code = 32
char = a;code = 97
char = c;code = 99
char = i;code = 105
char = d;code = 100
char = ;code = 32
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = (;code = 40
char = D;code = 68
char = A;code = 65
char = A;code = 65
char = O;code = 79
char = );code = 41
char = ;code = 32
char = (;code = 40
char = E;code = 69
char = C;code = 67
char = ;code = 32
char = 1;code = 49
char = .;code = 46
char = 4;code = 52
char = .;code = 46
char = 3;code = 51
char = .;code = 46
char = 3;code = 51
char = );code = 41
char = ;code = 32
char = f;code = 102
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = ;code = 32
char = R;code = 82
char = h;code = 104
char = o;code = 111
char = d;code = 100
char = o;code = 111
char = s;code = 115
char = p;code = 112
char = o;code = 111
char = r;code = 114
char = i;code = 105
char = d;code = 100
char = i;code = 105
char = u;code = 117
char = m;code = 109
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = r;code = 114
char = u;code = 117
char = l;code = 108
char = o;code = 111
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = w;code = 119
char = a;code = 97
char = s;code = 115
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = s;code = 115
char = i;code = 105
char = g;code = 103
char = n;code = 110
char = e;code = 101
char = d;code = 100
char = ,;code = 44
char = ;code = 32
char = t;code = 116
char = e;code = 101
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = v;code = 118
char = i;code = 105
char = t;code = 116
char = r;code = 114
char = o;code = 111
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = u;code = 117
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = e;code = 101
char = f;code = 102
char = f;code = 102
char = i;code = 105
char = c;code = 99
char = i;code = 105
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = i;code = 105
char = z;code = 122
char = e;code = 101
char = ;code = 32
char = D;code = 68
char = -;code = 45
char = g;code = 103
char = l;code = 108
char = u;code = 117
char = f;code = 102
char = o;code = 111
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = i;code = 105
char = t;code = 116
char = s;code = 115
char = ;code = 32
char = 2;code = 50
char = -;code = 45
char = o;code = 111
char = x;code = 120
char = o;code = 111
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = v;code = 118
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = v;code = 118
char = e;code = 101
char = ;code = 32
char = [;code = 91
char = 2;code = 50
char = -;code = 45
char = o;code = 111
char = x;code = 120
char = o;code = 111
char = -;code = 45
char = 4;code = 52
char = -;code = 45
char = (;code = 40
char = m;code = 109
char = e;code = 101
char = t;code = 116
char = h;code = 104
char = y;code = 121
char = l;code = 108
char = p;code = 112
char = h;code = 104
char = o;code = 111
char = s;code = 115
char = p;code = 112
char = h;code = 104
char = i;code = 105
char = n;code = 110
char = y;code = 121
char = l;code = 108
char = );code = 41
char = -;code = 45
char = b;code = 98
char = u;code = 117
char = t;code = 116
char = a;code = 97
char = n;code = 110
char = o;code = 111
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = a;code = 97
char = c;code = 99
char = i;code = 105
char = d;code = 100
char = ];code = 93
char = .;code = 46
char = ;code = 32
char = T;code = 84
char = o;code = 111
char = b;code = 98
char = a;code = 97
char = c;code = 99
char = c;code = 99
char = o;code = 111
char = ;code = 32
char = (;code = 40
char = N;code = 78
char = i;code = 105
char = c;code = 99
char = o;code = 111
char = t;code = 116
char = i;code = 105
char = a;code = 97
char = n;code = 110
char = a;code = 97
char = ;code = 32
char = t;code = 116
char = a;code = 97
char = b;code = 98
char = a;code = 97
char = c;code = 99
char = u;code = 117
char = m;code = 109
char = );code = 41
char = ;code = 32
char = p;code = 112
char = l;code = 108
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = s;code = 115
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = t;code = 116
char = r;code = 114
char = a;code = 97
char = n;code = 110
char = s;code = 115
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = m;code = 109
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = p;code = 112
char = r;code = 114
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = i;code = 105
char = s;code = 115
char = ;code = 32
char = m;code = 109
char = o;code = 111
char = d;code = 100
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = u;code = 117
char = n;code = 110
char = d;code = 100
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = n;code = 110
char = t;code = 116
char = r;code = 114
char = o;code = 111
char = l;code = 108
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = T;code = 84
char = A;code = 65
char = P;code = 80
char = 1;code = 49
char = ;code = 32
char = t;code = 116
char = a;code = 97
char = p;code = 112
char = e;code = 101
char = t;code = 116
char = u;code = 117
char = m;code = 109
char = -;code = 45
char = s;code = 115
char = p;code = 112
char = e;code = 101
char = c;code = 99
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = o;code = 111
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = .;code = 46
char = ;code = 32
char = A;code = 65
char = ;code = 32
char = n;code = 110
char = u;code = 117
char = m;code = 109
char = b;code = 98
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = s;code = 115
char = u;code = 117
char = l;code = 108
char = t;code = 116
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = ;code = 32
char = t;code = 116
char = r;code = 114
char = a;code = 97
char = n;code = 110
char = s;code = 115
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = l;code = 108
char = i;code = 105
char = n;code = 110
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = h;code = 104
char = i;code = 105
char = b;code = 98
char = i;code = 105
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = m;code = 109
char = p;code = 112
char = l;code = 108
char = e;code = 101
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = m;code = 109
char = a;code = 97
char = l;code = 108
char = e;code = 101
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = l;code = 108
char = i;code = 105
char = t;code = 116
char = y;code = 121
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = a;code = 97
char = t;code = 116
char = ;code = 32
char = p;code = 112
char = e;code = 101
char = r;code = 114
char = s;code = 115
char = i;code = 105
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = ;code = 32
char = t;code = 116
char = w;code = 119
char = o;code = 111
char = ;code = 32
char = o;code = 111
char = r;code = 114
char = ;code = 32
char = m;code = 109
char = o;code = 111
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = e;code = 101
char = k;code = 107
char = s;code = 115
char = ;code = 32
char = i;code = 105
char = m;code = 109
char = m;code = 109
char = e;code = 101
char = d;code = 100
char = i;code = 105
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = l;code = 108
char = l;code = 108
char = o;code = 111
char = w;code = 119
char = i;code = 105
char = n;code = 110
char = g;code = 103
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = l;code = 108
char = i;code = 105
char = a;code = 97
char = r;code = 114
char = ;code = 32
char = t;code = 116
char = r;code = 114
char = e;code = 101
char = a;code = 97
char = t;code = 116
char = m;code = 109
char = e;code = 101
char = n;code = 110
char = t;code = 116
char = ;code = 32
char = w;code = 119
char = i;code = 105
char = t;code = 116
char = h;code = 104
char = ;code = 32
char = 7;code = 55
char = 5;code = 53
char = ;code = 32
char = o;code = 111
char = r;code = 114
char = ;code = 32
char = 2;code = 50
char = 0;code = 48
char = 0;code = 48
char = ;code = 32
char = g;code = 103
char = /;code = 47
char = h;code = 104
char = a;code = 97
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = D;code = 68
char = -;code = 45
char = g;code = 103
char = l;code = 108
char = u;code = 117
char = f;code = 102
char = o;code = 111
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = w;code = 119
char = i;code = 105
char = t;code = 116
char = h;code = 104
char = o;code = 111
char = u;code = 117
char = t;code = 116
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = h;code = 104
char = i;code = 105
char = b;code = 98
char = i;code = 105
char = t;code = 116
char = i;code = 105
char = n;code = 110
char = g;code = 103
char = ;code = 32
char = o;code = 111
char = b;code = 98
char = v;code = 118
char = i;code = 105
char = o;code = 111
char = u;code = 117
char = s;code = 115
char = ;code = 32
char = p;code = 112
char = h;code = 104
char = y;code = 121
char = t;code = 116
char = o;code = 111
char = t;code = 116
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = s;code = 115
char = y;code = 121
char = m;code = 109
char = p;code = 112
char = t;code = 116
char = o;code = 111
char = m;code = 109
char = s;code = 115
char = ;code = 32
char = o;code = 111
char = r;code = 114
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = y;code = 121
char = ;code = 32
char = m;code = 109
char = e;code = 101
char = a;code = 97
char = s;code = 115
char = u;code = 117
char = r;code = 114
char = a;code = 97
char = b;code = 98
char = l;code = 108
char = e;code = 101
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = c;code = 99
char = l;code = 108
char = i;code = 105
char = n;code = 110
char = e;code = 101
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = f;code = 102
char = e;code = 101
char = m;code = 109
char = a;code = 97
char = l;code = 108
char = e;code = 101
char = ;code = 32
char = f;code = 102
char = e;code = 101
char = r;code = 114
char = t;code = 116
char = i;code = 105
char = l;code = 108
char = i;code = 105
char = t;code = 116
char = y;code = 121
char = .;code = 46
char = ;code = 32
char = S;code = 83
char = i;code = 105
char = m;code = 109
char = i;code = 105
char = l;code = 108
char = a;code = 97
char = r;code = 114
char = l;code = 108
char = y;code = 121
char = ,;code = 44
char = ;code = 32
char = p;code = 112
char = l;code = 108
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = s;code = 115
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = n;code = 110
char = t;code = 116
char = a;code = 97
char = i;code = 105
char = n;code = 110
char = i;code = 105
char = n;code = 110
char = g;code = 103
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = s;code = 115
char = a;code = 97
char = m;code = 109
char = e;code = 101
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = n;code = 110
char = s;code = 115
char = t;code = 116
char = r;code = 114
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ,;code = 44
char = ;code = 32
char = a;code = 97
char = d;code = 100
char = d;code = 100
char = i;code = 105
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = a;code = 97
char = l;code = 108
char = l;code = 108
char = y;code = 121
char = ,;code = 44
char = ;code = 32
char = a;code = 97
char = ;code = 32
char = P;code = 80
char = A;code = 65
char = T;code = 84
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = p;code = 112
char = r;code = 114
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = f;code = 102
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = ;code = 32
char = a;code = 97
char = ;code = 32
char = p;code = 112
char = l;code = 108
char = a;code = 97
char = s;code = 115
char = t;code = 116
char = o;code = 111
char = c;code = 99
char = y;code = 121
char = a;code = 97
char = n;code = 110
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = o;code = 111
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = h;code = 104
char = i;code = 105
char = b;code = 98
char = i;code = 105
char = t;code = 116
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = s;code = 115
char = i;code = 105
char = g;code = 103
char = n;code = 110
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = d;code = 100
char = u;code = 117
char = c;code = 99
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = m;code = 109
char = a;code = 97
char = l;code = 108
char = e;code = 101
char = ;code = 32
char = f;code = 102
char = e;code = 101
char = r;code = 114
char = t;code = 116
char = i;code = 105
char = l;code = 108
char = i;code = 105
char = t;code = 116
char = y;code = 121
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = n;code = 110
char = o;code = 111
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = d;code = 100
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = f;code = 102
char = e;code = 101
char = m;code = 109
char = a;code = 97
char = l;code = 108
char = e;code = 101
char = ;code = 32
char = f;code = 102
char = e;code = 101
char = r;code = 114
char = t;code = 116
char = i;code = 105
char = l;code = 108
char = i;code = 105
char = t;code = 116
char = y;code = 121
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = l;code = 108
char = l;code = 108
char = o;code = 111
char = w;code = 119
char = i;code = 105
char = n;code = 110
char = g;code = 103
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = l;code = 108
char = i;code = 105
char = a;code = 97
char = r;code = 114
char = ;code = 32
char = a;code = 97
char = p;code = 112
char = p;code = 112
char = l;code = 108
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = r;code = 114
char = a;code = 97
char = c;code = 99
char = e;code = 101
char = m;code = 109
char = i;code = 105
char = c;code = 99
char = ;code = 32
char = g;code = 103
char = l;code = 108
char = u;code = 117
char = f;code = 102
char = o;code = 111
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = .;code = 46
char = ;code = 32
char = T;code = 84
char = h;code = 104
char = u;code = 117
char = s;code = 115
char = ,;code = 44
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = l;code = 108
char = i;code = 105
char = a;code = 97
char = r;code = 114
char = ;code = 32
char = a;code = 97
char = p;code = 112
char = p;code = 112
char = l;code = 108
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = d;code = 100
char = -;code = 45
char = g;code = 103
char = l;code = 108
char = u;code = 117
char = f;code = 102
char = o;code = 111
char = s;code = 115
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = e;code = 101
char = i;code = 105
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = p;code = 112
char = u;code = 117
char = r;code = 114
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = o;code = 111
char = r;code = 114
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = m;code = 109
char = m;code = 109
char = e;code = 101
char = r;code = 114
char = c;code = 99
char = i;code = 105
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = h;code = 104
char = e;code = 101
char = r;code = 114
char = b;code = 98
char = i;code = 105
char = c;code = 99
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = ,;code = 44
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = m;code = 109
char = b;code = 98
char = i;code = 105
char = n;code = 110
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = w;code = 119
char = i;code = 105
char = t;code = 116
char = h;code = 104
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = e;code = 101
char = x;code = 120
char = p;code = 112
char = r;code = 114
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = a;code = 97
char = ;code = 32
char = m;code = 109
char = o;code = 111
char = d;code = 100
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = D;code = 68
char = A;code = 65
char = A;code = 65
char = O;code = 79
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = i;code = 105
char = s;code = 115
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = o;code = 111
char = v;code = 118
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = s;code = 115
char = t;code = 116
char = -;code = 45
char = e;code = 101
char = f;code = 102
char = f;code = 102
char = e;code = 101
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = v;code = 118
char = e;code = 101
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = n;code = 110
char = d;code = 100
char = i;code = 105
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = c;code = 99
char = h;code = 104
char = e;code = 101
char = m;code = 109
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = m;code = 109
char = a;code = 97
char = l;code = 108
char = e;code = 101
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = l;code = 108
char = i;code = 105
char = t;code = 116
char = y;code = 121
char = ;code = 32
char = s;code = 115
char = y;code = 121
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = m;code = 109
char = ;code = 32
char = w;code = 119
char = i;code = 105
char = t;code = 116
char = h;code = 104
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = c;code = 99
char = h;code = 104
char = a;code = 97
char = r;code = 114
char = a;code = 97
char = c;code = 99
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = s;code = 115
char = t;code = 116
char = i;code = 105
char = c;code = 99
char = s;code = 115
char = ;code = 32
char = n;code = 110
char = e;code = 101
char = c;code = 99
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = a;code = 97
char = r;code = 114
char = y;code = 121
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = a;code = 97
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = F;code = 70
char = &;code = 38
char = #;code = 35
char = 8;code = 56
char = 3;code = 51
char = 2;code = 50
char = 1;code = 49
char = ;;code = 59
char = ;code = 32
char = h;code = 104
char = y;code = 121
char = b;code = 98
char = r;code = 114
char = i;code = 105
char = d;code = 100
char = ;code = 32
char = s;code = 115
char = e;code = 101
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = o;code = 111
char = d;code = 100
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = .;code = 46
char = <;code = 60
char = b;code = 98
char = r;code = 114
char = /;code = 47
char = >;code = 62
result sql = A chemical male sterility system based on anther-localized conversion of the inactive D-enantiomer of the herbicide, glufosinate (2-amino-4-(methylphosphinyl)-butanoate) to the phytotoxic L is described. Highly pure D-glufosinate was isolated in >98% enantiomeric excess from the racemate via fermentation with a strain of Escherichia coli expressing the PAT (L-glufosinate N-acetyl transferase) gene and purification of the unreacted D-enantiomer from the broth by ion exchange. A modified (F58K, M213S) form of the D-amino acid oxidase (DAAO) (EC 1.4.3.3) from Rhodosporidium toruloides was designed, tested in vitro and found to efficiently oxidize D-glufosinate to its 2-oxo derivative [2-oxo-4-(methylphosphinyl)-butanoic acid]. Tobacco (Nicotiana tabacum) plants were transformed to express this modified oxidase under control of the TAP1 tapetum-specific promoter. A number of the resultant transgenic lines exhibited complete male sterility that persisted for two or more weeks immediately following foliar treatment with 75 or 200 g/ha of D-glufosinate without exhibiting obvious phytotoxic symptoms or any measurable decline in female fertility. Similarly, plants containing the same construct and, additionally, a PAT gene expressed from a plastocyanin promoter exhibited significantly reduced male fertility and no reduction in female fertility following foliar application of racemic glufosinate. Thus, foliar application of d-glufosinate either purified or as the commercial herbicide, combined with anther expression of a modified DAAO promises to provide a cost-effective conditional chemical male sterility system with the characteristics necessary for practical F₁ hybrid seed production.
Publication: Plant Biotechnol. J. 2011 Apr ;9(3)::301-14. :doi: 10.1111/j.1467-7652.2010.00549.x.
Publication Date: 2011 Apr
Study Author(s): Hawkes, Tim;Pline-Srnic, Wendy;Dale, Richard;Friend, Emma;Hollinshead, Tricia;Howe, Peter;Thompson, Paul;Viner, Russell;Greenland, Andy;
Institution: Syngenta, Jealott's Hill Research Centre, Bracknell, Berks, UK. [email protected]
Shortcut link to this study:
Publication Date: 2011 Apr
Study Author(s): Hawkes, Tim;Pline-Srnic, Wendy;Dale, Richard;Friend, Emma;Hollinshead, Tricia;Howe, Peter;Thompson, Paul;Viner, Russell;Greenland, Andy;
Institution: Syngenta, Jealott's Hill Research Centre, Bracknell, Berks, UK. [email protected]
Shortcut link to this study:
A chemical male sterility system based on anther-localized conversion of the inactive D-enantiomer of the herbicide, glufosinate (2-amino-4-(methylphosphinyl)-butanoate) to the phytotoxic L is described. Highly pure D-glufosinate was isolated in >98% enantiomeric excess from the racemate via fermentation with a strain of Escherichia coli expressing the PAT (L-glufosinate N-acetyl transferase) gene and purification of the unreacted D-enantiomer from the broth by ion exchange. A modified (F58K, M213S) form of the D-amino acid oxidase (DAAO) (EC 1.4.3.3) from Rhodosporidium toruloides was designed, tested in vitro and found to efficiently oxidize D-glufosinate to its 2-oxo derivative [2-oxo-4-(methylphosphinyl)-butanoic acid]. Tobacco (Nicotiana tabacum) plants were transformed to express this modified oxidase under control of the TAP1 tapetum-specific promoter. A number of the resultant transgenic lines exhibited complete male sterility that persisted for two or more weeks immediately following foliar treatment with 75 or 200 g/ha of D-glufosinate without exhibiting obvious phytotoxic symptoms or any measurable decline in female fertility. Similarly, plants containing the same construct and, additionally, a PAT gene expressed from a plastocyanin promoter exhibited significantly reduced male fertility and no reduction in female fertility following foliar application of racemic glufosinate. Thus, foliar application of d-glufosinate either purified or as the commercial herbicide, combined with anther expression of a modified DAAO promises to provide a cost-effective conditional chemical male sterility system with the characteristics necessary for practical F₁ hybrid seed production.
PMID: 20678098
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