Soybean plants with dominant selectable trait for herbicide resistance

The utilization of a positive selection seed screening process to isolate rate variants with resistance to a herbicide is described. The ability to screen large numbers of mutagenized seed has made it feasible to pursue and isolate plants with low frequency dominant herbicide-resistance mutations. The dominant herbicide resistance mutations are useful for many applications including expanding the utility of sulfonylurea herbicides for soybean weed control, production of F.sub.1 and F.sub.2 seeds, and as selectable markers for efficient seed purification.

FIELD OF THE INVENTION 
This invention relates to selection of variants including low frequency 
dominant mutations conferring resistance to both pre- and post-emergent 
applications of certain herbicidal acetolactate synthase inhibitors. 
BACKGROUND OF THE INVENTION 
Mutation breeding techniques can be applied to develop new germplasm having 
desirable agronomic characteristics. Mutation breeding is initiated by 
generating a Population of plants with increased genetic variability 
through mutagenesis. The resulting population is then subjected to 
selective conditions which identify individuals expressing a desired trait 
or characteristic. Gottshalk et al., Induced Mutations in Plant Breeding, 
(Springer-Verlag, New York, 1983, PP. 16-17), and Simmonds, Principles of 
Crop Improvement, (Longman, N.Y., 1979, pp 297-303) are general references 
covering various mutation breeding techniques. 
Mutations created using such procedures can occur at widely different 
frequencies, with highly desired traits often induced at extremely low 
rates. Where mutation frequencies are reasonably high, screening of the 
population to identify new traits has been accomplished by (1) exposure of 
progeny of the mutagenized population to a chemical treatment which kills 
unmodified specimens, or (2) inspection of plants germinated from the 
mutagenized seed for evidence of the new property. For example, Pinthus et 
al., Science. 177:715 (1972) described mutation breeding experiments 
involving herbicides. In these experiments, M.sub.2 seed Populations of 
tomato and wheat were generated by soaking wheat and tomato seeds in 8 mM 
ethyl methanesulfonate (EMS). The treated seeds were then screened by 
sowing in soil containing the herbicides diphenamid or terbutryn at 
concentrations inhibitory to growth of the normal parental variety. The 
tolerant tomato lines obtained showed a 25% reduction in seedling weight 
in response to treatment with diphenamid, while the original cultivar 
exhibited a 40% reduction in seedling weight. Increased tolerance to 
herbicide was also reported for certain mutant wheat lines, although 
quantitative results were not provided. 
Raut et al., Indian J. Genet. Plant Breed, 42:265-270 (1982), utilized 
chemical mutagenesis to induce and select mutations for a different seed 
coat color. Chamberlain and Bernard, Crop Science, 8:728-729 (1968) report 
the failure to obtain resistance to brown stem rot through mutagenesis 
despite the fact that such resistance can be found in nature. 
Agrichemical Age, June 1984, p. 20, reports that USDA scientists W. L. 
Barrentine and E. E. Hartwig discovered variation with respect to 
tolerance for the herbicide metribuzin within the soybean cultivar Glycine 
max vc. "Tracy". Tolerant individuals were present at approximately a 5% 
frequency in the "Tracy" population, and were not mutants resulting from 
mutation breeding techniques. 
There have been no reports of mutant soybean plants with altered 
acetolactate synthase (ALS) function associated with herbicide resistance. 
Where mutations are extremely rare, many more seeds must be mutagenized and 
screened to obtain the new trait. It is well known that dominant mutations 
occur much less frequently than recessive mutations. In fact, to date, no 
one has ever selected a dominant mutation in soybean. Gottschalk et al., 
in their extensive review of mutation breeding, state that "about 1% of 
all induced mutations are dominant ones". This means that approximately 
100 times as many individuals must be screened to find a dominant mutation 
as to find a recessive mutation. Using Arabidopsis thaliana as a model 
system, (Somerville, Trends in Genetics. 2: 89-93 (1986)) states that "for 
many loci, a mutation resulting in loss of function" (which is generally 
synonymous with a recessive mutation) "can be recovered by screening 
approximately 2000 M2 plants". Using these sources as guidelines, 
isolation of a dominant mutation in Arabidopsis could conceivably require 
screening of at least 200,000 M2 individuals depending on the locus in 
question and the vagaries of sampling the M2 population. The search for a 
dominant mutation, therefore, requires 100 times the work of a search for 
a recessive mutation. Such experiment size or labor is totally 
impractical. Due to the minute seed size of Arabidopsis thaliana, 
Somerville (Mol. Gen. Genet. (1986) 204: 430-434) was, however, able to 
isolate dominant chlorsulfuron-resistant mutants by screening up to 10,000 
M2 seeds on a single 90 mm petri plate. Hardcastle [Hardcastle W. S. 1979. 
Soybean (Glycine max) cultivar response to metribuzin in solution culture. 
Weed Science (27):278-279] discloses the use of hydroponics to study the 
response of small numbers of soybean cultivars to a single concentration 
of metribuzin. Feenstra and Jacobsen, (TAG 58:39-42) disclose the use of 
hydroponics to select a recessive pea mutant lacking nitrate reductase 
(NR) activity. Their selection system consisted of growing seedlings for 7 
days in moist vermiculite, removing the cotyledons, and trans- planting 
the seedlings to a small pan (22.5.times.22.5.times.5.5 cm) containing 
vermiculite and a nutrient solution. Five days later (when the plants were 
12 days old), the nutrient solution was eventually supplemented with a 
chlorate solution. Since NR reduces chlorate to chlorite, which is toxic 
to plants, plants lacking NR could be selected based on their lack of 
chlorate damage. Since the plants were not treated with chlorate until 
they were seedlings of considerable age (12 days) and size, plant spacing 
to permit normal growth and visual observation must have limited the 
density (Plants Per unit area) at which seedlings were screened. Feenstra 
and Jacobsen screened 12 M2 seedlings from each of 1090 fertile plants. 
Although this procedure enabled them to screen enough M2 plants (roughly 
13,080) to find one recessive NR mutant, transplanting and plant spacing 
would make it extremely laborious and space consuming to screen the 
population size required to find a dominant mutation. 
One highly desirable agronomic characteristic currently sought in elite, 
commercial germplasm of a number of crop plants is true herbicide 
resistance. If selected within an elite soybean cultivar, a dominant 
soybean mutation conferring herbicide resistance could be immediately 
incorporated into an agronomic breeding program. Such mutations would be 
expected to be extremely rare, however, and no dominant mutation of any 
type has been discovered through seed mutagenesis and reported for 
soybean. Specifically, a dominant mutation that decreased the sensitivity 
of the target enzyme acetolactate synthase (ALS) to compounds that are 
herbicidal due to the inhibition of ALS was desired. Such resistance would 
require a specific change that renders the ALS enzyme resistant to the 
herbicide to insure that a cultivar would sustain little or no injury when 
exposed to the herbicidal compound, either in screening operations or in 
the field for weed control. It has been found that seed mutagenesis and 
mutant selection from a population of up to one million seeds is required 
to isolate a dominant, herbicide resistant plant mutation. A clear need 
exists for such dominant mutant plants as a basis to develop commercial 
soybean cultivars efficiently without sacrificing existing agronomic 
traits. 
SUMMARY OF THE INVENTION 
Applicant has utilized mutation breeding and hydroponic positive selection 
screening methods to discover soybean plants containing at least one 
dominant mutation capable of being expressed in subsequent generations of 
said plant and conferring resistance to pre- and post-emergent application 
of ALS-inhibiting herbicides, such as sulfonylaureas, triazolopyrimidine 
sulfonamides, imidazolinones or heteroaryl ethers. 
Preferred are soybean plants, selected by mutation breeding, resistant to 
herbicidal sulfonylureas of Formula I: 
##STR1## 
wherein R is H or CH.sub.3 ; 
J is 
##STR2## 
R.sub.1 is Cl, Br, NO.sub.2, C.sub.1 -C.sub.4 alkyl, C.sub.2 -C.sub.4 
alkenyl, CF.sub.3, C.sub.1 -C.sub.4 alkoxy, C.sub.1 -C.sub.4 haloalkoxy, 
C.sub.3 -C.sub.4 alkenyloxy, C.sub.2 -C.sub.4 haloalkenyloxy, C.sub.3 
-C.sub.4 alkynyloxy, CO.sub.2 R.sub.9, CONR.sub.10 R.sub.11, 
S(O)mR.sub.12, OSO.sub.2 R.sub.12, phenyl, SO.sub.2 N(OCH.sub.3)CH.sub.3, 
SO.sub.2 NR.sub.10 R.sub.11, 
##STR3## 
R.sub.2 is H, Cl, Br, F, CH.sub.3, NO.sub.2, SCH.sub.3, OCF.sub.2 H, 
OCH.sub.2 CF.sub.3 or OCH.sub.3 : 
R.sub.3 is Cl, NO.sub.2, CO.sub.2 CH.sub.3, CO.sub.2 C.sub.2 H.sub.5, 
SO.sub.2 N(CH.sub.3).sub.2, SO.sub.2 CH.sub.3 or SO.sub.2 C.sub.2 H.sub.5 
: 
R.sub.4 is C.sub.1 -C.sub.3 alkyl, Cl, Br, NO.sub.2, CO.sub.2 R.sub.9, 
CON(CH.sub.3).sub.2, SO.sub.2 N(CH.sub.3).sub.2, SO.sub.2 
N(OCH.sub.3)CH.sub.3 or S(O).sub.m R.sub.12 : 
R.sub.5 is C.sub.1 -C.sub.3 alkyl, C.sub.4 -C.sub.5 cycloalkylcarbonyl, F, 
Cl, Br, NO.sub.2, CO.sub.2 R.sub.14, SO.sub.2 N(CH.sub.3).sub.2, SO.sub.2 
R.sub.12 or phenyl; 
R.sub.6 is H, C.sub.1 -C.sub.3 alkyl or CH.sub.2 CH.dbd.CH.sub.2 ; 
R.sub.7 is H, CH.sub.3, OCH.sub.3, Cl or Br; 
R.sub.8 is H, F, Cl, Br, CH.sub.3, OCH.sub.3, CF.sub.3, SCH.sub.3 or 
OCF.sub.2 H; 
R.sub.9 is C.sub.1 -C.sub.4 alkyl, C.sub.3 -C.sub.4 alkenyl or CH.sub.2 
-CH.sub.2 Cl; 
R.sub.10 is H or C.sub.1 -C.sub.3 alkyl; 
R.sub.11 is H or C.sub.1 -C.sub.2 alkyl; 
R.sub.12 is C.sub.1 -C.sub.3 alkyl; 
R.sub.13 is H or CH.sub.3 ; 
R.sub.14 is C.sub.1 -C.sub.3 alkyl or CH.sub.2 CH.dbd.CH.sub.2 ; 
m is 0, 1 or 2; 
n is 1 or 2; 
Q is CH.sub.2, CHCH.sub.3 or NR.sub.15 ; 
R.sub.15 is H or C.sub.1 -C.sub.4 alkyl; 
P is O or CH.sub.2 ; 
R.sub.16 is H or CH.sub.3 ; 
R.sub.17 is C(O)NR.sub.18 R.sub.19, CF.sub.3, COOCH.sub.3 or SO.sub.2 
CH.sub.2 CH.sub.3 ; 
R.sub.18 is H or CH.sub.3 ; 
R.sub.19 is CH.sub.3 ; 
R.sub.20 is H, Cl, F, Br, CH.sub.3, CF.sub.3, OCH.sub.3 or OCF.sub.2 H; 
R.sub.21 is H or CH.sub.3 ; 
X is CH.sub.3, OCH.sub.3, OC.sub.2 H.sub.5 or NHCH.sub.3 ; 
Y is CH.sub.3, C.sub.2 H.sub.5, OCH.sub.3, OC.sub.2 H.sub.5, OCF.sub.2 H, 
OCH.sub.2 CF.sub.3, Cl, CH.sub.2 OCH.sub.3 or cyclopropyl; 
Z is CH or N; 
and their agriculturally suitable salts; provided that 
a) when Y is Cl, then Z is CH and X is OCH.sub.3 ; 
b) when Y is OCF.sub.2 H, then Z is CH; 
c) when J is J-1 and R.sub.1 is OSO.sub.2 R.sub.12 or phenyl, then Y is 
other than OCF.sub.2 H; 
d) when J is J-2, then Y is other than OCF.sub.2 H or OCH.sub.2 CF.sub.3 ; 
and 
e) when J is J-3 and R.sub.4 is S(O).sub.m R.sub.12, then Y is other than 
OCH.sub.2 CF.sub.3. 
Sulfonylurea herbicides to which the soybean lines or cultivars are 
particularly resistant include: 
1) Compounds of Formula I where 
J is J-1; 
R.sub.1 is Cl, CH.sub.3, C.sub.1 -C.sub.4 alkoxy, C.sub.1 -C.sub.2 
haloalkoxy allyloxy, propargyloxy, CO.sub.2 R.sub.9, CONR.sub.10 R.sub.11, 
SO.sub.2 N(OCH.sub.3)CH.sub.3, SO.sub.2 NR.sub.10 R.sub.11, S(O).sub.m 
R.sub.12, OSO.sub.2 R.sub.12, phenyl or 
##STR4## 
2) Compounds of Formula I where J is J-2; 
R is H; and 
R.sub.3 is SO.sub.2 N(CH.sub.3).sub.2, CO.sub.2 CH.sub.3 or CO.sub.2 
C.sub.2 H.sub.5 
3) Compounds of Formula I where 
J is J-3 
R is H; and 
R.sub.4 is CO.sub.2 CH.sub.3 or CO.sub.2 C.sub.2 H.sub.5 ; 
4) Compounds of Formula I where 
J is J-4; 
R is H; 
R.sub.5 is Cl, Br, CO.sub.2 CH.sub.3, CO.sub.2 C.sub.2 H.sub.5 or 
##STR5## 
R.sub.6 is CH.sub.3 ; and R.sub.7 is H, Cl or OCH.sub.3 ; 
5) Compounds of Formula I where 
J is J-5; 
R is H; 
R.sub.5 is CO.sub.2 CH.sub.3 or CO.sub.2 C.sub.2 H.sub.5 ; and 
R.sub.7 is H or CH.sub.3. 
6) Compounds of Formula I where 
J is J-6; 
Q is CHCH.sub.3 or NR.sub.15 ; 
R is H; and 
R.sub.8 is H, F, Cl, CH.sub.3, OCH.sub.3, CF.sub.3 or SCH.sub.3. 
7) Compounds of Formula I where 
J is J-7; 
R is H; and 
R.sub.8 is H, F, Cl, CH.sub.3, OCH.sub.3, CF.sub.3 or SCH.sub.3. 
8) Compounds of Formula I where 
J is J-8; 
R is H: 
R.sub.16 is CH.sub.3 ; and 
R.sub.8 is H, F, Cl, CH.sub.3, OCH.sub.3, CF.sub.3 or SCH.sub.3. 
9) Compounds of Formula I where 
J is J-9; 
R is H; and 
R.sub.17 is C(O)N(CH.sub.3).sub.2, CF.sub.3, COOCH.sub.3 or SO.sub.2 
CH.sub.2 CH.sub.3. 
10) Compounds of Formula I where 
R is H; 
R.sub.1 is Cl, C.sub.1 -C.sub.4 alkoxy, OCF.sub.2 H, OCH.sub.2 CH.sub.2 Cl, 
CO.sub.2 R.sub.9, CON(CH.sub.3).sub.2, SO.sub.2 N(CH.sub.3).sub.2, 
SO.sub.2 R.sub.12 or OSO.sub.2 R.sub.12 ; and 
R.sub.2 is H, Cl, CH.sub.3, or OCH.sub.3. 
Sulfonylurea herbicides to which the soybean lines or cultivars are more 
particularly resistant include: 
2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl) 
aminocarbonyl]benzenesulfonamide, 
methyl 
2-[[[[(4,6-dimethyl-2-pyrimidinyl)-amino]carbonyl]amino]sulfonyl]benzoate, 
methyl 
2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]sulfonyl]-benz 
oate, 
2-[[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)-N-methylaminocarbonyl]aminosu 
lfonyl]-benzoic acid, methyl ester, 
ethyl 
2-[[[[(4-chloro-6-methoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]benz 
oate, 
2-[[(4-ethoxy-6-methylamino-1,3,5-triazin-2-yl)aminocarbonyl]aminosulfonyl] 
benzoic acid, methyl ester, 
2-[[(4,6-dimethoxy-1,3,5-triazin-2-yl)aminocarbonyl]aminosulfonyl]-4-(2,2,2 
-trifluoroethoxy)benzoic acid, ethyl ester, 
4-chloro-2-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]aminosulf 
onyl]-benzoic acid, isopropyl ester, 
3-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)-amino]carbonyl]amino]sulfonyl 
]-2-thiophene carboxylic acid, methyl ester, 
methyl 
2-[[[[(4-6-dimethoxy-2-pyrimidinyl)-amino]carbonyl]amino]sulfonyl]methylbe 
nzoate, 
2-[[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]aminosulfonyl]-N,N-dimethyl- 
3-pyridinecarboxamide, 
2-[[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]aminosulfonyl]-3-pyridinecar 
boxylic acid, methyl ester, 
N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-3-(ethylsulfonyl)-2-pyridine 
sulfonamide, 
N-[4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-2,3-dihydro-2-methyl-benzo(b) 
thiophene-7-sulfonamide, 1,1 dioxide, 
2[[[[(4,6-bis(difluoromethoxy)-2-pyrimidinyl]-amino]carbonyl]amino]sulfonyl 
]benzoic acid, methyl ester, 
ethyl 
5-[3-(4,6-dimethoxypyrimidin-2-yl)ureidosulfonyl]-1-methylpyrazole-4-carbo 
xylate, 
N-[(6-methoxy-4-methyl-1,3,5-triazin-2-yl)aminocarbonyl]-2-(2-chloroethoxy) 
benzene sulfonamide, and 
N-[(4,6-dimethoxy-1,3,5-triazin-2-yl)aminocarbonyl]-3-trifluoromethyl-2-pyr 
idinesulfonamide. 
Most preferred are those soybean plants bearing a dominant mutation 
conferring resistance to 
methyl2-[[[[(4,6-dimethyl-2-pyrimidinyl)-amino]carbonyl]sulfonyl]benzoate. 
Also preferred are soybean lines or cultivars, isolated by mutation 
breeding, resistant to herbicidal triazolopyrimidine sulfonamides of 
Formula II: 
##STR6## 
wherein Ar is 
##STR7## 
R.sub.a is C.sub.1 -C.sub.4 alkyl, F, Cl, Br, I, NO.sub.2, S(O).sub.p 
R.sub.d, COOR.sub.e or CF.sub.3 ; 
R.sub.b is H, F, Cl, Br, I, C.sub.1 -C.sub.4 alkyl, CF.sub.2 or COOR.sub.e 
; 
R.sub.c is H, C.sub.1 -C.sub.4 alkyl, F, Cl, Br, I, CH.sub.2 OR.sub.d, 
CF.sub.2, phenyl, NO.sub.2 or COOR.sub.e ; 
R.sub.d is C.sub.1 -C.sub.4 alkyl; 
R.sub.e is C.sub.1 -C.sub.4 alkenyl, C.sub.1 -C.sub.4 alkynyl, or .sub.2 
-ethoxyethyl; 
V is H, C.sub.1 -C.sub.3 alkyl, allyl, propargyl, benzyl or C.sub.1 
-C.sub.3 alkylcarbonyl; 
X.sub.1, Y.sub.1, and Z.sub.1, are independently H, F, Cl, Br, I, C.sub.1 
-C.sub.4 alkyl C.sub.1 -C.sub.2 alkylthio or C.sub.1 -C.sub.4 alkoxy; and 
p is 0, 1 or 2. 
Triazolopyrimidine sulfonamide herbicides include: 
1) Compounds of Formula II 
2) Compounds of Preferred 1 where 
X.sub.1 is H or CH.sub.3 ; 
Y.sub.1 is H; 
Z.sub.1 is CH.sub.3 ; and R.sub.a and R.sub.c are not simultaneously H. 
These triazolopyrimidine sulfonamide compounds are known inhibitors of ALS 
and are very similar to sulfonylureas in structure/activity relationships. 
Also preferred are soybean lines or cultivars, isolated by mutation 
breeding, resistant to herbicidal imidazolones of Formula III: 
##STR8## 
wherein A is 
##STR9## 
R.sub.f is C.sub.1 -C.sub.4 alkyl; R.sub.g is C.sub.1 -C.sub.4 alkyl or 
C.sub.3 -C.sub.6 cycloalkyl; 
A.sub.1 is COOR.sub.i, CH.sub.2 OH or CHO; 
R.sub.i is H; C.sub.1 -C.sub.12 alkyl optionally substituted by C.sub.1 
-C.sub.3 alkyl, C.sub.3 -C.sub.6 cycloalkyl or phenyl; C.sub.3 -C.sub.5 
alkenyl optionally substituted by phenyl or 1-2 C.sub.1 -C.sub.3 alkyl, F, 
C.sub.1, Br or I; or C.sub.3 -C.sub.5 alkynyl optionally substituted by 
phenyl or 1-2 C.sub.1 -C.sub.3 alkyl, F, Cl, Br or I; 
B is H; C(O)C.sub.1 -C.sub.6 alkyl or C(O)phenyl optionally substituted by 
Cl, NO.sub.2 or OCH.sub.3 ; 
X.sub.2 is H, F, Cl, Br, I, OH or CH.sub.3 ; 
Y.sub.2 and Z.sub.2 are independently H, C.sub.1 -C.sub.6 alkyl, C.sub.1 
-C.sub.6 alkoxy, F, Cl, Br, I, phenyl, NO.sub.2, CN, CF.sub.3 or SO.sub.2 
CH.sub.3 ; or 
Y.sub.2 and Z.sub.2 together with the carbon atoms to which they are 
attached form a 5- or 6-membered saturated or unsaturated ring containing 
1-3 heteroatoms selected from the group consisting of 0-2 oxygen, 0-2 
sulfur and 0-3 nitrogen atoms; said ring may be unsubstituted or 
substituted on carbon or nitrogen wherein the substituents are selected 
from the group consisting of C.sub.1 -C.sub.4 alkyl, C.sub.1 -C.sub.4 
haloalkyl, C.sub.3 -C.sub.6 alkenyl, C.sub.3 -C.sub.6 haloalkenyl, C.sub.3 
-C.sub.4 alkynyl, C.sub.3 -C.sub.4 haloalkynyl, C.sub.1 -C.sub.4 alkoxy, 
C.sub.1 -C.sub.4 haloalkoxy, C.sub.1 -C.sub.4 alkoxycarbonyl, phenyl, 
C.sub.1 -C.sub.4 dialkylamino and C.sub.1 -C.sub.4 alkylsulfonyl; 
L is M, Q and Rh are independently H, F, Cl, Br, I, CH.sub.3, OCH.sub.3, 
NO.sub.2, CF.sub.3, CN, N(CH.sub.3).sub.2, NH.sub.2, SCH.sub.3 or SO.sub.2 
CH.sub.3 provided that only one of M or Q may be a substituent other than 
H, F, Cl, Br, I, CH.sub.3 or OCH.sub.3. 
Preferred are the above wherein: 
B is H; and 
A.sub.1 is COOR.sub.i. 
Most preferred are the above wherein: 
R.sub.f is CH.sub.3 ; 
R.sub.g is CH(CH.sub.3).sub.2 ; 
X.sub.2 is H; 
Y.sub.2 is H or C.sub.1 -C.sub.3 alkyl or OCH.sub.3 ; 
Z.sub.2 is H; 
X.sub.3 is H, CH.sub.3, Cl or NO.sub.2 ; and 
L, M, Q and R.sub.h are H. 
These herbicidal imidiazolinones are also known inhibitors of ALS. 
This invention also relates to seed obtained by growing the soybean plants 
of the invention. 
Another embodiment of this invention involves a method for controlling the 
growth of undesired vegetation growing at the locus where a soybean plant 
of the invention has been cultivated comprising applying to the locus an 
effective amount of a compound of Formula I-III. Preferred is the method 
wherein the compound applied is methyl 
2-[[[[(4,6-dimethyl-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]benzoate.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention describes plants with rare dominant 
herbicide-resistance mutations obtained by a hydroponic seed selection 
process. The ideal method for screening and selecting the products of a 
mutation breeding program should be uniform, quick, nondestructive, labor 
saving, space efficient, and should approximate the actual field 
conditions that must be tolerated. These qualities are especially 
important when looking for a mutation that is extremely rare. Most methods 
of herbicide treatment lack one or more of the above qualities. For 
example, field treatments lack the uniformity of treatment necessary to 
prevent "escapes" or plants that were not fully exposed to the intended 
dose of herbicide. Escapes can result from shading of one plant by another 
during foliar application, emergence of late germinators after a foliar 
treatment, nonuniform soil incorporation of a preemergence-applied 
herbicide, uneven watering of herbicide-treated soil, or malfunctions in 
the spraying apparatus. Greenhouse pot treatments generally have better 
uniformity than field treatments but are labor intensive and space 
consuming. A seed soak treatment for selection is possible, but does not 
closely approximate field treatment conditions since the plants are only 
exposed to the herbicide for several hours. Although some useful variants 
can be identified with a seed soak procedure, many false leads are also 
selected which require rechallenges with herbicide or genetic tests to 
weed out the escapes. For example, a genetic variation that prevents 
movement of a herbicide through the seed coat would not protect the plant 
once the seed coat is broken by the germinating seedling. 
It is believed that an extremely efficient and effective positive selection 
screening method is provided by a hydroponic system resulting from the 
combination of five features. 
1. an inert planting medium to permit quick direct planting of seeds, 
physical support, and adequate aeration and uniform germination of densely 
planted seeds; 
2 a closed-loop irrigation system consisting of a water soluble toxin 
solution reservoir used to irrigate and receive drainage from a planting 
tank; 
3. automated or manual irrigation to ensure periodic flooding of the 
planting medium with a known concentration of the water soluble toxin; 
4. irrigation and soaking of a level planting tank to ensure uniform 
treatment of seedlings with a known toxin concentration; and 
5. periodic drainage of solution from the planting medium to provide proper 
aeration for healthy growth. 
The present hydroponic system invention can rapidly and uniformly screen at 
least 16,000 soybean seedlings per square meter of greenhouse bench space 
in less than 10 days. With this system, one can screen 1,000,000 soybean 
seedlings in about 60 days with 10 square meters of greenhouse bench 
space. For small seeded crops, much larger populations could be screened 
per unit greenhouse space. This system has permitted the successful 
isolation of the novel soy-bean mutant plants described herein. 
In the context of this disclosure, a number of terms shall be utilized. As 
used herein, the term "variant" refers to any individual that differs from 
the general population in terms of expressing a specific trait. A variant 
can result from an induced or spontaneous mutation or from selection 
within any collection of germplasm. "Mutation" refers to a detectable and 
heritable genetic change not caused by segregation or genetic 
recombination. A "dominant mutation" refers to a genetic change that is 
expressed in the heterozygous condition. Such dominant mutations are 
"completely dominant" if expressed to the same degree as in the homozygous 
state, and are "semidominant" when partially expressed in the hetero- 
zygous condition. "Recessive mutation" refers to a genetic change that is 
only expressed when in the homozygous condition. A "low frequency 
mutation" refers to a genetic change that retains and/or improves a gene's 
biochemical capacity; usually inherited as a dominant or semidominant 
trait and usually much less frequent than a recessive mutation. In 
contrast, a "high frequency mutation" refers to a genetic change that 
causes the loss of a gene and/or the gene's resulting biochemical 
capacity; usually inherited as a recessive trait and much more frequent 
than a dominant mutation. The term "tolerance" means the heritable ability 
of a plant to sustain less damage than other individuals of a given 
species in the presence of an injurious concentration of a toxin or 
pathogen, while "resistance" refers to a heritable ability to survive 
(with agronomically acceptable injury) a concentration of toxin or 
pathogen that is normally lethal or severely injurious to individuals of 
given species. 
As used herein, the term "mutant" refers to an individual or lineage of 
individuals possessing a genetic mutation. "Plants" are individuals of any 
species in the Plantae Kingdom, while "cultivar" means a line that is 
fully characterized, named, and intended for use by farmers. Cultivar is 
synonymous with "variety". The term "mutation breeding" refers to the use 
of a mutagenic agent to induce random genetic mutations within a 
population of individuals. The treated population or subsequent 
generations of that population are then screened for desirable mutations 
than can be used in a breeding program. A "population" is any group of 
individuals which share a common gene pool. In the instant case, this 
includes Ml, M2, M3, M4, Fl, F2, and F3 populations. As used herein, and 
"Ml population" is the seeds (and resulting plants) exposed to a mutagenic 
agent, while "M2 population" is the progeny of self-pollinated Ml plants, 
"M3 population" is the progeny of self-pollinated M2 plants, "M4 
population" is the progeny of self-pollinated M3 plants, and generally "Mn 
population" is the progeny of self-pollinated Mn-1 plants. The term 
"mutagen-treated population" refers to any population resulting from a 
mutagenic treatment (including the Ml and subsequent generations), while a 
"line" is a group of individuals from a common ancestry. For example, a 
mutant line is the progeny of a selected ancestor which all possess the 
heritable mutant trait of that ancestor. 
In that context of this disclosure the term "positive selection" refers to 
a selection procedure in which only the desired individuals survive (or 
develop normally) in the presence of a selective toxin, while "negative 
selection" means a procedure in which the desired individuals are selected 
as abnormal, delayed, or inhibited in growth in the presence of the 
selective toxin. The term "hydroponic" refers to culture of plants in 
water (in the absence of soil); in the present application, the plants are 
supported by an inert planting medium (e.g., vermiculite) that is 
irrigated with water (with or without added nutrients). The term "inert 
planting medium" refers to a medium that does not significantly alter or 
chemically react with the solution that the medium supports; used 
primarily for physical support of the seeds and resulting plants. As used 
herein, "irrigation" means flushing the hydroponic system with a solution 
channeled from a reservoir, while "subirrigated" refers to irrigation by 
increasing the water level from below and up through the suspended 
planting medium. The term "aeration" means to provide plant roots with a 
supply of air (containing oxygen) that permeates the planting medium after 
nonadsorbed irrigation water is drained from the planting medium. As used 
herein, "closely-packed or densely-planted seeds" refers to the planting 
of seeds as densely as possible (touching) in a single horizontal layer. 
Actual density per unit area depends on seed size. 
Hydroponic Positive Selection Method 
The hydroponic selection system can be used to efficiently screen any 
seed-propagated plant species for induced or natural genetic variation for 
tolerance/resistance to any water soluble toxin that exerts an observable 
effect on seeds or developing seedlings. Seed-propagated plant species 
suitable for use with the hydroponic method include any species in the 
Kingdom Plantae that produces seeds that are capable of germination, 
emergence, and further development when densely planted in an inert medium 
that is both aerated and moistened with water or an aqueous nutrient 
solution, including; corn, alfalfa, oats, millet, wheat, rice, barley, 
sorghum, soybean, petunia, cotton sugarbeets, sunflower, carrot, celery, 
flax, cabbage, cucumber, pepper, canola, tomato, potato, lentil, broccoli, 
tobacco, amaranth, bean, asparagus, lettuce, rape, and other crop plants. 
Efficiency of the screen is limited only by the density at which seeds of a 
given species can be planted in a single horizontal layer and the time 
required for that species to develop to the point where the herbicide's 
effect can be observed. Efficiency of the screen can therefore be defined 
as the number of individuals that can be screened per unit area per unit 
time. 
Induced genetic variation includes mutations resulting from any 
deliberately applied mutagenic agent including physical mutagens such as 
X-rays, gamma rays, fast or thermal neutrons, protons, and chemical 
mutagens such as ethyl methane sulfonate (EMS), diethyl sulfate (DES), 
ethylene imine (EI), propane sulfone, N-methyl-N-nitroso urethane (MNU), 
nitrosomethyl urea (NMU), ethylnitrosourea (ENU), and other chemical 
mutagens. 
Natural variation includes germplasm collections, mixed seed lots, a 
collection of lines or segregating populations generated from a breeding 
program, or spontaneous mutations within any line or population. 
Herbicide resistance represents an important trait in crop plants in light 
of current weed-control strategies. Representative examples of herbicides 
which are useful for mutant selection, either individually or in 
combination with any other herbicide, are those of the sulfonylurea, 
triazine, triazole, uracil, urea, amide, diphenyl ether, carbamate, 
imidazolinone, cineole and bipyridylium types. 
In addition, a number of compounds found to inhibit acetolactate synthase 
(ALS) have proven useful as herbicides and would also be useful 
individually or in combination with other herbicides as water soluble 
toxins in the selection of mutants. Mutant plants of the present invention 
are resistant to ALS-inhibiting herbicides such as sulfonylureas, 
triazolopyrimidine sulfonamides, imidazolinones and heteroaryl ethers. 
These herbicides are disclosed in the following patents and published 
patent applications as follows: 
______________________________________ 
Sulfonylureas 
______________________________________ 
U.S. Pat. No. 4,127,405 
U.S. Pat. No. 4,383,113 
U.S. Pat. No. 4,169,719 
U.S. Pat. No. 4,394,153 
U.S. Pat. No. 4,190,432 
U.S. Pat. No. 4,394,506 
U.S. Pat. No. 4,214,890 
U.S. Pat. No. 4,420,325 
U.S. Pat. No. 4,225,337 
U.S. Pat. No. 4,452,628 
U.S. Pat. No. 4,231,784 
U.S. Pat. No. 4,481,029 
U.S. Pat. No. 4,257,802 
U.S. Pat. No. 4,586,950 
U.S. Pat. No. 4,310,346 
U.S. Pat. No. 4,435,206 
U.S. Pat. No. 4,544,401 
U.S. Pat. No. 4,514,212 
U.S. Pat. No. 4,435,206 
U.S. Pat. No. 4,634,465 
______________________________________ 
Triazolopyrimidine sulfonamides 
EP-A 150,974 
South African Application 84/8844 (published 5/14/85) 
Imidazolinones 
U.S. Pat. No. 4,188,487 
EP-A 41,623 
Heteroaryl Ethers 
EP-A 249,707 
EP-A 249-708 
Other ALS-inhibiting herbicides 
U.S. Pat. No. 4,838,925 (heterocyclic aryl sulfonamides) 
U.S. Pat. No. 4,761,173 (heterocyclic sulfonamides) 
Table I lists a number of herbicidal compounds specifically utilized in 
developing applicant's soy-bean plant invention. 
TABLE I 
Herbicidal Compounds For Soybean Mutant Characterization 
Compound 1: 
2-chloro-N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]benzene 
sulfonamide 
Compound 2: Methyl 
3-[[[[(4-methoxy-6-methyl-1,3,5-triazine-2-yl)amino]carbonyl]amino]sulfony 
l]-2-thiophenecarboxylate 
Compound 3: Methyl 
2-[[[[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)N-methylamino]carbonyl]amin 
o]sulfonyl]benzoate 
Compound 4: Methyl 
2-[[[[(4,6-dimethyl-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]benzoate 
Compound 5: Ethyl 
2[[[[(4-chloro-6-methoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]benzo 
ate 
Compound 6: 
2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]N,N-dimet 
hyl-3-pyridinecarboxamide 
Compound 7: 
N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(ethylsulfonyl)-2-pyrid 
inesulfonamide 
Compound 8: Methyl 
2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-3-pyridi 
necarboxylate 
Compound 9: 
2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-Yl]-3-quinol 
inecarboxylic acid 
Compound 10: 
5-ethyl-2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidaxol-2-yl]- 
3-quinolinecarboxylic acid 
Compound 11: 
2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridi 
necarboxylic acid, 1-methylethanamine salt 
Compound 12: 
N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-2,3,-dihydro-2-methylbenz 
o [B] thiophene-7-sulfonamide, 1,1-dioxide 
The hydroponic nutrient solution can consist merely of distilled water or 
tap water if seed reserves of the species of interest are capable of 
supporting germination and development to the point where the selective 
herbicides effect can be observed. Additional soluble nutrients can be 
added to the nutrient solution in cases where such nutrients are needed to 
achieve germination, emergence, and development of the seedlings to the 
point where the selective herbicides effect can be observed. Pilot runs of 
the hydroponic system in the absence of the selective herbicides can be 
used to determine how far development can proceed in the absence or 
presence of water soluble nutrients. Once these conditions are 
established, a lethal concentration of the herbicides can be included as a 
component of the nutrient solution so that only resistant individuals will 
develop normally. 
Small-Scale prototype for Hydroponic Screening System 
A number of small-scale hydroponic systems are commercially available. 
These systems were designed to promote quick lush growth of transplanted 
seedlings and not intended for use as a screen of germinating seedlings. 
It became immediately apparent that the commercial systems, if operated as 
advertised, lacked two key features necessary for an efficient screen: 
direct seeding and dense planting. However, the commercially available 
systems provided a convenient framework for modification with these 
features. The applicant utilized two such systems called the Hydropot Solo 
and the Hydropot Quad from Applied Hydroponics (San Rafael, CA, 94901). 
Since both systems operate by the same principles, only one system, the 
Hydropot Solo will be described in detail. 
The Hydropot Solo consists of a plastic reservoir (48 cm.times.38 
cm.times.13 cm) which supplies nutrient solution to a plastic planting 
tank (48 cm.times.35 cm.times.18 cm) that is placed directly on top of the 
reservoir. A small electric pump is used to pump nutrient solution from 
the reservoir up through a hole in the bottom of the planting tank. 
Applied Hydroponics also supplies the Hydropot Solo system with an inert 
soil substitute called Geolite which is used to fill the planting tank. 
Geolite is a porous rock-like material (pebbles approximately 1 cm in 
diameter) that is adequate to hold the moisture supplied by the reservoir 
and provide physical support for established plants. Irrigation of the 
planting tank is controlled by a 24-hour programmable electric timer that 
turns the pump on and off at desired preset time intervals. When the pump 
is activated, nutrient solution is pumped through plastic tubing up 
through a hole in the bottom of the planting tank. The solution level 
continues to rise within the planting tank until it reaches a depth of 9 
cm. At this depth a drain valve returns the excess nutrient solution to 
the reservoir. The water level will remain at the level of the drain valve 
until the pump is deactivated. The remainder of the nonadsorbed nutrient 
solution then drains from the planting tank into the reservoir through a 
small hole in the bottom of the planting tank. The Hydrofarm Quad system 
is identical to the Hydrofarm Solo in concept. However, in the Quad 
system, a larger reservoir is used to irrigate four planting tanks instead 
of just one. To develop an efficient system, the hydropot system had to be 
modified to allow direct planting of densely-planted seeds. 
The intended use of the Hydropot systems is to transplant established 
seedlings into the Geolite medium and grow the seedlings to maturity--not 
to screen germinating seeds. The Geolite stones were not suitable to 
permit direct seeding of crop plants. The stones were too large (approx. 1 
cm in diameter) to prevent the seeds from falling down in between the 
stones; this would have resulted in seeds planted at nonuniform depth. To 
permit direct seeding, the Geolite pebbles had to be replaced with a 
planting medium that was both inert and of fine enough texture to support 
seeds in a single level horizontal layer. This would promote uniform 
emergence and uniform toxin exposure. A pilot study showed that 
vermiculite (particle size approx. 2 mm) was an ideal planting medium 
since it was lightweight, inert, and promoted quick seedling emergence and 
growth in the absence of toxin. The chosen design for future experiments 
consisted of the planting tank filled first with 3 cm of pea gravel or 
geolite and then covered with 13 cm of vermiculite. Seeds were planted 2 
cm deep into the vermiculite. The lower layer of pea gravel served to 
distribute the influx of irrigation solution horizontally first (because 
of the large pore size) and then vertically by capillary action as the 
solution level reached the finer vermiculite. 
A pilot study was conducted to determine whether the modified Hydropot Solo 
system could be used to consistently differentiate between previously 
isolated (Sebastian and Chaleff, 1987. Crop Science 27:948-952) 
SU-tolerant mutants and wild type soybean plants. Four Hydropot Solo units 
were prepared with each planting tank containing a lower layer of pea 
gravel and an upper layer of fine vermiculite as described previously. 
Each reservoir was filled with tap water supplemented with 1/4 tablespoon 
of Miracle Grow soluble fertilizer per gallon. One hydroponic unit served 
as an untreated control while the nutrient solution of each of the other 
three was supplemented with either 100, 300, or 1000 ppb of an 
experimental sulfonylurea herbicide Compound 12. Ten seeds each of 
Williams 82 (wild type soybean cultivar), mutant 1-184A, mutant 1-183A, 
mutant 1-166A, and mutant 1-126A were then planted 2 cm deep into the 
vermiculite layer of each of the four hydroponic units. The programmable 
timer was set to irrigate the planting tanks for four 30-minute intervals 
during each 24-hour period. At 10 days after planting, a consistent and 
clear difference between wild type and mutant soybean lines was observed 
in all three systems irrigated with Compound 12. At 100 ppb Compound 12, 
mutant plants were able to form both unifoliolate leaves and secondary 
roots while Williams 82 could not. Differences between mutant and wild 
type became less dramatic as the herbicide concentration increased to 1000 
ppb. Prolonged exposure (20 to 28 days) to 100 ppb Compound 12 prevented 
further development of even the tolerant mutants. These observations 
confirmed that the modified Hydropot system could be used to select for 
herbicide tolerance. It also became apparent that the herbicide 
concentration and exposure time could be used to differentiate between 
different levels of herbicide tolerance/resistance. 
Large-Scale Hydroponic Screening System 
Using the modified Hydropot system as a prototype, two large hydroponic 
systems were constructed to screen soybean populations for resistance to 
sulfonylurea herbicides. Each system consists of a transparent plexiglass 
planting tank (1.77 m long.times.0.85 m wide.times.0.27 m deep) which 
rests on a level greenhouse bench. Each planting tank is irrigated with 
herbicide solution from a 200 1 polyethylene reservoir that can be stored 
directly under or beside the greenhouse bench. Irrigation is initiated by 
a programmable timer that energizes a pump connecting the reservoir with 
the planting tank via plastic tubing. The pump is deenergized by an 
adjustable float level switch in the planting tank. The float level switch 
is adjusted so that it turns off the pump when the solution level reaches 
the desired level in the planting tank. Drain valves on the planting tank 
are also controlled by the programmable timer so that the irrigation 
solution (once filled to the level specified by the adjustable float) can 
be retained in the planting tank for any desired length of time. The 
desired soaking time is manually entered into the programmable timer at 
the beginning of each screening cycle. After the desired soaking time, 
drain valves on the planting tank are automatically opened and the 
irrigation solution is allowed to drain back into the reservoir by 
gravity. 
The inside of each planting tank is divided with a horizontal (level) 
perforated metal platform to create an upper and lower chamber. The 
platform is supported with plexiglass beams that span the width and length 
of the lower chamber. These beams have regular gaps to allow free movement 
of solution within the lower chamber. The upper chamber is first covered 
with nylon mesh (to retain the planting medium) and then filled 7 cm deep 
with granular vermiculite which serves as the inert planting medium and 
support for developing plants. This layer of vermiculite is then leveled 
by dragging a thin straight board over the surface using the level top 
edge of the planting tank as a guide. The population of seeds to be 
screened is then scattered by hand over the level vermiculite. Seeding can 
be sparse or dense enough so that the seeds form a solid layer (about 
12,000 to 13,000 seeds per square meter for soybean). Small-seeded soybean 
populations or other small-seeded crop species can be seeded at much 
greater densities. After seeding, the seeds are then covered with another 
2.5 cm layer of vermiculite that is also leveled with a thin straight 
board. Once seeded, the planting tank is then ready to be irrigated with 
the nutrient solution. The lower chamber is flooded from the reservoir 
through portals at one end of the lower chamber. As the lower chamber 
fills, the water level gradually approaches the perforated metal platform 
which supports the planting medium. The upper chamber (which contains the 
planting medium) is eventually flooded to a uniform depth determined by 
the adjustable float switch that deenergizes the pump when the solution 
level reaches the desired level. The lower chamber, therefore, serves two 
purposes. First, it protects the planting medium from swift influx of 
irrigation solution. Secondly, it allows the irrigation solution to enter 
the planting medium as a surface (from below) rather than radially from 
one point source. This prevents the establishment of a herbicide 
concentration gradient in the planting medium and promotes uniformity of 
herbicide treatment across the planting tank. 
To effectively screen for resistance to a herbicide, one must first 
establish the threshhold herbicide concentration that will consistently 
inhibit development of sensitive plants yet allow the development of 
mutants or variants with significant levels of tolerance or resistance. 
Pilot studies were conducted that showed a concentration of 100 mg/L of 
Compound 1 or Compound 12 (used to irrigate the hydroponic system) will 
consistently stunt or inhibit leaf and root development of wild type 
soybeans yet allow unifoliolate (but not trifoliolate) formation of 
mutants that have heritable sulfonylurea tolerance. The same sulfonylurea 
concentrations would select only highly resistant plants. 
Another pilot study demonstrated that sulfonylurea tolerant mutants could 
be distinguished from wild type when the seeds are densely-planted (12,000 
to 13,000 seeds per square meter) in the large hydroponic system when 
irrigated with a solution containing either 100 or 300 mg/1 Compound 12. 
Hence, the large hydroponics units demonstrate the two key components of 
the hydroponic screening system: 1) the ability to screen seeds that are 
planted directly into the hydroponic system which eliminates the labor and 
time for transplanting established seedlings and 2) the ability to screen 
densely planted seeds which saves greenhouse space and materials. 
Source of Mutagenized Soybean Populations 
A sample of eight different M2 populations were screened for SU resistance. 
These populations differed in either the variety used as starting 
material, the chemical mutagen used, or the soaking regimes used in the 
mutagen treatment. All three parental varieties (`Williams`, `Williams 
82`, and `A3205`) are agronomically acceptable but have 
herbicide-sensitive ALS activities. A detailed protocol for the generation 
of one M2 population (A3205-EMS) is outlined to illustrate the general 
procedure. This is followed by a table listing the variations used to 
generate the other seven populations. 
Approximately 50,000 seeds (8.6 kg) of the variety `A3205` were poured into 
a 50 1 carboy filled with 45 1 of tap water to "pre-soak" the seeds. After 
8 h of soaking under continuous aeration, the excess tap water was drained 
and the swelled seeds were added to a second carboy containing 32 1 of 25 
mM ethyl methane sulfonate (EMS) in 0.1 M potassium phosphate buffer (pH 
5.6). The seeds were then soaked in the presence of the mutagen under 
continuous aeration for three hours. Treated seeds were washed of 
exogenous mutagen by first draining the EMS solution and then filling and 
draining the carboy twice with 30 1 of tap water. A third volume of 30 1 
tap water was added to the carboy and retained as a "post-wash" to soak 
the seeds for 7 h under continuous aeration. Following the post-wash 
treatment, seeds were again rinsed with three batches of 20 1 tap water. 
After the final rinse, seeds were decanted onto flat cardboard sheets to 
drain. After drainage, the seeds were field-planted 2 cm deep in rows 
spaced 76 cm apart with a density of approximately 30 seeds per meter 
within the row. The resulting Ml plants were allowed to reach maturity and 
produce M2 seeds, M2 seeds were harvested, bulked and thoroughly mixed to 
randomly distribute the progeny of any given Ml plant. Table II lists 
relevant deviations from the above procedure used to generate the eight M2 
populations screened for SU resistance. 
TABLE II 
__________________________________________________________________________ 
MUTAGEN, DOSAGE, SOAKING TIMES, AND POSTWASH TIMES 
USED TO GENERATE EIGHT DIFFERENT SOYBEAN 
M2 POPULATIONS 
Estimated 
Exposure 
Postwash 
Number of 
number of 
Population 
Mutagen 
time time M1 seeds 
M1 
code dose (hours) 
(hours) 
treated 
Survivors 
__________________________________________________________________________ 
A3205-EMS 25 mM 3 7 50,000 
40,000 
Williams-EMS-1 
50 mM 9 9 10,000 
7,700 
Williams-EMS-2 
50 mM 9 5 10,000 
7,500 
Williams-NMU-1 
2.5 
mM 3 9 10,000 
6,200 
Williams 2.5 
mM 5 3 2,500 
1,500 
82-NMU-A 
Williams 2.5 
mM 5.5 2.5 2,500 
1,250 
82-NMU-B 
Williams 2.5 
mM 6 2 2,500 
1,000 
82-NMU-C 
Williams 2.5 
mM 6 2 2,500 
82-NMU-D 
750 
__________________________________________________________________________ 
Population code includes (parental variety)(mutagen)-(numeral or letter 
code for designated soaking regime). 
EMS = ethyl methane sulfonate NMU = nitroso methyl urea 
Survival rate of Ml plants was based on a visual estimate of percent 
emergence in field plots or on a subsample of seed observed for emergence 
frequency. 
Table 3 summarizes the results of selection for sulfonylurea resistance 
within the eight M2 populations. It should be noted that a total of 
approximately 379,000 M2 individuals were screened for sulfonylurea 
resistance. Of the eight M2 populations screened, only two (Williams-EMS-2 
and Williams-NMU-1) yielded resistant mutants. Examples of selections from 
these two populations are described herein. Although 21 resistant 
individuals were selected from the Williams-NMU-1 M2 population, these 
individuals are probably the result of only one or two mutational events 
(i.e. the progeny of one or two Ml plants), considering the rarity of 
dominant mutations. Since all M2 populations were harvested in bulk, it is 
impossible to make conclusions about mutation frequency. It is also 
important to note that many putatively "tolerant" mutants were not saved 
especially after the first putatively "resistant" mutants were selected. 
Therefore, the number of tolerant versus resistant mutants shown in Table 
III does not represent the relative frequency of these two types of 
mutants. The sulfonylurea resistant mutants selected with the applicant's 
novel hydroponic selection system are the first recorded examples of 
dominant mutations in soybean; this fact alone testifies to the rarity of 
such a mutation. 
TABLE III 
______________________________________ 
Number and Type of Soybean Mutants 
Selected from Eight M2 Populations 
Estimated 
Mean 
Number of 
number of Number of Putative 
M2 Plants 
M2 per Mutants saved 
Population code 
Screened M1 plant Resistant 
Tolerant 
______________________________________ 
A3205-EMS 100,000 2.5 0 3 
Williams-EMS-1 
42,000 5.4 0 6 
Williams-EMS-2 
88,000 11.7 2 10 
Williams-NMU-1 
55,000 8.9 21 6 
Williams 26,000 17.3 0 2 
82-NMU-A 
Williams 30,000 24.0 0 0 
82-NMU-B 
Williams 18,000 18.0 0 1 
82-NMU-C 
Williams 20,000 26.7 0 1 
82-NMU-D 
______________________________________ 
Once true-breeding herbicide resistant mutants were selected from the first 
round of mutagenesis, a seed increase of such mutants was obtained to 
generate starting material for a second round of mutagenesis. The intent 
was to cause a second mutation in the background of a soybean plant that 
already possessed one mutation for herbicide resistance. The resulting M2 
populations were then screened by exposing the population to an herbicide 
treatment that was lethal to the parental starting material. This screen 
identified a "second generation" mutant "W4-4" with herbicide resistance 
greater than that provided by the first mutation alone. 
Three different mutant lines (W4, W23, and W28) were shown to contain 
single dominant mutations for resistance to compound 1 (Table V and 
Examples 2, 5, and 6). Another mutant line W6 has a similar resistance 
phenotype (Table IV and Example 9). All four of these lines contain a 
herbicide resistance mutation that is allelic or closely linked to the W20 
mutation (Table V and Examples 2, 5, 6, and 9). This evidence, coupled 
with the fact that W4, W6, W23, and W28 were all selected from the same 
small M2 subpopulation as W20 (Table IV), make it highly probable that all 
five of these mutants are descendents of the same Ml plant (i.e. are 
identical). Hence, lines W4, W6, W23, and W28 were used as starting 
material for a second cycle of mutagenesis with the assumption that all 
four lines were essentially identical to W20 in both genotype and 
phenotype. Various quantities of seed of each line were mutagenized in a 
fashion similar to that described above. Significant variations from the 
A3205 mutagenesis protocol are listed in Table II-A. 
TABLE II-A 
__________________________________________________________________________ 
PROTOCOLS USED FOR SECOND CYCLE OF MUTAGENESIS 
Number 
Estimated 
Mutagen 
Exposure 
Postwash 
of number 
Number of 
Population 
NMU time time M1 seeds 
of M1 M2 plants 
code dose (hours) 
(hours) 
treated 
Survivors 
screened 
__________________________________________________________________________ 
W4-NMU-T3 
2.5 
mM 2 9 12,500 
5,480 73,250 
W6-NMU-T3 
2.5 
mM 2 9 12,500 
7,715 112,000 
W23-NMU-T1 
2.5 
mM 3 5 10,000 
509 41,000 
W23-NMU-T2 
2.5 
mM 3 8 10,000 
854 28,500 
W23-NMU-T3A 
2.5 
mM 2 9 10,000 
2,720 89,250 
W23-NMU-T3B 
2.5 
mM 2 9 14,000 
7,232 75,750 
W23-NMU-T1 
2.5 
mM 3 5 10,000 
1,396 41,000 
W23-NMU-T2 
2.5 
mM 3 8 10,000 
1,815 36,750 
W23-NMU-T3A 
2.5 
mM 2 9 10,000 
2,936 120,500 
W23-NMU-T3B 
2.5 
mM 2 9 11,500 
4,368 87,000 
TOTAL 110,500 
35,025 
705,000 
__________________________________________________________________________ 
Two screening protocols were used to isolate mutants that had a higher 
level of herbicide resistance than the previously selected mutants. Both 
protocols utilized an experimental sulfonylurea (Compound 8) that was 
toxic to previously selected mutants when sprayed postemergence at a rate 
of only 2 g/ha (Table VII). Pilot studies were conducted to discover rates 
of Compound 8 that would consistently inhibit growth of mutant W20 in both 
the hydroponic screening system and a seed soak selection system. W20 is a 
mutant line representative of the class of mutants selected from the first 
cycle of mutagenesis. W20 contains a single dominant mutation for 
resistance to many ALS-inhibiting herbicides. By using a screening 
protocol that uniformly inhibits development of W20, the applicant 
selected for mutants with herbicide resistance superior even to that of 
W20 and similar mutants (including W4, W6, W23, and W28). 
The first screening protocol used was identical to the "Large-Scale 
Hydroponic Screening System" described previously except for the fact that 
300 ppb of Compound 8 was used as the selective agent instead of 100 ppb 
of Compound 1. Pilot studies demonstrated that when 300 ppb of Compound 8 
was used to irrigate the hydroponic system, W20 seedlings would germinate, 
emerge, and expand cotyledons but would not develop true leaves. Rare 
seedlings developing true leaves became very obvious against the 
background of inhibited seedlings. These rare seedlings were selected as 
potentially superior to the parental starting material in terms of 
heritable herbicide resistance. 
The second screening protocol was carried out as follows: samples of seed 
from W20 were tested for preemergence tolerance to Compound 8 by soaking 
seeds in a 2.0 ppm buffered solution of Compound 8 for 16 hours, rinsing, 
and planting in flats in a greenhouse. Pilot studies demonstrated that 2 
ppm of Compound 8 was sufficient to uniformly inhibit growth of W20 beyond 
the stage of emergence and cotyledon expansion. Plants developing beyond 
this stage were selected as potentially superior to the parental starting 
material in terms of heritable herbicide resistance. 
Using these two screening systems, a total of 234 putatively mutant plants 
were selected from a population of 705,000 M2 plants (Table IIA). Selected 
plants were transplanted into a standard peat-based soil in 20 cm pots at 
7 to 12 days of age. After transplantation, it was obvious that some of 
the selected plants recovered better than others after exposure to 
Compound 8. The healthiest-looking plants were suspected of being truly 
more resistant to Compound 8 than were the mutants with single dominant 
mutations for sulfonylurea resistance. Leaf samples from the 30 healthiest 
plant selections were then assayed for their ability to retain in vitro 
ALS activity in the presence of solutions containing Compound 8 (a potent 
sulfonylurea) and was compared to that of wild type Williams and 
previously selected mutant W20. W20 is similar (and probably identical) to 
mutants W4, W23, and W28 (and presumably W6) in terms of herbicide 
resistance spectrum (Table VII). 
TABLE II-B 
__________________________________________________________________________ 
RESULTS OF SELECTION FOR RESISTANCE TO COMPOUND 8 
Number of 
Number plants with 
of M2 
Number of 
Number of plants 
superior ALS re- 
Population 
plants 
M2 plants 
assayed at ALS 
sistance to 
code screened 
selected 
level with CMPD 8 
Compound 8 
__________________________________________________________________________ 
W4-NMU-T3 
73,250 
15 5 1 
W6-NMU-T3 
112,000 
23 7 0 
W23- NMU-T1 
41,000 
18 0 0 
W23-NMU-T2 
28,500 
15 0 0 
W23-NMU-T3A 
89,500 
60 8 0 
W23-NMU-T3B 
75,750 
14 4 0 
W23-NMU-TI 
41,000 
17 0 0 
W23-NMU-T2 
36,750 
10 1 0 
W23-NMU-T3A 
120,500 
52 5 0 
W23-NMU-T3B 
87,000 
10 0 0 
TOTAL 705,000 
234 30 1 
__________________________________________________________________________ 
The present invention is further defined in the following examples, in 
which all parts and percentages are by weight and degrees are Celsius, 
unless otherwise stated. It should be understood that these examples, 
while indicating preferred embodiments of the invention, are given by way 
of illustration only. From the above discussion and these examples, one 
skilled in the art can ascertain the essential characteristics of this 
invention, and without departing from the spirit and scope thereof, can 
make various changes and modifications of the invention to adapt it to 
various needed usages and conditions. 
EXAMPLE 1 
Sulfonylurea Resistant Mutant W20 
A. Selection 
Four Modified Hydropot Solo units were prepared as described in the 
Detailed Description by filling the planting tanks with a 5 cm layer of 
pea gravel followed by a 10 cm layer of fine vermiculite. M2 seeds of the 
William-NMU-1 were densely planted (approx. 16,600 seeds per square meter) 
by placing the seeds in a single horizontal layer on top of the 
vermiculite and then covering the seeds with a second level 2.5 cm layer 
of vermiculite. The nutrient solution reservoir was filled with tap water 
supplemented with 100 ppb of Compound 1 as the selective agent. Plant 
tanks were irrigated with the Compound 1 solution for four 30 minute 
periods per day beginning at 12 midnight, 8 AM, 12 noon, and 4 pm. At 
twelve days after planting, putative mutant "W20" was selected from one of 
the Modified Hydropot Solo units based on its vigorous growth in the 
presence of Compound 1 concentration that was lethal to the surrounding 
seedlings. The W20 plant was gently removed from the from the hydroponic 
planting tank and transplanted into a 20 cm pot containing a standard 
peat-based potting mixture. After transplantation, W20 showed no obvious 
signs of sulfonylurea injury when compared to wild type. This observation 
led the applicant to believe that W20 represented a new class of resistant 
mutants. W20 was allowed to reach maturity under greenhouse conditions and 
produce an M3 line that was also referred to as W20. 
Genetic Stability of Sulfonylurea Resistance in W20 
B. Characterization 
Thirty M3 seeds of the W20 line were then rescreened for chlorsulfuron 
resistance by exposing the seeds to 100 ppb Compound 1 using a hydroponic 
screening system similar to the one described in A. Under these 
conditions, Compound 1 "sensitivity" was defined as the inability to form 
leaves, "tolerance" was defined as the ability to form unifoliolate leaves 
but no subsequent shoot development, "high tolerance" was defined as the 
ability to form trifoliolates that were somewhat abnormal, and 
"resistance" was defined as the ability to form both unifoliolate and 
normal trifoliolate leaves. Twenty-seven of the 30 M3 seeds germinated and 
all were resistant to Compound 1 (Table IV). Based on this result, the W20 
line was classified as homozygous resistant to Compound 1. 
Seed Increase of W20 for Future Testing 
Another sample of M3 seeds of W20 were field planted and allowed to 
self-pollinate to increase the number of seeds of the W20 line. In all 
subsequent repeats where W20 was exposed to Compound 1 or other 
sulfonylureas, the line showed uniformity in response to the herbicides 
and was therefore considered nonsegregating or true-breeding for 
sulfonylurea resistance. All subsequent generations of self-pollinated 
(inbred) W20 seeds and plants were also referred to as W20. A seed deposit 
of W20 was made at the American Type Culture Collection (12301 Parklawn 
Drive, Rockville, Md., 20852) on July 5, 1988. The deposit was identified 
as ATCC designation 40467. 
Inheritance of Sulfonylurea Resistance in W20 
Using standard hand pollination procedures, F1 seeds of the cross 
W20.times.Williams 82 (wild type) were produced in the field. Five Fl 
seeds were tested for resistance to Compound 1 by hydroponic exposure to 
100 ppb Compound 1 for 19 days. All five Fl seeds and resulting seedlings 
were resistant to Compound 1 (Table V) indicating that W20's resistance 
was a dominant trait. After 19 days of exposure to Compound 1, F1 
seedlings were transplanted to individual greenhouse pots, allowed to self 
pollinate and produce F2 seeds. A sample of F2 seeds segregated 22 
resistant to 10 sensitive (Table V) when screened hydroponically for 
Compound 1 reaction. Based on chi-square analysis, this segregation (chi 
square value=0.67) is not significantly different (at alpha=0.05) from a 
3:1 ratio expected for segregation of a single dominant allele. It was 
therefore concluded that W20's resistance was due to a single dominant 
mutation conditioning resistance to Compound 1. 
EXAMPLE 2 
Selection and Characterization of Sulfonylurea Resistant Mutant W4 
M2 seeds of the Williams-NMU-1 population were screened for sulfonylurea 
resistance as described in EXAMPLE 1 except that one of the custom-built 
large hydroponic units described in the DETAILED DESCRIPTION was used 
instead of the small modified Hydropot Solo system. 
M2 seeds of the Williams-NMU-1 treatment (Table II) were densely planted 
(approximately 16,600 seeds Per m2) on top of a level 7 cm layer of 
granular vermiculite supported by the upper chamber of the planting tank 
of the previously described large hydroponic selection system. The seeds 
were then covered with a second level 2.5 cm layer of granular 
vermiculite. The nutrient solution reservoir was then filled with 200 1 of 
a solution containing tap water plus 100 ppb Compound 1. The float valve 
in the planting tank was adjusted to allow the solution level to reach the 
upper vermiculite layer. The programmable timer was programmed to enforce 
four 30-minute soakings of the planting medium per day: 8 to 8:30 AM, 12 
noon to 12:30 PM, 4 to 4:30 PM, and 12 midnight to 12:30 AM. The remainder 
of each day was programmed for drainage and aeration of the planting 
medium. The irrigation cycle continued for 10 days. At 8 days after 
planting, the background of wild type seedlings were uniformly inhibited 
after emergence and expansion of the cotyledons. Putative mutants could 
have been selected at 8 days after planting, but were kept in the 
hydroponic system for two more days to help identify the most resistant of 
the putative mutants. At 10 days after planting, putative mutant "W4" was 
selected based on its vigorous growth in the presence of the chlorsulfuron 
concentration that was lethal to the surrounding seedlings. The W4 plant 
was transplanted and allowed to produce an M3 and subsequent generations 
of seed as described in EXAMPLE 1. W4 also was resistant to compound 1 
based on its ability to grow normally after exposure to normally toxic 
levels of chlorsulfuron. A seed deposit of W4-4 was made at the American 
Type Culture Collection (12307 Parklawn Drive, Rockville, Md., 20852) on 
Sept. 1, 1989. The deposit was identified as ATCC designation 40650. 
Genetic stability of W4's Compound 1 resistance and inheritance of W4's 
resistance were tested as described in Example 1. The results are 
indicated in Tables IV and V. 
Linkage Analysis of Mutant W4 With Mutant W20 
Homozygous resistant W4 plants were also crossed with homozygous resistant 
W20 plants to determine if the mutation in W4 is allelic with, linked 
with, or at a separate genetic locus than the W20 mutation. This linkage 
test was conducted by exposing approximately 64 F2 progeny from this cross 
to 100 ppb of Compound 1 in the large hydroponic screening system. At 
approximately 8 days after planting, the F2 progeny were scored as either 
resistant or sensitive to Compound 1. If the two mutants contained 
mutations at unlinked loci, 1/16 of the F2 progeny l(4 out of 64 plants) 
would be expected to be sensitive to Compound 1. If the two mutants 
contained mutations in the same genetic locus or two tightly linked loci, 
one would expect all F2 progeny to be resistant. 
All F2 progeny from the cross between W4 and W20 were resistant to Compound 
1 (Table V). This indicates that W4 has a mutation at either the same 
locus as the W20 mutation or a tightly linked locus. Since W4 was obtained 
from the same small M2 subpopulation as W20 (Table IV), it is highly 
likely that W4 and W20 trace back to the same M1 plant and the same 
mutational event. 
EXAMPLE 3 
Selection and Characterization of Sulfonylurea Resistant Mutant W17 
Mutant "W17" was selected from the Williams-NMU-1 M2 population the same as 
W20 as described in EXAMPLE 1. Seed of the W17 line was increased as 
described for W20 in EXAMPLE 1. 
Genetic stability of W17's chlorsulfuron resistance and inheritance of 
W17's resistance were tested as described in EXAMPLE 1. 
The results are indicated in Tables IV and V. 
Linkage Analysis of Mutant W17 With Mutant W20 
Homozygous resistant W17 plants were also crossed with homozygous resistant 
W20 plants to determine if the mutation in W17 is allelic with, linked 
with, or at a separate genetic locus than the W20 mutation. This linkage 
test was conducted by exposing approximately 64 F2 progeny from this cross 
to 100 ppb of Compound 1 in the large hydroponic screening system. At 
approximately 8 days after planting, the F2 progeny were scored as either 
resistant or sensitive to Compound 1. If the two mutants contained 
mutations at unlinked loci, 1/16 of the F2 progeny (4 out of 64 plants) 
would be expected to be sensitive to Compound 1. If the two mutants 
contained mutations in the same genetic locus or two tightly linked loci, 
one would expect all F2 progeny to be resistant. 
All F2 progeny from the cross between W17 and W20 were resistant to 
Compound 1 (Table V). This indicates that W17 has a mutation at either the 
same locus as the W20 mutation or a tightly linked locus. Since W17 was 
obtained from the same small M2 subpopulation as W20 (Table IV), it is 
highly likely that W17 and W20 trace back to the same M1 plant and the 
same mutational event. 
EXAMPLE 4 
Selection and Characterization of Sulfonylurea Resistant Mutant W19 
Mutant "W19" was selected from the Williams-NMU-1 M2 population the same as 
W20 as described in EXAMPLE 1. Seed of the W19 line was increased as 
described for W20 in EXAMPLE 1. 
Genetic stability of W19's chlorsulfuron resistance, inheritance of W19's 
resistance were tested as described in EXAMPLE 1. The results are 
indicated in Tables IV and V. 
Linkage Analysis of Mutant W19 With mutant W20 
Homozygous resistant W19 plants were also crossed with homozygous resistant 
W20 plants to determine if the mutation in W19 is allelic with, linked 
with, or at a separate genetic locus than the W20 mutation. This linkage 
test was conducted by exposing approximately 64 F2 progeny from this cross 
to 100 ppb of Compound 1 in the large hydroponic screening system. At 
approximately 8 days after planting, the F2 progeny were scored as either 
resistant or sensitive to Compound 1. If the two mutants contained 
mutations at unlinked loci, 1/16 of the F2 progeny (4 out of 64 plants) 
would be expected to be sensitive to Compound 1. If the two mutants 
contained mutations in the same genetic locus or two tightly linked loci, 
one would expect all F2 progeny to be resistant. 
All F2 progeny from the cross between W19 and W20 were resistant to 
Compound 1 (Table V). This indicates that W19 has a mutation at either the 
same locus as the W20 mutation or a tightly linked locus. Since W19 was 
obtained from the same small M2 subpopulation as W20 (Table IV), it is 
highly likely that W19 and W20 trace back to the same Ml plant and the 
same mutational event. 
EXAMPLE 5 
Selection and Characterization of Sulfonylurea Resistant Mutant W23 
Mutant "W23" was selected from the Williams-NMU-1 M2 population the same as 
W20 as described in EXAMPLE 1. Seed of the W23 line was increased as 
described for W20 in EXAMPLE 1. 
Genetic stability of W23's chlorsulfuron resistance and inheritance of 
W23's resistance were tested as described in EXAMPLE 1. 
The results are indicated Tables IV and V. 
Linkage Analysis of Mutant W23 With Mutant W20 
Homozygous resistant W23 plants were also crossed with homozygous resistant 
W20 plants to determine if the mutation in W23 is allelic with, linked 
with, or at a separate genetic locus than the W20 mutation. This linkage 
test was conducted by exposing approximately 64 F2 progeny from this cross 
to 100 ppb of Compound 1 in the large hydroponic screening system. At 
approximately 8 days after planting, the F2 progeny were scored as either 
resistant or sensitive to Compound 1. If the two mutants contained 
mutations at unlinked loci, 1/16 of the F2 progeny (4 out of 64 plants) 
would be expected to be sensitive to Compound 1. If the two mutants 
contained mutations in the same genetic locus or two tightly linked loci, 
one would expect all F2 progeny to be resistant. 
All F2 progeny from the cross between W23 and W20 were resistant to 
Compound 1 (Table V). This indicates that W23 has a mutation at either the 
same locus as the W20 mutation or a tightly linked locus. Since W23 was 
obtained from the same small M2 subpopulation as W20 (Table IV), it is 
highly likely that W23 and W20 trace back to the same Ml plant and the 
same mutational event. 
EXAMPLE 6 
Selection and Characterization of Sulfonylurea Resistant Mutant W28 
Mutant "W28" was selected from the Williams-NMU-1 M2 population the same as 
W20 as described in EXAMPLE 1. Seed of the W28 line was increased as 
described for W20 in EXAMPLE 1. 
Genetic stability of W28's chlorsulfuron resistance and inheritance of 
W28's resistance were tested as described in EXAMPLE 1. 
The results are indicated in Tables IV and V. 
Linkage Analysis of Mutant W28 With Mutant W20 
Homozygous resistant W28 plants were also crossed with homozygous resistant 
W20 plants to determine if the mutation in W28 is allelic with, linked 
with, or at a separate genetic locus than the W20 mutation. This linkage 
test was conducted by exposing approximately 64 F2 progeny from this cross 
to 100 ppb of Compound 1 in the large hydroponic screening system. At 
approximately 8 days after planting, the F2 progeny were scored as either 
resistant or sensitive to Compound 1. If the two mutants contained 
mutations at unlinked loci, 1/16 of the F2 progeny (4 out of 64 plants) 
would be expected to be sensitive to Compound 1. If the two mutants 
contained mutations in the same genetic locus or two tightly linked loci, 
one would expect all F2 progeny to be resistant. 
All F2 Progeny from the cross between W28 and W20 were resistant to 
Compound 1 (Table V). This indicates that W28 has a mutation at either the 
same locus as the W20 mutation or a tightly linked locus. Since W28 was 
obtained from the same small M2 subpopulation as W20 (Table Iv), it is 
highly likely that W28 and W20 trace back to the same Ml plant and the 
same mutational event. 
EXAMPLE 7 
Selection of Sulfonylurea Resistant Mutant W36 
Mutant "W36" was selected from the Williams-EMS-2 M2 population the same as 
W20 was selected from the Williams-NMU-1 M2 population as described in 
EXAMPLE 1. M3 seed of the W36 line was increased as described for W20 in 
EXAMPLE 1. 
Genetic Stability of Sulfonylurea Resistance in W36 
Genetic stability of W36's chlorsulfuron resistance was tested as W20 in 
Example 1 except that different results were obtained as shown in Table 
IV. The W36 M3 line (derived from the self-pollinated W36 M2 selection) 
segregated 25 resistant to 5 sensitive. This segregation ratio is not 
significantly different than a 3:1 ratio expected for segregation of a 
single dominant allele for chlorsulfuron resistance. It was therefore 
concluded that the original M2 selection of W36 was heterozygous for a 
single dominant mutation conferring chlorsulfuron resistance. 
Establishment of the W36 Mutation in the Homozygous Condition 
A sample of the M3 family of W36 was field-planted as described in EXAMPLE 
1. Instead of harvesting the M3 plants in bulk (as done for homozygous 
mutants such as W20), individual M3 plants were harvested and the 
resulting M4 families were kept separate. A 15 to 20 seed sample of each 
of 100 M4 families was then screened for resistance to 100 ppb 
chlorsulfuron using the large hydroponics unit as described in Example 2. 
In this case, however, each M4 family was planted in a defined area within 
the planting tank so that the response of each M4 family could be 
monitored. Remnant seed of the M4 families that were uniformly resistant 
to chlorsulfuron (homozygous for the W36 mutation) were then bulked to 
reconstitute the W36 line in a condition that was homozygous and 
true-breeding for the chlorsulfuron resistance mutation. 
Linkage Analysis of Mutant W36 With Mutant W20 
Homozygous resistant W36 plants were crossed with homozygous resistant W20 
plants to determine if the mutation in W36 is allelic with, linked with, 
or at a separate genetic locus than the W20 mutation. This linkage test 
was conducted by exposing approximately 60 F2 progeny from this cross to 
100 ppb of Compound 1 in the large hydroponic screening system. At 
approximately 8 days after planting, the F2 progeny were scored as either 
resistant or sensitive to Compound 1. If the two mutants contained 
mutations at unlinked loci, 1/16 of the F2 progeny (approximately 4 out of 
60 plants) would be expected to be sensitive to Compound 1. If the two 
mutants contained mutations in the same genetic locus or two tightly 
linked loci, one would expect all F2 progeny to be resistant. 
All F2 progeny from the cross between W36 and W20 were resistant to 
Compound 1 (Table V). This indicates that W36 has a mutation at either the 
same locus as the W20 mutation or a tightly linked locus. Although the two 
mutations may be similar or even identical, they were induced 
independently since W36 was obtained from a different M2 subpopulation 
than W20 (Table IV). 
EXAMPLE 8 
Selection of Sulfonylurea Resistant Mutant W40 
Mutant "W40" was selected from the Williams-EMS-2 M2 population the same as 
W20 was selected from the Williams-NMU-1 M2 population as described in 
EXAMPLE 1. M3 Seed of the W40 line was increased as described for W20 in 
EXAMPLE 1. 
Linkage Analysis of Mutant W40 With Mutant W20 
Homozygous resistant W40 plants were crossed with homozygous resistant W20 
plants to determine if the mutation in W40 is allelic with, linked with, 
or at a separate genetic locus than the W20 mutation. This linkage test 
was conducted by exposing approximately 54 F2 progeny from this cross to 
100 ppb of Compound 1 in the large hydroponic screening system. At 
approximately 8 days after planting, the F2 progeny were scored as either 
resistant or sensitive to Compound 1. If the two mutants contained 
mutations at unlinked loci, 1/16 of the F2 progeny (approximately 3 out of 
54 plants) would be expected to be sensitive to Compound 1. If the two 
mutants contained mutations in the same genetic locus or two tightly 
linked loci, one would expect all F2 progeny to be resistant. 
Out of the 54 F2 progeny from the cross between W40 and W20, 4 were 
sensitive to Compound 1 (Table V). Four sensitive plants out of 54 is 
extremely close to the expected theoretical ratio for segregation of two 
unlinked dominant genes. This indicates that W40 has a mutation at a 
different locus than the W20 mutation. Although 54 plants do not provide 
enough data to claim independent segregation of the two loci, W40 defines 
the existence of a second soybean locus that controls reaction to 
sulfonylurea herbicides. The fact that the W40 mutation resides at a 
separate locus than the W20 mutation provides the opportunity to breed 
plants with herbicide resistance genes at both loci. Presumably such 
plants could have a level of herbicide resistance that is superior to that 
of either single mutant. 
Genetic Stability of Sulfonylurea Resistance in W40 
Genetic stability of W40's chlorsulfuron resistance was tested as described 
for W20 in EXAMPLE 1 except that different results were obtain as shown in 
Table IV. The W40 M3 line (derived from the self-pollinated W40 M2 
selection) segregated 20 resistant to 8 sensitive. This segregation ratio 
is not significantly different than a 3:1 ratio expected for segregation 
of a single dominant mutation conferring chlorsulfuron resistance. 
Establishment of the W40 Mutation in the Homozygous Condition 
W40 was obtained in a condition homozygous for its dominant chlorsulfuron 
resistance mutation in the same way as described for mutant W36 in EXAMPLE 
7. 
TABLE IV 
__________________________________________________________________________ 
M3 Family Segregation for Compound 1 
Reaction of Resistant and Tolerant Soybean Mutants 
M3 Chi- 
M2 Segrega- 
square 
Classification of M2 Plant 
M2 Popula- tion value Gene 
Plant 
tion R,Ht,T,S 
for 3:1 
Genotype 
Phenotype 
Action 
__________________________________________________________________________ 
W2 WM EMS-2 0,0,3,0 ? TOL ? 
W4 WM NMU-1 8,0,0,0 HOMO RESIST 
? 
W5 WM NMU-1 11,15,0,4 
2.18 
HETERO 
RESIST 
DOM 
W6 WM NMU-1 17,3,0,0 HOMO RESIST 
? 
W7 WM EMS-1 0,0,12,0 HOMO TOL ? 
W12 
WM NMU-1 12,20,0,8 
0.53 
HETERO 
RESIST 
DOM 
W13 
WM NMU-1 16,21,0,8 
1.25 
HETERO 
RESIST 
DOM 
W14 
WM NMU-1 16,25,0,15 
0.10 
HETERO 
RESIST 
DOM 
W15 
WM NMU-1 23,21,0,11 
0.73 
HETERO 
RESIST 
DOM 
W16 
WM NMU-1 0,19,11,16 
2.35 
HETERO 
RESIST 
DOM 
W17 
WM NMU-1 26,0,0,0 HOMO RESIST 
? 
W18 
WM NMU-1 12,26,0,12 
0.03 
HETERO 
RESIST 
DOM 
W19 
WM NMU-1 29,0,0,0 HOMO RESIST 
? 
W20 
WM NMU-1 27,0,0,0 HOMO RESIST 
? 
W21 
WM NMU-1 0,8,27,0 HOMO TOL ? 
W22 
WM NMU-1 0,0,52,0 HOMO TOL ? 
W23 
WM NMU-1 19,24,0,0 
HOMO RESIST 
? 
W24 
WM NMU-1 19,16,0,17 
1.64 
HETERO 
RESIST 
DOM 
W25 
WM NMU-1 0,0,38,0 HOMO TOL ? 
W26 
WM NMU-1 6,32,0,7 
2.14 
HETERO 
RESIST 
DOM 
W27 
WM NMU-1 0,18,5,4 
1.49 
HETERO 
TOL DOM 
W28 
WM NMU-1 30,15,0,0 
HOMO RESIST 
? 
W29 
WM NMU-1 0,0,34,0 HOMO TOL ? 
W30 
WM NMU-1 28,20,5,0 
HOMO RESIST 
? 
W31 
WM NMU-1 20,18,0,15 
0.31 
HETERO 
RESIST 
DOM 
W32 
WM NMU-1 28,19,4,0 
HOMO RESIST 
? 
W33 
WM NMU-1 28,21,4,0 
HOMO RESIST 
? 
W34 
WM NMU-1 20,18,0,13 
0.01 
HETERO 
RESIST 
DOM 
W35 
WM NMU-1 16,16,1,13 
0.26 
HETERO 
RESIST 
DOM 
W36 
WM EMS-2 25,0,0,5 
1.11 
HETERO 
RESIST 
DOM 
W37 
WM EMS-2 0,30,20,0 
HOMO TOL ? 
W39 
WM EMS-2 0,21,0,9 
0.40 
HETERO 
TOL DOM 
W40 
WM EMS-2 20,0,0,8 
0.19 
HETERO 
RESIST 
DOM 
W41 
WM EMS-2 0,29,0,0 HOMO TOL ? 
W42 
WM EMS-z 0,23,2,0 HOMO TOL ? 
W43 
WM EMS-2 0,28,1,0 HOMO TOL ? 
W44 
WM EMS-2 0,29,0,0 HOMO TOL ? 
W45 
WM EMS-2 0,16,14,0 
HOMO TOL ? 
W46 
WM EMS-2 0,23,6,0 HOMO TOL ? 
W48 
WM EMS-2 0,1,24,0 HOMO TOL ? 
W48 
WM EMS-2 0,24,0,0 HOMO TOL ? 
W50 
WM EMS-2 0,29,0,0 HOMO TOL ? 
W52 
WM EMS-2 0,0,25,0 HOMO TOL ? 
W53 
WM EMS-2 0,7,18,0 HOMO TOL ? 
W55 
WM EMS-2 0,7,22,0 HOMO TOL ? 
W56 
WM 82 NMU-A 
0,0,13,0 HOMO TOL ? 
W57 
WM 82 NMU-A 
0,0,26,0 HOMO TOL ? 
W60 
WM 82-NMU-C 
0,0,24,0 HOMO TOL ? 
W62 
WM 82-NMU-D 
0,0,18,0 HOMO TOL ? 
A1 A3205 EMS 0,8,7,0 HOMO TOL ? 
A2 A3205 EMS 0,0,8,0 HOMO TOL ? 
A3 A3205 EMS 0,0,1,0 ? TOL ? 
__________________________________________________________________________ 
R = resistant 
Ht = highly tolerant (an intermediate class) 
T = tolerant 
S = sensitive to chlorsulfuron at 100 mg L1 in hydroponics 
Chisquare values of less than 3.84 indicate that observed segregation 
ratio is not significantly different than 3:1 at alpha = 0.05. Ht and/or 
individuals were pooled with R individuals for chisquare testing of 
segregating families. 
HOMO = homozygous/true breeding 
HETERO = heterozygous/segregating progeny 
? = not enough data 
DOM = resistance or tolerance is segregating as a dominant allele at a 
single locus 
? = no segregation data 
TABLE V 
__________________________________________________________________________ 
Compound 1 Reaction of F1 and F2 Progenies From 
Crosses Between Chlorsulfuron Resistant (Mutant) 
and Chlorsulfuron Sensitive (Williams 82) Soybean Lines 
Number of Plants 
Chi-square Value* 
Resistant for Fit to 3:1 Ratio or 15:1 
or Highly in Segregating F2 
Line or Cross 
Tolerant 
Sensitive 
Progenies 
__________________________________________________________________________ 
Williams 82 0 4 3:1 15:1 
W4 33 0 0.10 3.33 
W4 .times. Williams 82 F1 
4 0 
W4 .times. Williams 82 F2 
23 8 
W4 .times. W20 F2*** 
63 0 
W17 30 0 4.77* 0.04 
W17 .times. Williams 82 F1 
2 0 
W17 .times. Williams 82 F2 
26 2 
W17 .times. W20 F2*** 
61 0 
W19 26 0 0.49 32.58** 
W19 .times. Williams 82 F1 
1 0 
W19 .times. Williams 82 F2 
23 10 
W19 .times. W2 F2*** 
64 0 
W20 29 0 0.67 34.13** 
W20 .times. Williams 82 F1 
5 0 
W20 .times. Williams 82 F2 
22 10 
W23 33 0 3.89 0.62 
W23 .times. Williams 82 F1 
1 0 
W23 .times. Williams 82 F2 
28 3 
W23 .times. W20 F2*** 
66 0 
W28 35 0 0.89 30.04** 
W28 .times. Williams 82 F1 
1 0 
W28 .times. Williams 82 F2 
16 8 
W28 .times. W20 F2*** 
66 0 
W6 .times. W20 F2 
57 0 8.92** 0.12 
W36 .times. W20 F2*** 
60 0 
W40 .times. W20 F2*** 
50 4 
__________________________________________________________________________ 
*At a = 0.05, a chisquare value of 3.84 is significant. 
**At a = 0.025, a chisquare value of 5.02 is significant. 
***This F2 data includes pooled data from the reciprocol cross also. 
EXAMPLE 9 
Selection and Characterization of Sulfonylurea Resistant Mutant W6 
Mutant "W6" was selected from the Williams-NMU-1 M2 population the same as 
W4 as described in Example 2. Genetic stability of W6.s chlorsulfuron 
resistance was tested as described in Example 1. Since none of the M3 
seeds from W6 were sensitive to chlorsulfuron (Table IV), W6 appears to 
breed true for resistance to chlorsulfuron. Since W6 was selected from the 
same M2 population as W4, W17, W19, W20, W23, and W28 (which probably 
trace back to the same mutational event), it was presumed that W6 was 
similar to W20 in both genotype and herbicide resistance phenotype. 
Although W6 was not crossed to wild type Williams plants to confirm 
monogenic inheritance, the probability of W6 containing any more than a 
single mutation for sulfonylurea resistance is extremely small. This 
assumption coupled with the following linkage test provide strong evidence 
that W6 is identical to W20 in terms of herbicide resistance. 
Linkage Analysis of Mutant W6 With Mutant W20 
Homozygous resistant W6 plants were crossed with homozygous resistant W20 
plants to determine if the mutation in W6 is allelic with, linked with, or 
at a separate genetic locus than the W20 mutation. This linkage test was 
conducted by exposing approximately 57 F2 progeny from this cross to 300 
ppb of Compound 1 in the large hydroponic screening system. At 
approximately 14 days after planting, the F2 progeny were scored as either 
resistant or sensitive to Compound 1. If the two mutants contained 
mutations at unlinked loci, 1/16 of the F2 progeny (4 out of 64 plants) 
would be expected to be sensitive to Compound 1. If the two mutants 
contained mutations in-the same genetic locus or two tightly linked loci, 
one would expect all F2 progeny to be resistant. 
All F2 progeny from the cross between W6 and W20 were resistant to Compound 
1 (Table V). This indicates that W6 has a mutation at either the same 
locus as the W20 mutation or a tightly linked locus. Since W6 was obtained 
from the same small M2 subpopulation as W20 (Table IV), it is highly 
likely that W6 and W20 trace back to the same Ml plant and the same 
mutational event. 
EXAMPLE 10 
Selection and Characterization of Mutant W4-4 
Approximately 25,000 M2 seeds (4500 grams) from each of three M2 
populations (W4-NMU-T3, W6-NMU-T3, and W23-NMU-T3B) were screened for 
resistance to Compound 8 using a seed soak selection procedure similar to 
that described in CR-8362. Each population sample was placed in a separate 
immersible mesh bag. The bags were then immersed and soaked for 16 hours 
in an aqueous solution containing 2 ppm of Compound 8. The solution was 
kept under continuous aeration through the use of an "air stone" supplied 
with air from a standard aquarium pump. During the soaking period, the 
seeds imbibed both water and the dissolved herbicide. After the soaking 
period, the seeds were washed in running tap water for two minutes to 
remove exogenous herbicide solution. The applicant employed existing 
greenhouse pallets with flat horizontal surfaces constructed from a 
perforated metal grating which allowed for excellent drainage. The metal 
grating was then covered with a single layer of cheese cloth to prevent 
soil from falling through the perforations. A planting bed was created by 
filling the greenhouse pallets with a peat-based soil mixture. The soil 
mixture was leveled at 7 cm deep by dragging a flat board across the 
surface of the mixture. Prior to planting, the 7 cm soil mixture layer was 
thoroughly moistened by sprinkling the upper surface with tap water. 
Excess water drained through the perforated greenhouse bench. The 
herbicide-treated seeds were then planted out in a single horizontal layer 
at a density of approximately 12,000 seeds per square meter on the level 
planting bed. The seeds were then covered with a 2.5 cm level layer of 
coarse vermiculite. The described planting medium was kept moist by 
periodically sprinkling the upper surface of the planting medium with 
plain tap water. At 8 days after planting, all of the viable seeds had 
germinated, emerged and expanded their cotyledons. However, only a few of 
the seedlings had proceeded to develop true leaves. These few seedlings 
were easily detectable because of the presence of their leaves against a 
background of uniformly inhibited seedlings. The healthiest looking 
seedlings were selected and transplanted into separate 20 cm pots. 
Selected seedlings were named according to the parental source material 
and the order in which the were selected. Individual M2 plant selections 
from this screen included W4-4, W4-5, and W4-6 from the W4-NMU-T3 
population; W6-1, W6-2, W6-3, and W6-4 from the W6-NMU-T3 population; and 
W23-9, W23-10, and W23-11 from the W23-NMU-T3B population. 
At approximately 1 month after transplantation, some of the selections 
appeared much healthier than others. For example, W4-4, W4-6, W6-1, W6-2, 
W6-4, W23-10, and W23-11 recovered very well from the seed soak treatment 
with Compound 8. These plants were suspected of having a higher level of 
ALS resistance to Compound 8 than did the parental material from which 
they were derived. The applicant was also interested to see if any of 
these selections were cross resistant to an imidazolinone herbicide such 
as Compound 9. To test this hypothesis, leaf tissue of the one-month-old 
M2 plants was sampled and assayed in vitro for ALS activity in the 
presence of Compound 8 and Compound 9. The ALS assay procedure was 
essentially identical to the previously described ALS assay except that 
the herbicide concentrations tested were 100 ppb Compound 8 and 10 ppm 
Compound 9. Williams and mutant W20 soybean plants were also assayed as 
control genotypes for comparison. Williams contains no genes conferring 
the ALS-based sulfonylurea resistance while W20 is homozygous for a single 
mutation conferring ALS-based sulfonylurea resistance. 
At the ALS enzyme level, only mutant W4-4 appears to be significantly more 
resistant than W20 to Compounds 8 and 9 (Table V-A). 
TABLE V-A 
______________________________________ 
Percentage of ALS Activity Remaining 
in the Presence of Compounds 8 and 9 
Compound 8 
Compound 9 
100 ppb 10 ppm 
uninhibited ALS activity as 
Genotype % of control 
______________________________________ 
Williams -0.3 14.0 
W20 10.7 18.4 
W4-4 37.6* 44.3* 
W4-5 7.1 19.3 
W4-6 4.0 10.7 
W6-1 8.5 16.2 
W6-2 9.7 11.7 
W6-4 10.5 18.7 
W23-10 7.9 19.5 
W23-11 3.8 13.0 
LSD (0.05) 2.9 5.1 
______________________________________ 
*Significantly higher ALS resistance than W20. 
Production of a True-breeding Resistant W4-4 Line 
The original W4-4 M2 plant was allowed to self pollinate, mature, and 
produced a family of M3 seeds. An M3 progeny test was then conducted to 
determine if the new resistance phenotype was heritable and if the M3 
family was true-breeding or segregating for the new resistance trait. The 
M3 progeny of W4-4 were screened for resistance with the same seed soak 
technique used to select the original W4-4 M2 plant. Twenty seeds each of 
the W4-4 M3 family, W20 line, and Williams line were soaked for 16 hours 
in a solution of 2 ppm of Compound 8. Twenty additional seeds of Williams 
were soaked for 16 hours in plain tap water as a control treatment. 
Treated seeds were then washed for two minutes in running water. Each seed 
was then planted in a separate pot for ease of observation. Within two 
weeks of planting, it was observed that all of the Williams and W20 plants 
had emerged and expanded cotyledons but had not started to form true 
leaves. However, all twenty W4-4 plants had developed both unifoliolate 
and trifoliolate leaves and were essentially identical to the control 
plants in vigor. This demonstrated that the W4-4 resistance phenotype was 
heritable and clearly superior to the W20 resistance phenotype. The 
uniform resistance of all M3 progeny demonstrated that the original W4-4 
M2 plant was homozygous for the new resistance mutation and that the new 
line of W4-4 plants were true-breeding for the new resistance phenotype. 
Utility 
The new class of sulfonylurea-resistant soybean mutants can now be used as 
a source of herbicide resistance in soybean breeding programs. Since the 
mutations are present in an acceptable agronomic background (Williams), 
the monogenic dominant resistance can be transferred quickly and 
efficiently through conventional means without sacrificing agronomic 
traits or without the need for extensive backcrossing. The use of 
sulfonylurea-resistant soybean varieties will greatly expand the utility 
of sulfonylurea herbicides and provide the soybean farmer with more 
options for weed control; herbicides previously excluded from soybean 
application (due to poor crop safety) could be used for soybean weed 
control. Sulfonylurea resistance will also increase the safety margin for 
application of sulfonylurea herbicides that are currently registered for 
use on soybean. With less concern for crop safety constraints, 
sulfonylurea resistance also provides an opportunity to combine herbicides 
that have complementary weed control spectrums to enable the farmer to 
control additional weed species. 
Dominant sulfonylurea herbicide resistance could also be used to produce 
experimental or commercial quantities of pure Fl hybrid seeds. In such an 
application, a herbicide-resistant line (that is rendered male sterile 
through genetic, chemical, and/or manual means) can be planted (either 
interplanted or in separate rows) in the same field with a male fertile 
but herbicide sensitive line. After pollination, the male parent can be 
removed from the field with a sulfonylurea herbicide treatment that is 
selectively lethal to the male parent. The entire field (containing Fl 
seeds borne by the sulfonylurea-resistant female line) can then be bulk 
harvested without seed contamination from the male line. 
Dominant sulfonylurea resistance could also be a useful tool in the 
experimental or commercial production of F2 varieties. In such an 
application, F1 hybrid seed would be produced on a herbicide sensitive 
female parent (that could be rendered at least partially male sterile 
through genetic, chemical, and/or manual means) that is fertilized by 
pollen from a parent with dominant homozygous herbicide resistance. Male 
and female parents would be planted in separated rows to facilitate 
mechanical harvest of seed from the female parent. Since the resulting F1 
seeds/plants will be herbicide resistant (heterozygous), undesirable 
seeds/plants resulting from self-pollination of the female parent can then 
be rogued from a population of Fl seeds/plants with a herbicide treatment 
that is selectively lethal to the sensitive female line. This would result 
in a pure stand of F1 plants that could be bulk harvested for the 
production of a pure F2 seed population. 
A selectively lethal sulfonylurea treatment could be used to rogue 
sensitive plants from sulfonylurea-resistant populations that have been 
contaminated through careless seed handling operations. Currently, the use 
of other dominant markers (such as purple hypocotyls and tawny pubescence) 
requires visual inspection of each plant for expression of the marker and 
hand roguing of undesirable types. Such labor makes these visual markers 
impractical for commercial-scale purification of inbred lines or hybrids. 
With dominant herbicide resistance, large seed production fields can be 
easily rogued by spraying the entire field with a herbicide treatment that 
is lethal to herbicide-sensitive plants. 
Demonstration of Resistance to Postemergence Chlorsulfuron Application and 
Cross-Resistance to Other Sulfonylureas 
In addition to isolation and confirmation of heritable sulfonylurea 
resistance, the hydroponic screen demonstrated resistance to preemergence 
application of Compound 1. Subsequent tests were conducted to demonstrate 
resistance of true breeding mutant lines (M3 families) to foliar 
(postemergence) applications of Compound 1 and other sulfonylurea 
herbicide compounds (see Table I). The following examples are 
representative of tests that repeatedly confirm the sulfonylurea 
resistance phenotype of soybean mutants isolated using the applicant's 
hydroponic selection technique. 
Test A 
In the first post-emergence test, two true-breeding resistant mutant lines, 
W19 and W20, were compared to previously isolated tolerant mutants and 
wild-type Williams 82. Eighteen pots (20 cm diameter) of each soybean line 
(2 seeds per pot) were planted using a standard sterile potting mix. When 
the seedlings reached the second trifoliolate stage, plants were thinned 
back to one plant per pot. Each pot was then sprayed with a specific 
herbicide treatment using a conveyor belt spray apparatus to emulate field 
application. The nine different herbicide treatments included one rate (8 
g/ha) of Compound 1 and two rates (8 and 32 g/ha) each of four other 
sulfonylurea herbicides (Compounds 2,3,7 and 8) (see Table VI) using a 
factorial treatment design with 2 replications. 
TABLE VI 
Comparison of "Williams 82", Mutant 1-184A, and New Mutants W19 and W20 in 
Terms to response to Postemergence Application of Five Sulfonylurea 
Herbicides 
TABLE VI 
______________________________________ 
Comparison of "Williams 82", Mutant 
1-184A, and New Mutants W19 and W20 
in Terms to response to Postemergence 
Application of Five Sulfonylurea Herbicides 
WM 82 1-184A W19 W20 LSD 
Herbicide 
g/ha mean % injury (0.05) 
______________________________________ 
CMPD 1 8 97 97 12 13 5 
CMPD 2 8 13 13 0 0 11 
32 63 58 7 6 11 
CMPD 3 8 92 90 14 8 6 
32 95 91 63 63 6 
CMPD 7 8 95 93 65 65 19 
32 93 93 80 84 19 
CMPD 8 8 95 93 84 93 8 
32 95 92 95 97 8 
______________________________________ 
Mean % injury = average injury of two replications rated at 3 dates. 
Test B 
In the second postemergence test, six true-breeding resistant mutant lines 
(W4, W17, W19, W20, W23, and W28) were compared to Williams 82 for cross 
resistance to a broad range of ALS inhibitors including eight 
sulfonylureas and three imidazolinones. Two seeds were planted in each 10 
cm pot and allowed to grow to the second trifoliolate stage. Pots were not 
thinned back if both plants were at the proper stage. For each of 11 
herbicides, two rates (Table VII) were selected based on previous data: 
the low rate was expected to cause significant injury (but not death) to 
Williams 82 and the high rate was expected to severely injure or kill 
Williams 82. All but a few line x rate treatments were replicated twice. 
In both postemergence tests, plants were returned to the greenhouse after 
treatment and provided with ample light, moisture, and nutrients to 
support healthy plant growth. At regular intervals (7 to 12 days), each 
pot was rated for percent herbicide injury according to the scale shown in 
Table VIII. Two to three ratings per pot were recorded over a three-week 
period and averaged to obtain a single value for each pot For each 
herbicide, analysis of variance on injury ratings was performed 
separately. Fisher's least significant difference (LSD) was used to 
determine the significance of line mean differences at each herbicide rate 
using the 95% level of confidence. 
"Resistance" was defined as "the ability to survive, with agronomically 
acceptable injury, a concentration of herbicide that is normally lethal or 
extremely injurious to individuals of a given species". 
Based on herbicide trials, at 1 to 3 weeks after treatment, 30% injury 
(according to Table VIII) is generally considered the threshhold between 
commercially acceptable and unacceptable levels of soybean injury. For the 
purpose of interpreting the postemergence herbicide test results, a 
soybean line was considered "resistant" to a particular treatment if less 
than 30% injury was observed during the first 3 weeks after application. 
If injury was greater than 30% but significantly less than the injury 
displayed by wild-type soybean plants, the soybean line was considered 
more "tolerant" of that treatment than wild-type. 
Previously isolated mutants were not significantly different from Williams 
82 in terms of reaction to postemergence treatment of the tested 
sulfonylurea herbicides. However, it is clear that mutants W19 and W20 
(representatives of the new class of mutants) are resistant to 
postemergence rates of Compounds 1, 2 and 3 that severely injure or kill 
both Williams 82 and 1-184A. Postemergence sulfonylurea resistance can 
clearly be used to differentiate the new class of resistant soybean 
mutants from the previously isolated sulfonylurea-tolerant soybean 
mutants. W19 and W20 were also more tolerant than Williams 82 of 
postemergence application of Compound 7 at 8 g/ha. Obviously, these 
mutants do not display the same level of resistance to all sulfonylureas. 
Rates of Compound 7 and Compound 8 were decreased for the subsequent test 
to study the resistance phenotype at rates that are sublethal to wild-type 
soybeans. 
From the results of the second post-emergence test (Table VII), it is clear 
that all mutant lines possess a higher level of tolerance or resistance 
(compared to Williams 82) to all postemergence sulfonylurea treatments. 
These mutants, however, are not more tolerant of the imidazolinones, 
Compounds 9, 10, and 11 than Williams 82. In response to the third 
imidazolinone, Compound 11, only mutants W17, W19, and W20 were slightly 
more tolerant than Williams 82. Apparently, the resistance afforded by 
these mutants is not equally effective against all ALS inhibitors. Within 
the sulfonylurea herbicides, there is considerable variation in the degree 
of resistance or tolerance afforded by these mutants. The six mutants 
studied seem to be very similar in reaction to the herbicides tested. 
Since all six were selected from the same M2 population, these mutants may 
trace back to the same mutational event (same M12 plant). 
TABLE VII 
__________________________________________________________________________ 
Responses of "Williams 82" and Mutant 
Soybean Lines to Eight Sulfonylurea and 
Three Imidazolinone Herbicides Applied 
Post-emergence at Second Trifoliolate Stage 
Herbi- 
Rate 
Wm 82 
W4 W17 
W19 W20 
W23 
W28 LSD 
cide g/ha 
mean % injury (0.05) 
__________________________________________________________________________ 
CMPD 1 
2 98 0 0 0 --.sup.a 
--.sup.a 
0 --.sup.a 
8 100 0 0 13 0 0 0 --.sup.a 
CMPD 2 
16 70 0 0 0 5 0 0 28 
64 91 5 3 6 5 8 5 28 
CMPD 3 
2 60 3 0 5 0 0 8 18 
8 94 18 25 29 15 45 21 18 
CMPD 4 
0.5 
58 10 3 3 0 8 0 16 
2.0 
95 8 0 18 6 11 18 16 
CMPD 5 
16 40 6 0 0 0 0 0 10 
64 58 0 0 20 0 10 0 10 
CMPD 6 
16 25 4 0 8 3 5 0 12 
64 66 43 40 48 38 54 45 12 
CMPD 7 
1 61 21 16 19 28 31 20 11 
4 98 69 55 65 66 60 54 11 
CMPD 8 
0.5 
93 39 26 29 19 21 53 11 
2.0 
98 81 83 85 89 70 84 11 
CMPD 9 
250 
8 13 0 9 5 9 9 10 
1000 
13 13 8 9 15 13 15 10 
CMPD 10 
250 
16 25 13 19 20 18 18 25 
1000 
51 61 46 46 54 51 54 25 
CMPD 11 
16 49 39 39 41 44 41 45 10 
64 75 73 61 60 61 74 68 10 
__________________________________________________________________________ 
Mean % injury = average injury of two replications rated at two dates. 
.sup.a No data. 
Increased Sulfonylurea Resistance and Cross Resistance of W4-4 to Other 
Classes of AlS 
Inhibitors at the Whole Plant Level 
Because W4-4 was confirmed as having a higher level of sulfonylurea 
resistance than W20 and cross resistance to an imidazolinone at the ALS 
enzyme level, it was suspected that W4-4 would also demonstrate such 
resistance at the whole plant level. To investigate this hypothesis, a 
postemergence herbicide spray test was conducted to compare the reaction 
of W4-4 to both W20 and wild-type Williams. The applicant was particularly 
interested in the resistance of W4-4 to herbicide treatments that were 
injurious to both Williams and W20 in a previous test (Table VII). 
Herbicide treatments that were significantly injurious (greater than 30% 
injury) to W20 were selected to test the resistance of W4-4. Hence, 
applications of Compounds 6, 7, and 8 were selected to test W4-4's 
reaction to sulfonylureas and applications of Compound 11 were selected to 
test W4-4's reaction to an imidazolinone. Twenty-four plants each of W4-4, 
W20, and Williams were planted in separate 10 cm pots and allowed to grow 
to the second trifoliolate stage. Each pot constituted a single 
experimental unit receiving a single herbicide treatment. Herbicide 
treatments (Table VIIA) were applied as described for the first 
postemergence test and were replicated twice. Control plants were sprayed 
with the spray carrier solution only (water with 0.25% X77 surfactant). At 
two weeks after treatment, each plant was rated for % herbicide injury as 
described in Table VIII. Analysis of variance on injury ratings was 
performed separately for each herbicide to calculate treatment means and 
standard errors. Fisher's least significant difference (LSD) was used to 
determine the significance of line means differences at each herbicide 
rate using the 95% level of confidence. 
TABLE VII-A 
______________________________________ 
Comparison of Mutant W4-4 to Mutant W20 and 
Williams 82 in terms of Response to Postemergence 
Application of Four ALS Inhibitor Herbicides 
Rate WM82 W20 W4-4 LSD (0.05) 
Herbicide 
g/ha mean % injury 
______________________________________ 
CMPD 6 64 60 10 0 14.9 
125 68 28 8 14.9 
CMPD 7 4 83 28 10 6.8 
16 93 43 30 6.8 
32 90 83 43 6.8 
CMPD 8 4 90 90 48 4.9 
16 95 93 48 4.9 
32 95 95 65 4.9 
CMPD 11 64 73 48 38 7.6 
125 83 80 53 7.6 
______________________________________ 
The preceding test in Table VII-A shows some resistance of W20 and W4-4 
soybeans to Compound 11, an imidizaloninone, at higher rates. The field 
use rate test shown in Table VIIB provides an even more impressive 
demonstration of the superior resistance to Compound 11 of W20 relative to 
Williams, and of W4-4 relative to W20. 
TABLE VII-B 
______________________________________ 
Rate WM82 W20 W4-4 
Herbicide g/ha mean % injury 
______________________________________ 
CMPD 11 8 50 20 0 
17.5 60 30 5 
35 75 60 25 
______________________________________ 
TABLE VIII 
______________________________________ 
Injury Scale for Rating Plants 
Treated With Post-emergence Applied 
Sulfonylurea or Imidazolinone Herbicides 
Rating 
(% injury) 
Visual Symptoms 
______________________________________ 
0 No apparent injury compared to untreated controls. 
10 Slightly stunted. 
20 Noticeable stunting and/or slight reddening of 
pulvini. 
30 Stunted with reddening, and chlorosis/reddening 
of apex. 
40 More stunting, reddening, and chlorosis/reddening 
of apex. 
50 Stunting which reduces plant to 1/2 of the size 
of control plants, severe reddening and 
inhibition of apical bud. 
60 Severe stunting and vein reddening but recovery 
is probable. 
70 Severe stunting and leaf necrosis evident. 
Recovery possible but plant will be very weak. 
80 Severe stunting and necrosis of apex, upper 
leaves, and stem. Recovery doubtful. 
90 Very little growth and most of plant tissue is 
necrotic. 
100 No shoot growth and/or plant is completely 
necrotic. 
______________________________________ 
Test C 
Demonstration of Resistance of Preemergence (Soil) Sulfonylurea Application 
After demonstration of postemergence sulfonylurea resistance, tests were 
conducted to determine whether mutant W20 (representative of the new class 
of dominant mutants) could also resist applications of sulfonylureas 
applied to soil in which the mutant seeds were planted (preemergence 
application). The following example is representative of the preemergence 
sulfonylurea resistance displayed by soybean mutants with dominant 
sulfonylurea resistance. 
W20, a line homozygous for a dominant sulfonylurea-resistance mutation, was 
compared to Williams 82 in terms of response to preemergence application 
of three sulfonylurea herbicides. The experimental unit consisted of a 
single 18 cm pot filled with soil (Sassafras loamy sand with 0.8% organic 
matter and pH 6.7) in which 6 seeds of a given soybean line were planted 2 
cm deep. After planting, each pot was sprayed (using a conveyor belt spray 
apparatus to emulate field application) with one of 22 different 
treatments including a check sprayed with AGWT (90% acetone, 4% glycerol, 
4% water, 2% Tween-20), and 21 different herbicide treatments using AGWT 
as a carrier. The herbicide treatments included 7 different concentrations 
(Table IX) each of three sulfonyl-ureas: Compound 5, Compound 4, and 
Compound 1. Each herbicide x line treatment was replicated three times. 
After herbicide treatment, pots were transferred to a greenhouse and 
arranged in a randomized complete block design. Nineteen days after 
treatment, each pot was rated visually for herbicide injury using the 
scale described previously. For each herbicide, analysis of variance on 
injury ratings was performed separately. Fisher's least significant 
difference (LSD) was used to determine the significance of line mean 
differences at each rate using the 95% level of confidence. 
Based on responses to increasing preemergence sulfonylurea rates, 10 to 30 
times as much herbicide was required to injure W20 as compared to Williams 
82 (Table IX). W20 showed agronomically acceptable levels of herbicide 
injury (less than 30%) to many of the preemergence sulfonylurea treatments 
that severely injured Williams 82 (Table IX). W20 is clearly resistant to 
some of the sulfonylurea rates and clearly more tolerant than Williams 82 
to all treatments except the highest rate of Compound 4. 
Results of all phenotypic characterization tests demonstrate the high 
degree of preemergence and postemergence sulfonylurea resistance provided 
by the applicant's new class of soybean mutants. 
TABLE IX 
______________________________________ 
Mean Injury of Williams 82 and W20 
Soybean Plants at 19 Days After Preemergence 
Treatment With Three Sulfonylurea Herbicides 
Rate % Injury 
Herbicide g/ha WM 82 W20 
______________________________________ 
Compound 5 1 0 0 
3 0 0 
10 0 0 
30 15 3 
100 47 10 
300 68 15 
1000 92 35 
LSD* 10 10 
Compound 4 0.3 33 0 
1 55 0 
3 88 0 
10 97 17 
30 98 68 
100 100 90 
300 100 95 
LSD* 9 9 
Compound 1 0.3 30 0 
1 50 0 
3 73 3 
10 95 3 
30 100 18 
100 100 63 
300 100 65 
LSD* 6 6 
______________________________________ 
*LSD = Fisher's least significant difference at .alpha. = 0.05 
Test D 
Demonstration of Sulfonylureas Resistance at the ALS Enzyme Level 
ALS assays were conducted to determine whether the new class of soybean 
mutants have sulfonylurea resistance at the ALS enzyme level. 
Acetolactate synthase (ALS) was extracted from leaves of soybean plants 
growing vegetatively. The leaves were homogenized in 4 volumes of buffer 
containing 100 mM hepes (pH 7.5), 1 mM sodium pyruvate, 0.5 mM magnesium 
chloride, 0.5 mM thiamine pyrophosphate, 10 mM FAD, 10% glycerol, 5% PVPP, 
and 0.2% mercaptoethanol. The homogenate was filtered through cheese cloth 
and centrifuged at 20,000 g for 20 min. ALS was precipitated from the 
supernatant with ammonium sulfate. The enzyme was collected between 20 and 
60% saturation by centrifugation at 20,000 g for 20 min. The pellet was 
resuspended in buffer containing 100 mM Hepes, 1 mM sodium pyruvate, and 
0.5 mM magnesium chloride, and desalted on PD-10 columns (Pharmacia) 
equilibrated with the same buffer. ALS assays were carried out in a final 
volume of 0.5 mL at 30.degree. C. The final reaction mixture contained 100 
mM Hepes (pH 7.5), 1 mM magnesium chloride, 60 mM sodium pyruvate, 0.4 mM 
thiaminepyrophosphate, 40 mM FAD, and various concentrations of 
chlorsulfuron. The assays were initiated with the addition of enzyme (100 
mL) and terminated with the addition of 50 mL of 6N sulfuric acid. The 
acidified reaction mixture was heated to 60.degree. C. for 15 min, after 
which 0.5 mL of 0.5% (W/V) creatine and 0.5 mL of 5% (W/V) napthol 
(freshly prepared in 2.5 N sodium hydroxide) were added. The reaction 
mixtures were heated for an additional 15 min at 60.degree. C., and the 
absorbance was read at 525 nm. Data are expressed in terms of % control 
(minus Compound 1) activity and are the average of two replications. 
Fisher's least significant difference (LSD) value (at the 95% level of 
confidence) was calculated for each Compound 1 concentration to determine 
the significance of genotype mean differences. 
The ALS activity from the leaves of sulfonylurea-resistant mutant lines was 
significantly higher than the activity of ALS from Williams 82 at all 
concentrations of Compound 1 (Table X). These results indicate a Compound 
1-resistant form of ALS in the leaves of the resistant mutants. The 
previously described whole-plant cross resistance tests imply that the 
mutant soybean ALS is less sensitive to sulfonylureas in general. 
TABLE X 
______________________________________ 
Percentage of ALS Activity Remaining 
at Different Concentration of Chlorsulfuron 
Chlor- 
sulfuron 
WM 82 W19 W20 W23 W28 LDS* 
mg/l-l uninhibited ALS activity (% of control) 
______________________________________ 
10 41.1 60.7 50.6 55.3 54.6 3.0 
20 26.3 54.5 35.2 46.3 46.1 7.2 
50 15.0 48.7 31.1 38.9 42.8 7.4 
100 11.1 45.7 20.8 37.4 36.4 5.1 
200 8.5 40.8 19.0 34.0 34.3 3.6 
______________________________________ 
*LSD = Least significant difference at 95% confidence level.