U.S. patent number RE35,661 [Application Number 08/414,449] was granted by the patent office on 1997-11-11 for sulfonylurea herbicide resistance in plants.
This patent grant is currently assigned to Idaho Research Foundation, Inc.. Invention is credited to Donald C. Thill.
United States Patent |
RE35,661 |
Thill |
November 11, 1997 |
Sulfonylurea herbicide resistance in plants
Abstract
Plants, plant tissues and plant seeds which are resistant to
inhibition by sulfonylurea and/or imidazolinone herbicides are
provided. In particular, domestic lettuce varieties having
resistance to herbicides which target the enzyme acetolactate
synthase are provided. The resistant plants find use in areas where
weed growth is controlled by sulfonylurea and/or imidazolinone
herbicides.
Inventors: |
Thill; Donald C. (Moscow,
ID) |
Assignee: |
Idaho Research Foundation, Inc.
(Moscow, ID)
|
Family
ID: |
24126216 |
Appl.
No.: |
08/414,449 |
Filed: |
March 30, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
533497 |
Jun 5, 1990 |
05198599 |
Mar 30, 1993 |
|
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Current U.S.
Class: |
800/300;
435/418 |
Current CPC
Class: |
A01H
5/12 (20130101); C12N 9/88 (20130101) |
Current International
Class: |
A01H
5/12 (20060101); A01H 005/00 (); A01H 005/10 ();
C12N 005/04 () |
Field of
Search: |
;800/200,230,255,DIG.13,DIG.71 ;47/58 ;435/418 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Alcocer-Ruthling et al., Weed Technology (1992) 6:303-309. .
Alcocer-Ruthling et al., Weed Technology (1992) 6:858-864. .
Amor, R., Plant Protection Quarterly (1986) 1:103-105. .
Donn et al., J. Mol. Appl. Genet. (1984) 2:621-635. .
Eberlein et al., Weed Science (1996): in review. .
Guttieri et al., Weed Science (1992) 40:670-676. .
Haughn et al., Mol. Gen. Genet. (1988) 21:266-271. .
Mallory-Smith et al., Weed Tech. (1990) 4:163-168. .
Mallory-Smith et al., Hort. Science (1993) 28:63-64. .
Mazur et al., Nato ASI Ser. A., vol. 140, Plant Mol. Biol., eds.,
pp. 339-349. .
Michelmore et al., Plant Cell Reports (1987) 6:439-442. .
Reed et al., Brighton Crop. Prot. Conf.-Weeds (1989) 1:295-300.
.
Saari et al., in:Herbicide Resistant Plants: Biology and
Biochemistry, eds. S. Powles & J. Holtum (1994), pp.
83-139..
|
Primary Examiner: Fox; David T.
Attorney, Agent or Firm: Rae-Venter Law Group, P.C.
Claims
What is claimed is:
1. .[.A.]. .Iadd.An herbicide resistant .Iaddend.Lactuca
.[.sativa.]. plant selected from the group consisting of (1) a
plant .[.retardant.]. .Iadd.resistant .Iaddend.to sulfonylurea
herbicides, (2) a plant resistant to imidazolinone herbicides, and
(3) .Iadd.sulfonylurea or imidazolinone herbicide resistant
.Iaddend.progeny plants of either of plant (1) or plant (2).[.;.].
wherein said plant or .[.a parent.]. .Iadd.an ancestor .Iaddend.of
said plant was grown from seed obtained by crossbreeding a Lactuca
serriola plant resistant to an herbicide selected from the group
consisting of (1) sulfonylurea herbicides and (2) imidazolinone
herbicides, with a Lactuca sativa plant.
2. The .Iadd.herbicide resistant Lactuca .Iaddend.plant according
to claim 1, wherein said Lactuca sativa plant is of the variety
Bibb, Grand Rapids, Prizehead, Vanguard, Ithaca, Empire or
Salinas.
3. A seed of .[.a.]. .Iadd.an herbicide resistant .Iaddend.Lactuca
.[.sativa.]. plant, said seed obtained from a plant selected from
the group consisting of (1) .[.a.]. .Iadd.an herbicide resistant
.Iaddend.plant according to claim 1 and (2) progeny of .[.a.].
.Iadd.an herbicide resistant .Iaddend.plant according to claim
1.
4. The seed according to claim 3, wherein said Lactuca sativa plant
is of the variety Bibb, Grand Rapids, Prizehead, Vanguard, Ithaca,
Empire or Salinas.
5. A plant cell of .[.a.]. .Iadd.an herbicide resistant
.Iaddend.Lactuca .[.sativa.]. plant and progeny cells of said
.[.cells.]. .Iadd.plant cell.Iaddend., said plant cell obtained
from a plant selected from the group consisting of (1) .[.a.].
.Iadd.an herbicide resistant Lactuca .Iaddend.plant according to
claim 1 and (2) progeny of .[.a.]. .Iadd.an herbicide resistant
Lactuca .Iaddend.plant according to claim 1.
6. The plant cell according to claim 5, wherein said Lactuca sativa
is of the variety Bibb, Grand Rapids, Prizehead, Vanguard, Ithaca,
Empire or Salinas.
7. A plant comprising cells according to claim 5, and progeny
plants derived from said plant.
8. Seeds from .[.a.]. .Iadd.an herbicide resistant Lactuca
.Iaddend.plant according to claim .[.2.]. .Iadd.1. .Iaddend.
Description
INTRODUCTION
Technical Field
This invention relates to plants, plant tissues and seeds, and
methods for their preparation, having increased tolerance to
herbicides. In particular, the invention involves production of
crops which are resistant to sulfonylurea and/or imidazolinone
herbicides.
BACKGROUND
Selective herbicides are routinely applied to control weeds among
crop plants. The weeds would otherwise compete for available
nutrients, water, and light, and thus reduce crop yield and
quality. Selective herbicides which show low toxicity to crop
species, while playing an important role in the control of weeds in
modern agriculture, are often available for only the major crop
species because of the high cost of development.
An alternative to the identification of new selective herbicides
for use with particular crop species is the genetic modification of
susceptible crop species so that they are resistant to
non-selective herbicides. One method of achieving this is through
the genetic transformation of plants to herbicide resistance. The
prerequisites for such an approach are the ability to transform the
species of interest, and availability of a gene which confers
resistance to the herbicide of interest.
Resistance to a specific herbicide may be a result of introduction
into a plant of a gene conferring resistance to the herbicide or
may be as a result of long periods of exposure to the herbicide.
The resistance may be the result of changes in enzymes which are
involved in particular biosynthetic pathways. For example, the
broad spectrum weed killer glyphosate (phosphonomethylglycine) acts
by inhibiting the enzyme 5-enolpyruvyl-3-phosphoshikimate
synthetase that converts phosphoenolpyruvate and 3-phosphoshikimaic
acid to 5-enolpyruvate-3-phosphoshikimaic acid in the shikimic acid
pathway in bacteria. Following mutagenesis of Salmonella
typhimurium, an altered synthetase enzyme resistant to glyphosate
has been identified and introduced into plants where it confers
resistance to glyphosate.
It is of interest to identify other biosynthetic pathways which may
be affected by specific herbicides as a means of developing
herbicide resistant plants. Two unrelated classes of herbicides,
the sulfonylureas and the imidazolinones, notable for their high
herbicidal potencies and low mammalian toxicities, target the
enzyme acetolactate synthase, the first common step in the
biosynthesis of the essential amino acids isoleucine, leucine and
valine, and inhibit plant growth by inactivating the target enzyme.
The selective toxicity to weeds of these compounds or their analogs
is due to their metabolism by particular crop species but not by
most weed species. Thus, for those crop species sensitive to
sulfonylureas and imidazolines, it would be of interest to develop
crop hybrids or varieties having resistance to these
herbicides.
Relevant Literature
Chaleff et al., Molecular Strategies For Crop Protection (1987) pp:
415-425 (Ellen R. Liss Inc. 1987) is an overview of general methods
used for developing sulfonylurea herbicide resistant plant
varieties. Haughn et al., Molecular General Genetics (1988)
211:266-271 disclose transgenic tobacco plants having resistance to
chlorsulfuron as a result of transformation with a gene encoding
acetolactate synthase. Mazur et al., Plant Physiol. (1987)
85:1110-1117 and Lee et al., The EMBO Journal (1988) 7:1241-1248
disclose the isolation and characterization of plant genes coding
for acetolactate synthase. Chaleff et al., Molecular General
Genetics (1987) 210:33-38 disclose two isozymes of acetolactate
synthase in tobacco plants resistant to chlorsulfuron and
sulfometuron methyl. Ray, Plant Physiol. (1984) 15:827-831
discloses that the site of action of chlorsulfuron is the enzyme
acetolactate synthase. U.S. Pat. No. 4,761,373 discloses herbicide
resistant maize plants, plant tissues and plant seeds having
altered acetohydroxyacid synthase enzymes.
Sulfonylurea resistance has been reported in natural populations of
prickly lettuce (Lactuca serriola L.), kochia (Kochia scoparai L.),
Russian thistle (Salsola iberica Sennen and Pau, Thill et at. Proc.
Weed Sci. Soc. Am. (198) 29:132, and annual ryegrass (Lolium
rigidum Gaudin) (Heap et al., Aust. J. Agric. Res. (1986)
37:149-156). Tolerance to sulfonylurea herbicides due to an
increased rate of herbicide metabolism has been reported in corn
(Zea mays L.) (Eberlein et al. Weed Science (1989) 37:651-657).
Inheritance of sulfonylurea resistance in the bacterium Salmonella
typhimurium (LaRossa et at., J. Bacteriol. (1987) 169:1372-1378; in
yeast Saccharomyces cerevisiase (Falco et al., Genetics (1985)
109:21-35); and in the green alga, Chlamydomonas reinhardtii
(Hartnett et al., Plant Physiol. (1987) 85:898-901) is due to a
dominant mutation. In mutated tobacco (Nicotiana tabacum) plants,
sulfonylurea resistance is due to a single, semi-dominant, nuclear
mutation (Chaleff et al., Science (1984) 223:1148-1151). In
Arabiodopis thaliana, sulfonylurea resistance is due-to a single,
dominant, nuclear mutation (Haughn et al., Mol. Gen. Genet. (1986)
204:430-434. Sulfonylurea herbicide resistance in soybean mutants
has been reported to be a recessive trait (Sebastian et al., Crop
Sci. (1987) 27:948-952) and a dominant or semidominant trait
(Sebastian et al., Agronomy Abst. (1988) p. 95).
SUMMARY OF THE INVENTION
Novel plants and seeds, and methods for their preparation, are
provided which have enhanced resistance to herbicides which target
the enzyme acetolactate synthase. The plants are obtained by
introduction of a DNA sequence encoding an altered acetolactate
synthase into a plant of interest. The resulting transgenic plants
are resistant to the growth and development inhibition by said
herbicides at concentrations which normally inhibit the growth and
development of the plant of interest. The plants can also be grown
to produce seed having the resistance phenotype. The resistant
plants find use in areas where weed growth is controlled by
herbicides which target the enzyme acetalactate synthase.
DESCRIPTION OF SPECIFIC EMBODIMENTS
In accordance with the subject invention, plants, plant tissues,
and seeds as well as methods for their preparation are provided
which allow for enhanced resistance to herbicides which target the
enzyme acetolactate synthase. The gene encoding an altered
acetolactate synthase enzyme, which has decreased sensitivity to
inhibition by herbicides which target acetolactate synthase,
particularly herbicides characterized as sulfonylureas and
imidazolinones is transferred to a desired host plant using
conventional crossing techniques. The resultant transgenic plants
are then resistant to the sulfonylurea and imidazolinone herbicides
at concentrations where the herbicides are used selectively to
control weeds.
To incorporate the herbicide resistance into a desired crop plant,
a plant comprising an altered acetolactose synthase gene can be
crossbred with a susceptible crop plant wherein the resistant
biotype is capable of transferring genetic information to the
susceptible plant to produce a novel hybrid plant having the
desired herbicide resistance trait. In order to obtain transgenic
plants having the desired trait in a given plant, it is important
to determine the mechanism of genetic control of the herbicide
resistance. This requires crossing resistant plants with sensitive
plants in studying the pattern of inheritance in segregating
generations to ascertain whether the trait is expressed as dominant
or recessive, the number of genes involved and any possible
interactions between genes if more than one are required for
expression. This genetic analysis can be part of the initial
efforts to convert sensitive plants to resistant plants.
A conversion process (back crossing) is carried out by crossing an
original resistant plant to sensitive plants and crossing the
progeny back to the sensitive parent. The progeny from this cross
would segregate such that some plants carry the gene responsible
for resistance whereas some do not. Plants carrying such genes will
be crossed again to the sensitive parent resulting in progeny which
segregate for resistance and sensitivity once more. This is
repeated until the original sensitive parent has been converted to
a resistant plant yet possesses all other important attributes as
originally found in the sensitive parent. A separate back-crossing
program is implemented for each strain that is to be converted to
herbicide resistance.
Subsequent to the back crossing, the new resistant plants and the
appropriate combinations of strains which make good commercial
hybrids are evaluated for resistance as well as important agronomic
traits. Resistant strains and hybrids are produced which are true
to type of the original sensitive strains and hybrids. This
requires evaluation under a range of environmental conditions where
the strains or hybrids will generally be grown commercially. For
production of herbicide-resistant plants, it may be necessary that
both parents of the hybrid seed be homozygous for the resistant
trait. Parental lines of hybrids that perform satisfactorily are
increased and used from hybrid production using standard hybrid
seed production practices.
The source of plants for cross-breeding purposes is any resistant
plant capable of cross-breeding with the plant of interest. Known
resistant plants include prickly lettuce, kochia (Kochia scoparia
L.)/SCRAD), Russian thistle (Salsosa iberica, Sennen and Pau),
chickweed (Stellaria media L.) Vill). Other herbicide-resistant
plants which may be used for cross-breeding purposes may be
identified, for example, by herbicide failure. Herbicide failure is
often attributed to environmental conditions, plant growth stage,
and improper use or application of the herbicide. However, if these
factors are eliminated, herbicide resistance may explain the lack
of weed control.
"Herbicide resistance" is the ability of a biotype to survive
herbicide treatment to which the species is normally susceptible.
Thus, plants resistant to the herbicide of interest may be
identified by the lack of weed control in the presence of the
herbicide to which the species is normally susceptible. Resistance
is due to a heritable genetic trait in the population. Resistance
is not based on the herbicide dosage the resistant plant is able to
withstand, but rather the difference in response between the
response of the original susceptible population and the response of
the new biotype.
Alternatively, recombinant DNA techniques may be used for
developing resistant lines of plants. This can be achieved by
inserting a DNA sequence coding for an altered acetolactate
synthase into a plant cell by means of an expression cassette. The
expression cassette will include in the 5'-3' direction of
transcription, a transcriptional and translational initiation
region; a structural gene encoding an altered acetolactase
synthase; and a transcriptional and translational termination
regulatory region. The initiation and termination regulatory
regions are functional in the intended host plant cell and may be
either homologous (derived from the original host), or heterologous
(derived from a foreign source, or synthetic sequences).
Where recombinant DNA techniques are to be used to obtain the
resistant biotype, DNA sequences encoding an altered acetolactase
may be obtained in a variety of ways. They may be derived from
resistant plants (see above) but may be derived from other
eukaryotic sources such as the yeast Saccharomyces cerevisiae and
prokaryotes such as Salmonella typhimurium. The altered
acetolactate synthase structural gene may be derived from cDNA,
from chromosomal DNA or may be synthesized in whole or in part. For
the most part, some or all of the structural gene will be from a
natural source. Methods for identifying genes of interest have
found extensive exemplification in the literature, although in
individual situations different degrees of difficulty may be
encountered. Various techniques include the use of probes where
genomic or cDNA libraries may be searched for complementary
sequences.
The gene may be synthesized in whole or in part, particularly where
it may be desirable to modify all or a portion of the codons, for
example to enhance expression, by employing host-preferred codons.
Thus, all or a portion of the open reading frame encoding the
altered acetolactate synthase may be synthesized using codons
preferred by the plant host. Plant-preferred codons may be
determined from the codons of highest frequency and the proteins
expressed in the largest amount in the particular plant species of
interest.
Methods for synthesizing sequences and bringing the sequences
together are well established in the literature. Where a portion of
the open reading frame is synthesized, and a portion is derived
from natural sources, the synthesized portion may serve as a bridge
between two naturally-occurring portions, or may provide a
3'-terminus or a 5'-terminus. Particularly where the
transcriptional initiation region and the open reading frame
encoding the altered acetolactase synthase are derived from
different genes, synthetic adaptors commonly will be employed. In
other instances, polylinkers may be employed, where the various
fragments may be inserted at different restriction sites or
substituted for a sequence in the polylinker.
If the structural gene to be inserted is derived from prokaryotic
cells, it is desirable to minimize this 3' non-coding region of the
prokaryotic gene. The substantial absence of this region can have a
positive effect on the transcription, the stability, and/or
translation of the mRNA in the host plant cells. In order to have
expression of a gene other than a plant gene in a plant cell,
transcriptional and translational initiation regulatory regions
functional in a plant cell must be provided. Promoters and
translation initiation signals functional in plant cells include
those from genes which are present in the plant host or other plant
species, for example the ribulose biphosphate carboxylase small
subunit transcriptional initiation region, for example from
tobacco; those present in viruses, such as the cauliflower mosaic
virus (CaMV), for example the 35S transcriptional initiation
region; and those associated with T-DNA such as the opine synthase
transcriptional initiation regions, for example, octopine,
manopine, agropine, and the like.
Regulatory regions may be homologous or heterologous to the plant
host. In order to join the promoter to the structural gene, the
noncoding 5' region upstream from the structural gene may be
removed by endonuclease restriction. Alternatively, where a
convenient restriction site is present near the 5' terminus of the
structural gene, the structural gene may be restricted and an
adaptor employed for linking the structural gene to a promoter
region where the adaptor provides the missing nucleotides of the
structural gene.
The termination region may be derived from the 3' region of the
gene from which the initiation region was obtained or from a
different gene. The termination region may be derived from a plant
gene, particularly the tobacco ribulose biphosphate carboxylase
small subunit termination region; a gene associated with the
Ti-plasmid such as the octopine synthase termination region; or the
tml termination region.
In developing the expression cassette, the various fragments
comprising the regulatory regions and open reading frame may be
subjected to different processing conditions, such as ligation,
restriction enzyme digestion, resection, in vitro mutagenesis,
primary repair, use of linkers and adaptors, and the like. Thus,
nucleotide transitions, transversions, insertions, deletions or the
like, may be performed on the DNA which is employed in the
regulatory regions and/or reading frame. The expression thus may be
wholly or partially derived from natural sources, and either wholly
or partially derived from sources homologous to the host cell, or
heterologous to the host cell. Furthermore, the various DNA
constructs (DNA sequences, vectors, plasmids, expression cassettes)
of the invention are isolated and/or purified or synthesized and
thus are not "naturally occurring."
The expression cassette will normally be joined to a marker for
selection in plant cells. Conveniently, the marker may be
resistance to a biocide, particularly an antibiotic, such as
kanamycin, G418, bleomycin, hygromycin, chloramphenicol, or the
like. The particular marker employed will be one which will allow
for selection of transformed plant cells as compared to plant cells
lacking the DNA of interest.
During the construction of the expression cassette, the various
fragments of the DNA will usually be cloned in an appropriate
cloning vector, which allows for amplification of the DNA,
modification of the DNA or manipulation by joining or removing of
sequences, linkers or the like. Normally the vectors will be
capable of replication and at least a relatively high copy number
in E. coli.
Plants of interest include crops whose cells or explants can be
manipulated and subjected to selection and regeneration in tissue
culture. The appropriate candidates for transformation with a gene
conferring resistance to herbicides characterized as sulfonylureas
and imidazolinones. It is contemplated that any plant variety
having the above characteristics would constitute a suitable host
plant. Of particular interest are plants of the family composite
including sun flower lettuce, safflower.
A variety of techniques are available for the introduction of DNA
into the plant cell host. These techniques include transformation
with T-DNA. employing Agrobacterium tumefaciens or Agrobacterium
rhizogenes as the transforming agent, protoplast fusion, injection,
electroporation, etc. The transformation with agrobacteria is
commonly with plasmids which contain DNA homologous with the
Ti-plasmid, particularly T-DNA, which can be prepared in E
coli.
The plasmid may or may not be capable of replication in
Agrobacteria, that is, it may or may not have a broad spectrum
prokaryotic replication system, for example, rk290, depending in
part upon whether the expression cassette is to be integrated into
the Ti-plasmid or to be retained on an independent plasmid. By
means of a helper plasmid, the transcription construct may be
transferred to the Agrobacterium and the resulting transformed
organism used for transforming plant cells. The use of T-DNA for
transformation of plant cells has received extensive study and is,
for example, described in EPA Serial Number 120,516, Hockema, in:
The Binary Plant Vector System, Offsetdrukkerij Kanters BV,
Alblasserdam, 1985; Chapter V, Kanaf et al., "Genetic Analysis of
Host Range Expression by Agrobacterinm," in Molecular Genetics of
the Bacteria - Plant Interaction" Puhler A. Ed. Springer Verlag,
New York (1983) p 245, and An et al., EMBO Journal (1985)
4:277-284.
After transformation, the cell tissue (for example protoplasts,
explants, or cotyledons) is transferred to a regeneration medium,
such as Murashige-Skoog (MS) medium for plant tissue and cell
culture, for formation of a callus. Cells that have been
transformed may be grown into plants in accordance with
conventional ways. See for example, McCormick et al., Plant Cell
Reports (1986) 5:81-84.
Transformed plants may be analyzed to determine whether the desired
gene product is being produced in or a portion of the plant cells.
After expression of the desired product has been demonstrated in
the plant, these plants may then be grown, and either pollinated
with the same transformed strain or different strains, and the
resulting hybrid having the desired phenotypic characteristic(s)
identified. Two or more generations may be grown to ensure that the
subject phenotypic characteristic is still being maintained and
inherited. Seeds may then be harvested for use to provide plants
having the new phenotypic property, namely resistance to herbicides
which target the acetolactate synthase enzyme.
Various techniques exist for determining whether the desired DNA
sequences are present in the plant cell and are being transcribed.
Techniques such as the Northern Blot can be employed for detecting
messenger RNA which codes for rhiticide herbicide. In addition, the
presence of expression can be detected by identifying the altered
enzyme, including solution enzyme assay, estrin analysis and native
electrophoresis with activity staining. Furthermore, anti-bodies
specific for the altered enzyme may be employed.
The transgenic plants can be evaluated directly, for example,
transgenic plants can be evaluated for resistance to the herbicide
of interest, particularly sulfonylurea and imidazolinone
herbicides. By the ability of the plant to grow in the presence of
higher concentrations of the herbicide as compared to
non-transgenic plants, or plants transformed with other than an
expression cassette providing for herbicide resistance.
Plants engineered to produce an altered acetolactate synthase find
use in being able to grow under conditions where sulfonylurea and
imidazolinone herbicides are used to selectively control weeds.
The following examples are offered by way of illustration and not
by way of limitation.
EXPERIMENTAL
Sulfonylurea resistant prickly lettuce (Lactuca serriola) seeds
were deposited with the American Type Culture Collection, 12301
Parklawn Drive, Rockville, Md. 20852, on May 29, 1990 and have
received accession number 40815.
EXAMPLE 1
Identification of Sulfonylurea Herbicide-Resistant Prickly
Lettuce
In the spring of 1987, a field study was established 15 km south of
Lewiston, ID in response to a grower's complaint that .Iadd.in
.Iaddend.1986 a fall-applied 5:1 formulated mixture of
chlorsulfuson
[2-chloro-N-[[CH-methoxy-6-methyl-1,3,5-triazin-2-yl-amino]carbonyl]benzen
esulfonamide]:metsulfuron
[2-[(L-4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl] E. I.
dupont de Nemours & Company, Inc.) (26 g ai/ha) failed to
control prickly lettuce in winter wheat. The grower also noted
prickly lettuce had not been controlled effectively in the previous
winter wheat crop with 16 g/ha chlorosulfuron:metsulfuron
(DPX-G8311). The cropping system on this farm had been continuous
no-till winter wheat since 1983. Chemical fallow was used in
rotation with winter wheat on some of the fields, but fields with
shallow soil (<60 cm) were cropped each year.
Wheat had been the only crop produced for the past 30 years.
Average rainfall for the farm was 36 to 46 cm/year. The grower had
used sulfonylurea herbicides since 1982 for both chemical fallow
and weed control in winter wheat. Weed scientists from the
University of Idaho had experimented with herbicide efficacy on
this farm for more than 12 years. Sulfonylurea herbicides first
were applied in 1980 experiments. (Handly et al., "Wild Carrot and
Broadleaf Weed Control in Winter Wheat," Idaho Feed Control, Report
9 (1980).) Often the experimental rates, for example 137 g ai/ha
chlorsulfuron in the 1980 study (ibid), were much higher than the
final labeled rate; so actual amount of herbicide applied to some
fields was greater than was shown by the grower's field records.
The grower applied sulfonylurea herbicides at intervals ranging
from 6 to 14 months (See Table 1 below).
TABLE 1 ______________________________________ Field History and
Edaphic Information for the Farm Where Sulfonylurea (SU) Resistant
Prickly Lettuce Initially was Reported Soil First Field Organic
Year Maximum Num- Size Matter Treatment SU.sup.a Interval.sup.b ber
(ha pH (%) (SU) (g ai/ha) (months)
______________________________________ 1 167 5.7 3.2 1982 151 7 2
54 5.4 3.8 1982 153 9 3 88 6.7 3.8 1983 52 14 4 43 6.4 2.8 1983 100
9 5 97 5.7 3.1 1982 96 9 6 22 5.7 2.9 1982 114 8 7 23 64 2.7 1982
148 9 8 37 5.1 4.0 1982 137 7 9 90 6.2 4.1 1982 200 6
______________________________________ .sup.a Maximum ai/ha that
was applied in this field. All areas in the field may not have
received the maximum. .sup.b Months between herbicide
applications.
Field Studies
1. 1987 Study. Field plots were established Apr. 14, 1987, and Apr.
20 and 26, 1988 to determine if this prickly lettuce biotype would
resist sulfonylurea herbicides. The experimental design for all
studies was a randomized complete block with four replications.
Plots were 3 by 9 m. Treatments were applied with a CO.sub.2
pressurized backpack sprayer calibrated to deliver 90 L/ha at 275
kPA and 4.8 km/h. Sulfonylurea herbicides were applied with a
nonionic surfactant (octyl phenoxy polyethoxy ethanol, 90% ai) at
0.25% vv. The soil was a silt loam with a pH of 4.9, 3.9% organic
matter, and a cation exchange capacity (meg/100 g soil) of 21.
In 1987, the sulfonylurea treatments were Thifensulfuron DPX-G8311,
and a 2:1 formulated mixture of thifensulfuron (Dupont)
{3-[[[[(4-methoxy-6-methyl-1,3,5,triazin-2-yl)amino]carbonyl]amino]sulfony
l]-2-thiophenecarboxylic acid}:DPX-L5300 (Dupont)
{methyl-2-[[[[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)methylamino]carbony
l]amino]sulfonyl]benzoate} at 13, 26, and 52 g ai/ha.
Thifensulfuron:DPX-L5300 is numbered DPX-R9674 (Dupont). The other
two herbicide treatments were the butoxyethyl ester of 2,4-D
[(2,4-dichlorophenoxy)acetic acid] and the octanoic acid ester of
bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) plus the isooctyl
ester of MCPA [(4-chloro-2-methylphenoxy)acetic acid] at 840 and
430 g ae/ha, respectively. There were 200 to 400 prickly lettuce
plants per m.sup.2 at the time of herbicide application. Percent
control was evaluated visually five times during the growing
season. On July 6, plants in a 0.37 m.sup.2 quadrant were counted.
The shoots were harvested, dried for seven days at 60.degree. C.,
and then weighed.
Neither of the sulfonylurea herbicides at any application rate
controlled more than 10% of the prickly lettuce, while the 2,4-D
and bromoxynil plus MCPA treatments controlled 100% of the prickly
lettuce. The number of prickly lettuce plants (range 168 to 316
m.sup.2 or weight per prickly lettuce plant (range 1410 to 2270 mg)
in the sulfonylurea treated plots was not significantly different
from plant number (292 m.sup.2) or weight (1600 mg) in the control
plots.
2. 1988 Study. In 1988, two field studies similar to the 1987 study
were established in the same field. There were 100 prickly lettuce
plants per m.sup.2 at the time of treatment. Prickly lettuce
control was evaluated visually May 20.
The herbicides used in study 1 were DPX-G8311, DPX-R9674, 2,4-D,
bromoxynil, diuron [N'-(3,4-dichlorophenyl)-N,N-dimethylurea) and
CGA-131036
[N-(6-methoxy-4-methyl-1,3,5-triazin-2-yl-aminocarbonyl-2-(2-chloroethoxy)
benzene-sulfonamide]. DPX-G8311 and DPXR9674 were applied at 16 and
26 g/ha alone and in tank mixes with the other herbicides except
CGA-131036 which was applied alone at 26 g ai/ha. Diuron,
bromoxynil, and 2,4-D were applied alone at 670, 430, and 840 g/ha,
respectively. These same rates as well as diuron at 340 g/ha,
bromoxynil at 220 g/ha, and 2,4-D at 420 g/ha were included in the
tank mixes with 16 and 26 g/ha of both DPX-G8311 and DPX-R9674.
Study 2 also included herbicides alone and in Btank mixes.
Herbicides in this study were DPX-G8311, diuron, MCPA, CGA-131036,
the potassium salt of picloram
[4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid], the
dimethylamine salt of dicamba [3,6-dichloro-2-methoxybenzoic acid],
and the alkanolamine salt of clopyralid
[3,6-dichloro-2-pyridinecarboxylic acid] plus the alkanolamine salt
of 2,4-D. DPX-G8311 at 16 g/ha was applied alone and in a tank mix
with 18 g ae/ha picloram. Clopyralid plus 2,4-D was applied alone
at 450 and 670 g ae/ha and at 450 g/ha with 450 g/ha MCPA. MCPA and
2,4-D were each applied alone at 840 g/ha. They both were applied
at two rates, 280 and 420 g/ha, as tank mixes with 18 and 26 g/ha
picloram. Picloram at 18 and 26 g/ha was tank mixed with 70 and 140
g/ha dicamba.
The results of the 1988 field studies were the same as those of the
1987 study. In Study 1, neither DPX-G8311 nor DPX-R9674 controlled
prickly lettuce. CGA-131036 controlled only 30% of the prickly
lettuce, while all other herbicides controlled at least 86% of the
weed. When sulfonylurea herbicides were tank mixed with the other
broadleaf herbicides, prickly lettuce control did not increase.
In Study 2, DPX-G8311 alone did not control prickly lettuce, while
all other treatments, either alone or in tank mixes, controlled at
least 98% of the prickly lettuce. When DPX-G8311 was tank mixed
with picloram, the same level of control as achieved with picloram
alone was observed.
Results from these field experiments show this bio-type of prickly
lettuce was not controlled with sulfonylurea herbicides. In field
studies conducted during the 1983-1984 winter wheat growing season,
at the same location as the 1987 and 1988 field studies,
thifensulfuron plus either metsulfuron or chlorsulfuron controlled
100% of the prickly lettuce in the treated plots (Schaat et al.,
"Broadleaf weed control in winter wheat," Res. Prog. Rep. West.
Soc. Weed Sci. (1985) pp 320-321). The sulfonylurea-resistant
biotype of prickly lettuce likely was selected because sulfonylurea
herbicides were used frequently in a continuous winter wheat
production system. Other herbicides with different mechanisms of
action and known to control prickly lettuce control the resistant
biotype effectively.
Field Survey. On Apr. 3, 1988, 153 representative plants were
randomly selected from 51 locations in the 9 fields. Seedlings in
the 2 to 3 leaf stage were dug, transplanted into 580-ml styrofoam
cups, and then transported to the University of Idaho greenhouses.
Seedlings were treated Apr. 21, 1988, with 30 ml of 500 ppb w/v
metsulfuron as a combination foliar application and soil drench.
This concentration is five times the concentration required to
reduce plant growth of the susceptible biotype by 90% (see
Greenhouse Studies). Plants were evaluated visually as resistant
(alive with normal or near-normal growth) or susceptible (dead) May
2 and again May 9, 1988.
Of the 153 plants collected, 43% resisted 500 ppb metsulfuron
(Table 2).
TABLE 2 ______________________________________ The Percentage of
Prickly Lettuce Plants in the Field Survey Showing Resistance to
Metsulfuron at 500 ppb w/v Field.sup.a Plants (no.) Resistant (%)
______________________________________ 1 30 70 2 9 56 3 15 53 4 12
67 5 27 0 6 12 8 7 6 83 8 15 0 9 27 48 Total 153 mean = 43
______________________________________ .sup.a See Table 1 for field
description.
Only 2 of the 9 fields surveyed had no resistant plants, indicating
that the resistant biotype was spread over most of the cultivated
areas of the farm. Since prickly lettuce seeds can be dispersed by
wind, environmental and geographical factors may explain why no
resistant plants were found in Fields 5 and 8. Field 5 was located
at the west edge of the farm, and the prevailing winds are away
from the field. Field 8, near the middle of the farm, was isolated
by terrain. It was lower in elevation and had a rock bluff on the
west edge.
Noncultivated areas of the farm and areas bordering the farm are
being surveyed for the resistant biotype. Preliminary results of
this survey indicate the resistant biotype is contained on the
cultivated areas of the farm. However, resistant plants have been
found on roadsides and disturbed areas 2 km from the farm.
Greenhouse Studies. Seeds were gathered during August and September
1987 from three sites: the field plot area south of Lewiston and
untreated sites near Troy and Moscow, Id. All three selections were
ascertained to be prickly lettuce by a University of Idaho plant
taxonomist. Preliminary studies showed the Troy and Moscow
selections were equally susceptible to metsulfuron; therefore, the
selections were used interchangeably in the studies. Seeds were
planted in commercially prepared potting mix (Sunshine Mix #1.
Fisons West corp., 1212 W. Broadway, Vancouver, B.C. Canada B6H3B1)
in 440-ml styrofoam cups in the greenhouse. Seedlings were thinned
to two per cup at the 2- to 3-leaf stage and were treated with 30
ml of metsulfuron herbicide solution as a combination foliar
application and soil drench. A nonionic surfactant (same as in the
field studies) at 0.25% v/v was added to all herbicide treatments.
Metsulfuron concentrations were 1, 5, 10, 50, 100, 500, and 1000
ppb ai w/v. The experimental design was a randomized complete block
with four replications repeated three times. Ten days after
treatment, plant shoots were harvested and leaf areas measured with
a leaf area meter (Li-COR Model LI 3000. LI-COR, Inc., P.O. Box
4425, Lincoln, Nebr. 68504). The plant shoots then were dried for
48 hours at 50.degree. C. and weighed.
Two studies were conducted to determine resistance to seven other
sulfonylurea herbicides using the prickly lettuce seed collected
near Lewiston (resistant) and Troy (susceptible). Sulfonylurea
herbicides included in Study 1 were CGA-131036, chlorsulfuron, and
DPX-L5300. Study 2 included the sulfonylurea herbicides bensulfuron
{2-[[[[[(4-6-dimethoxy-2-pyrimidinyl)amino]-carbonyl]amino]sulfonyl]methyl
]benzoic acid}, chlorimuron
{2-[[[[(4-chloro-6-methoxy-2-pyrimidinyl)amino]carbonyl]-amino]sulfonyl]be
nzoic acid}, thifensulfuron, and sulfometuron
{2[[[[(4,6-dimethyl-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]benzoic
acid}. Metsulfuron was included as a reference in all experiments.
Herbicide concentrations used in these experiments were 10, 100,
and 1000 ppb ai w/v.
Resistance experiments were repeated twice. Experimental design was
a randomized complete block with three replications. Plants were
harvested, and leaf area and dry weight were determined as
described previously.
Cross resistance to three imidazolinone herbicides,
imazapyr={(+)-2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazole
-2-yl]-3-pyridinecarboxylic acid},
imazaquin={2-[4,5-dihydro-4-methyl-4-(1-mythelethyl)-5-oxo-1H-imidazole-2-
yl]-3-quinolinecarboxylic acid}, and
imazethapyr={(+)-2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidaz
ole-2-yl]-5-ethyl-3-pyridinecarboxylic acid)} was examined, since
this class of herbicides has the same site of action as the
sulfonylurea herbicides (Shaner et al., "Imidazolinones: Potent
inhibitors of acetohydroxyacid synthase," Plant Physiol. (1984)
76:545546). The experimental design was the same as the
sulfonylurea herbicide resistance studies, except only one seedling
was left per cup; and herbicide concentrations were 500, 1000,
2500, 5000, 10000, and 25000 ppb ai w/v. Plant shoots were
harvested 14 days after treatment, and leaf area and dry weight
were measured.
Regression analysis was used to compute a 50% growth reduction
(GR50). Analysis of variance was used to calculate Least
Significant Differences (LSD) in leaf area and dry weight as well
as to determine interactions among variables. Pearson correlation
coefficients (Snedecor et al., Statistical Methods, 7th Ed. Iowa
State University Press, Ames (1980) p 192) were computed between
leaf area and dry weight.
The Pearson correlation coefficient between leaf areas and dry
weight was high (0.9); therefore, only leaf area data are reported.
Herbicide concentrations required for GR.sub.50 values were
calculated using regression; however, in most cases, a GR.sub.50
was not reached for the resistant biotype.
TABLE 3 ______________________________________ Leaf Area and
Percent of Control Leaf Area for Susceptible and Resistant Biotypes
of Prickly Lettuce Treated with Eight Concentrations of Metsulfuron
in the Greenhouse Biotype Rate Susceptible Resistant (ppb w/v)
(cm.sup.2) (% of Control) (cm.sup.2) (% of Control)
______________________________________ 0 104 100 12 100 1 78 75 143
117 5 70 67 123 100 10 80 77 140 114 50 15 15 146 119 100 17 17 122
99 500 8 8 91 74 1000 7 7 60 49 LSD (0.05) 33 40 LSD (0.05) (rate
.times. biotype) 40 ______________________________________
TABLE 4 ______________________________________ Leaf Area and
Percent of Control Leaf Area for Susceptible and Re- sistant
Biotypes of Prickly Lettuce Treated with Four Sulfonylurea
Herbicides at Three Concentrations in the Greenhouse Biotype
Susceptible Resistant Rate (% of (% of Herbicide (ppb w/v)
(cm.sup.2) Control (cm.sup.2) Control)
______________________________________ CGA-131036 0 81 100 164 100
10 53 65 191 116 100 8 10 174 106 1000 8 10 106 65 LSD (0.05) 25 53
LSD (0.05) (rate .times. biotype) 21 Chlorsulfuron 0 85 100 181 100
10 61 72 196 108 100 17 20 194 107 1000 5 6 164 91 LSD (0.05) 18 ns
LSD (0.05) (rate .times. biotype) 34 DPX-L5300 0 86 100 172 100 100
68 79 176 102 100 11 13 180 105 1000 6 7 181 105 LSD (0.05) 22 ns
LSD (0.05) (rate .times. biotype) 33 Metsulfuron 0 88 100 176 100
10 42 48 178 101 100 8 9 170 97 1000 6 8 69 39 LSD (0.05) 33 43 LSD
(0.05) (rate .times. biotype) 37
______________________________________
TABLE 5 ______________________________________ Leaf Area and
Percent of Control Leaf Area for Susceptible and Re- sistant
Biotypes of Prickly Lettuce Treated with Five Sulfonylurea
Herbicides at Three Concentrations in the Greenhouse Biotype
Susceptible Resistant Rate (% of (% of Herbicide (ppb w/v)
(cm.sup.2) Control (cm.sup.2) Control)
______________________________________ Bensulfuron 0 128 100 152
100 10 132 103 137 90 100 122 95 149 98 1000 48 37 152 100 LSD
(0.05) 45 LSD (0.05) (rate .times. biotype) 43 Chlorimuron 0 126
100 146 100 10 102 81 138 94 100 45 35 112 75 1000 14 11 81 55 LSD
(0.05) 39 LSD (0.05) (rate .times. biotype) ns Thifen- 0 112 100
149 100 sulfuron 10 132 118 152 102 100 135 120 145 97 1000 26 23
133 89 LSD (0.05) 28 ns LSD (0.05) (rate .times. biotype) 40
Metsulfuron 0 131 100 143 100 10 137 105 148 103 100 57 44 125 87
1000 7 5 86 60 LSD (0.05) 26 ns LSD (0.05) (rate .times. biotype)
44 Sulfometuron 0 132 100 157 100 10 112 85 152 96 100 40 30 157
100 1000 9 7 147 93 LSD (0.05) 36 ns LSD (0.05) (rate .times.
biotype) 41 ______________________________________
Therefore, rather than extrapolate a concentration, actual leaf
area (cm.sup.2) are presented in Tables 3, 4, and 5 as well as leaf
area of the treated plants as a percent of the untreated check (0
ppb). LSD mean separation for herbicide concentration within each
biotype by herbicide are included in addition to the mean
separation for the biotype by concentration interaction.
Fifty percent growth reduction (GR.sub.50) on the resistant biotype
was reached with the highest concentration of metsulfuron, 1000 ppb
(Table 3), but not with the other sulfonylurea herbicides (Tables 4
and 5). The concentration required to produce a GR.sub.50 on the
susceptible biotype depended on the sulfonylurea herbicide (Tables
3, 4, and 5). For six of the eight sulfonylurea herbicides tested,
the GR.sub.50 value for the susceptible biotype was between 10 and
100 ppb. Prickly lettuce is less sensitive to bensulfuron and
thifensulfuron.
The prickly lettuce response to metsulfuron was similar in all
experiments (Tables 3, 4, and 5). For example, in Study 1 (Table
4), the GR.sub.50 for metsulfuron on the susceptible biotype was
between 0 and 10 ppb, while the GR.sub.50 for metsulfuron on the
resistant biotype was between 100 and 1000 ppb. In Study 2 (Table
5), the GR.sub.50 for the susceptible biotype was between 10 and
100 ppb, while a GR.sub.50 on the resistant biotype was not reached
with metsulfuron. Response to metsulfuron between the two
susceptible biotypes, Moscow and Troy did not differ.
The response of the resistant and susceptible biotypes varied among
imidazolinone herbicides (Table 6). The GR.sub.50 values for the
resistant biotype treated with imazapyr and imazethapyr were more
than 4 times the GR.sub.50 for the susceptible biotype. Both
biotypes were equally affected by imazaquin.
TABLE 6 ______________________________________ Leaf Area and
Percent of Control Leaf Area Susceptible and Resistant Biotypes of
Prickly Lettuce Treated with Three Imidazolinone Herbicides at Six
Rates in the Greenhouse Biotype Susceptible Resistant Rate (% of (%
of Herbicide (ppb w/v) (cm.sup.2) Control (cm.sup.2) Control)
______________________________________ Imazapyr 0 152 100 152 100
500 122 80 124 82 1000 99 65 128 84 2500 31 20 113 74 5000 13 9 91
60 10000 11 7 65 43 25000 9 6 23 15 LSD (0.05) 49 44 LSD (0.05)
(rate .times. biotype) 34 Imazaquin 0 137 100 107 100 500 118 86
119 111 1000 124 91 85 79 2500 86 63 51 48 5000 42 31 26 24 10000
23 17 21 20 25000 11 8 13 12 LSD (0.05) 44 25 LSD (0.05) (rate
.times. biotype) ns Imazethapyr 0 165 100 158 100 500 168 100 139
88 1000 137 83 121 77 2500 90 55 124 78 5000 83 50 108 68 10000 47
28 123 78 25000 28 17 73 46 LSD (0.05) 68 ns LSD (0.05) (rate
.times. biotype) ns ______________________________________
Results from these experiments showed the prickly lettuce biotype
from Lewiston was resistant to sulfonylurea herbicides. This
biotype showed cross resistance to the eight sulfonylurea
herbicides tested but not to broadleaf herbicides with different
sites of action which were tested. Resistance of a prickly lettuce
biotype to sulfonylurea herbicides may or may not indicate
resistance to an imidazolinone herbicide. These whole plant
responses are similar to results reported by Saxena et al.,
"Herbicide resistance in Datura Innoxia," Plant Physiol. (1988)
86:863-867 for Datura Innoxia cell culture.
EXAMPLE 2
Incorporating Sulfonylurea Herbicide Resistance into Domestic Bibb
Lettuce
1. Bibb Lettuce
Incorporation of sulfonylurea herbicide resistance into domestic
Bibb lettuce was accomplished as shown in the following schematic.
RPL=sulfonylurea herbicide resistant prickly lettuce; RF or RBC
indicate resistant progeny. ##STR1## Bibb lettuce seed (Lot No.
58387) were obtained from Charles H. Lilly Co., Portland, Oreg.
Method
Prickly lettuce seeds were gathered in the fall of 1987 from field
plots 15 km south of Lewiston, Id. treated with sulfonylurea
herbicides and also from an area near Troy, Id. that never had been
treated with sulfonylurea herbicides (Mallory-Smith, et al., Weed
Technol. (1990) H: (in press). Seeds were planted in a greenhouse
soil mix (75% Manitoba peat, 15% perlite, and 10% vermiculite) in
440-ml styrofoam cups and germinated in a growth chamber at
18.degree. C. Seedlings were transplanted into 4.4L containers when
they had 3 to 5 leaves. The plants were grown to maturity in a
greenhouse under the following conditions: 16-hr photoperiod with a
combination of high pressure sodium and multiple vapor halogen
lights, and a temperature range of 15.degree.-27.degree. C.
Subsequent generations were grown under the same greenhouse
conditions as the parents.
For crossing, a susceptible biotype or Bibb lettuce pollen acceptor
flowers were selected when the petals either were just visible or
had opened slightly. These flowers were sprayed two or three times
with a fine stream of distilled water to remove the pollen (Oliver,
"New methods of plant breeding," U.S. Bureau Plant Ind. (1910)
Bulletin 167). The flowers were air dried. An open flower from the
resistant biotype was used as the pollen donor. Pollen was placed
on the pollen acceptor flowers by brushing the anthers against the
stigma of the washed flower. Reciprocal crosses were made using the
susceptible biotype as the male parent. Mature seeds were collected
by hand from individual crosses and placed in dry storage at room
temperature.
Prickly lettuce is an obligate self-fertilizing species (Ryder,
"Lettuce breeding," Breeding Vegetable Crops, edited by M. J.
Bassett AVI Publishing Co., Westport, Conn. (1986) pp 436-472).
Since it was not possible to visually ascertain if a successful
cross had been made, it was necessary to treat the F.sub.1
seedlings with metsulfuron to determine if the resistance trait had
been crossed into the susceptible biotype. Those plants that
survived the treatment were considered to be successful crosses.
Since there was no marker, it was impossible to distinguish
successfully crossed individuals from self-fertilized individuals
in the reciprocal crosses (S biotype as pollen donor) and the
screening technique could not be used to determine if the cross had
been made because the selfed individuals would be resistant as well
as the hybrids.
In order to test the effects of herbicide treatment, plants were
grown under the conditions previously described. At least 100
plants were used in each study. A 30 ml 500 ppb ai w/v metsulfuron
foliar and soil drench treatment was applied at the 2 to 3 leaf
stage of growth to the parents (resistant=R, susceptible=S), the
F.sub.1 generation and the F.sub.2 population of the S.times.R
cross. The F.sub.2 population of the Bibb.times.R cross and the
F.sub.3 populations of both crosses were treated with 13 g ai/ha (3
times the commercial rate) of metsulfuron applied through a custom
built .[.CO2.]. .Iadd.CO.sub.2 .Iaddend.pressurized greenhouse
spray chamber. Treatments were applied at 275 kPa, 300 L/ha, and
2.5 km/h. A non-ionic surfactant (octyl phenoxy polyethoxy ethanol,
90% ai) at 0.5% v/v was added to all herbicide treatments.
The F.sub.1 plants that survived the herbicide treatment were
transplanted into 4.4L containers and allowed to self to produce
F.sub.2 seed. The F.sub.2 seedlings in the 3 to 5 leaf stage were
treated with herbicide as described previously. The plants were
scored as susceptible (S), intermediate (I) or resistant (R) in
their response to metsulfuron. The F.sub.2's scored as I or R were
transplanted into 4.4L containers and grown to maturity for F.sub.3
seed production. The herbicide treated F.sub.3 population was
evaluated for segregation of the resistance trait. The best fit for
Chi-Square analysis of the F.sub.2 generation of both crosses was a
1:2:1 ratio indicating the trait was controlled by a single nuclear
gene with incomplete dominance (Tables 7 and 8).
TABLE 7 ______________________________________ Chi-Square Analysis
for the F.sub.2 Generation Susceptible by Resistant Prickly Lettuce
Biotype Cross Ratio Exp Obs X.sup.2
______________________________________ 1 29 25 0.5517 2 58 66
1.1034 1 29 25 0.5517 X.sup.2 = 2.2068 0.25 < P < 0.50
______________________________________
TABLE 8 ______________________________________ Chi-Square Analysis
for the F.sub.2 Generation Bibb Lettuce by Resistant Prickly
Lettuce Biotype Cross Ratio Exp Obs X.sup.2
______________________________________ 1 20.75 22 0.0753 2 41.50 41
0.0060 1 20.75 20 0.0271 X.sup.2 = 0.1084 0.90 < P < 0.95
______________________________________
The F.sub.3 generations of both crosses were evaluated to confirm
the results of the F.sub.2 Chi-Square. Seeds from these F.sub.2
S.times.R prickly lettuce plants scored as resistant produced
seedlings that were resistant to the 13 g/ha treatment of
metsulfuron. Of the 116 seedlings treated, one showed symptoms and
one died. Prickly lettuce plants rated as intermediate in the
F.sub.2 generation produced seedlings which segregated as expected
with approximately one-fourth of the seedlings susceptible and
three-fourths of the seedlings intermediate or resistant (X.sup.2
=3.36, 0.05<P<0.10). The F.sub.3 generation of the
Bibb.times.R cross responded the same as the prickly lettuce
F.sub.3 generation with the seedlings from the R plants all
surviving with no symptoms and the seedlings from the I plants
segregating 1:2:1 (X.sup.2 =0.28, 0.75<P<0.90).
The response of the F.sup.3 generation supports the hypothesis that
the R plants were homozygous for the resistance trait and the I
plants were heterozygous for the trait. These results are similar
to those reported in the literature for resistant plants produced
by mutagenesis where the trait has been reported to be either
dominant (Haughn et al., "Sulfonylurea-resistant mutants of
Arabidopsis thaliana," Mol. Gen. Genet. (1986) 204:430-434) or
semidominant (Chaleff et al., "Herbicide-resistant mutants from
tobacco cell cultures," Science (1984) 223:1148-1151).
2. Prizehead Lettuce
Domestic head lettuce var. Prizehead (Lot No. 68319) seed were
obtained from Charles H. Lilly Co., Portland, Oreg. Incorporation
of sulfonylurea herbicide resistance into domestic prizehead
lettuce was accomplished as shown in the following schematic.
##STR2## The methods used to obtain transgenic plants were as
described above for Bibb lettuce.
3. Grand Rapids Lettuce
Domestic leaf lettuce var. Grand Rapids seed (Lot No. 68180) were
obtained from Charles H. Lily Co., Portland, Oreg. Incorporation of
sulfonylurea herbicide resistance into domestic Grand Rapids
lettuce was accomplished as shown in the following schematic.
##STR3## Grand Rapids seed (Lot 68180) were obtained from Charles
H. Lilly Co., Portland, Oreg.
The methods used to obtain transgenic plants were as described
above for Bibb lettuce.
4. Vanguard Lettuce
Incorporation of sulfonylurea herbicide resistance into domestic
Vanguard lettuce was accomplished as shown in the following
schematic. ##STR4##
Vanguard seed obtained from Asgrow Seed Company, P.O. Box L, San
Juan Bautista, Calif. 95045.
The methods used to obtain transgenic plants were as described
above for bibb lettuce.
5. Ithica Lettuce
Incorporation of sulfonylurea herbicide resistance into domestic
lettuce var. Ithica was accomplished as shown in the following
schematic.
Ithica seed were obtained from Asgrow Seed Company, P.O. Box L, San
Juan Bautista, Calif. 95045.
The methods used to obtain transgenic plants were as described
above for Bibb lettuce.
6. Empire Lettuce
Incorporation of sulfonylurea herbicide resistance into domestic
lettuce var. Empire was accomplished as shown in the following
schematic. ##STR5##
Empire seed were obtained from Asgrow Seed Company, P.O. Box L, San
Juan Bautista, Calif. 95045. The methods used to obtain transgenic
plants were as described above for Bibb lettuce.
7. Salinas Lettuce
Incorporation of sulfonylurea herbicide resistance into domestic
lettuce var. Empire was accomplished as shown in the following
schematic. ##STR6##
Salinas seed were obtained from Asgrow Seed Company, P.O. Box L,
San Juan Bautista, Calif. 94045.
The methods used to obtain transgenic plants were as described
above for Bibb lettuce.
EXAMPLE 3
Field Performance of Resistant Bibb Lettuce
A study was established in order to evaluate BC.sub.2
(Bibb.times.Sulfonylurea Herbicide Resistant Prickly Lettuce
crosses) and parent Bibb plants responses to DPX-R9674 in a field
situation. Transgenic plants and parent Bibb lettuce plants were
transplanted into the field in a randomized complete block design
with four replications. Each plot contained 10 plants. Two rates of
DPXR9674, 0.0313 and 0.0939 lb ai/a, were applied after the plants
became established in the field.
BC.sub.2 plants were evaluated visually and rated as susceptible or
resistant in their response to the herbicide (Table 9). The plants
segregated 3:1 as was expected based on previous greenhouse
studies. The resistance trait is inherited as a single, nuclear
gene with incomplete dominance. The plants responded the same in
the field as they had in the greenhouse.
TABLE 9 ______________________________________ Chi-Square Analysis
for the BC.sub.2 .times. Prickly Lettuce Cross Ratio Exp Obs
X.sup.2 ______________________________________ 1 17.5 19 0.057 3
52.5 51 0.019 0.076 0.7 < P < 0.9
______________________________________
Hybrid crop plants have been obtained having resistance to
sulfonylurea herbicides which will allow them to be grown in
rotation with crops for which sulfonylurea herbicides are the
treatment of choice for weed control. Additionally, seed producers
will be able to maintain varietal purity between resistant and
susceptible lettuce varieties.
All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
* * * * *