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IV. Genetic Improvement of Crop Tolerance to Herbicides

IV. Genetic Improvement of Crop Tolerance to Herbicides

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the development of resistant biotypes of a weed that was formerly susceptible to a given herbicide. The ability of these biotypes to grow in the presence

of a herbicide is termed resistance. In contrast to plant tolerance, however,

plant resistance is characterized by the ability of resistant biotypes to survive and grow at any feasible rate of application of a specific herbicide and

not only at its agriculturally recommended rates (Duessing, 1984).

In general, it is believed that herbicide tolerance and/or resistance is

naturally present in plant systems before the introduction of a particular

herbicide (Gressel, 1986; LeBaron, 1984). Moreover, physiological resistance and tolerance to any herbicide can only develop within the framework

of metabolic processes that are present in the plant cell (Duessing, 1984;

Gressel, 1986). Extensive biochemical investigations on the development of

plant resistance and tolerance to several herbicides has identified the involvement of five major mechanisms (Duessing, 1984; Hatzios and Penner,

1982). These mechanisms include the following:

1. altered uptake and translocation or compartmentation of the herbicide

in the resistant or tolerant plants

2. extensive metabolic detoxification of the herbicide in the resistant or

tolerant plants

3. modification of the target site of a herbicide in the resistant or tolerant


4. increased production of a target enzyme in the resistant or tolerant


5 . increased synthesis of substrates able to reverse the herbicide-induced

inhibition of growth in the resistant or tolerant plants

A brief discussion of these mechanisms including the citation of selected examples will be given in the following paragraphs. Our present knowledge of

the genetic basis of these mechanisms is also discussed. This information is

vital for a better understanding of the discussion on the application of

biotechnological techniques for the improvement of crop tolerance to herbicides that will be given later.

I . Altered Uptake, Translocation, or Compartmentation

Resistant or tolerant plants in this class survive by preventing the herbicide

from reaching its site of action (target site). Use of radiolabeled herbicides

has demonstrated modified translocation and vascular compartmentation

as the cause for the observed tolerance of cucumber (Cucumissativus L.) to

the herbicide atrazine (Werner and Putnam, 1980), horseweed [Conyza

linefolia (L.) Cronq.] to paraquat (Fuerst et al., 1985), and soybean to

metribuzin (Falb and Smith, 1984). Autoradiograms for all three plant



species clearly showed that herbicide translocation in atrazine-resistant

cucumber, paraquat-resistant horseweed, and metribuzin-resistant soybean

was limited to leaf veins and insufficient amounts of each herbicide reached

the mesophyll cells containing the target site (chloroplast) of these

photosynthesis-inhibiting herbicides. Alternative mechanisms conferring

tolerance to other plants against these herbicides are also available (Gressel,

1986; Hatzios and Penner, 1982).

2. Metabolic Detoxification

The selective activity of most of the currently marketed herbicides is dependent on their differential metabolism by tolerant and susceptible plant species

(Hatzios and Penner, 1982). Tolerant plants are able to render a phytotoxic

molecule inactive by enzymatic or nonenzymatic conversion before the active

herbicide reaches its site of action. Corn, for example, is tolerant to atrazine

because of its ability to metabolize this herbicide by conjugating it to the

tripeptide glutathione (GSH) (Frear and Swanson, 1970; Hatzios and Penner,

1982). The reaction is enzymatic, being catalyzed by an atrazine-specific

GSH-sulfotransferase (Frear and Swanson 1970; Guddewar and Dauterman,

1979). Most (but not all) of the available corn hybrids used today possess a

dominant gene for the biosynthesis of the atrazine-specific GSH-Stransferase. Additional mechanisms for the metabolic detoxification of

atrazine in corn or other plants include hydrolytic and dealkylation reactions,

and these may be the causes of the increased tolerance to atrazine in some of

the weed species that do not possess plastid level tolerance to this herbicide

(Gressel et al., 1983; Hatzios and Penner, 1982). Gressel et al., (1983)

reported that biotypes of the weed Senecio vulgaris which developed

resistance to atrazine at the chloroplast level had also coevolved much higher

levels of degradative enzymes catalyzing N-dealkylation of atrazine.

In addition to the atrazine-specific GSH-sulfotransferase, corn hybrids

possess a number of other GSH-sulfotransferase isozymes which are

specific for detoxifying the herbicides alachlor (Mozer et al., 1983) and

EPTC (Lay and Niland, 1985). The important function of glutathione and

GSH-S-transferase enzymes in herbicide metabolism by higher plants has

been reviewed (Hatzios and Penner, 1982). GSH-S-transferase (GST) enzymes have been identified and partially characterized from leaves and

roots of several plant species (Diesperger and Sandermann, 1979; Edwards

and Owen, 1986; Frear and Swanson, 1970; Guddewar and Dauterman,

1979; Mozer et al., 1983). At least three isozymes, designated as GST I,

GST 11, and GST 111, differing in their purification characteristics and

substrate preference, have been identified in etiolated corn tissue (Mozer et

al., 1983). GST I is expressed constitutively in corn roots and leaves and is



capable of conjugating glutathione to chloroacetanilide herbicides such as

alachlor. Treatment of corn seeds with chemical herbicide safeners used to

protect crop plants from injury, such as flurazole, increased the activity of

GST I and induced the appearance of a novel GST activity (GST 11) which

has greater detoxification activity on these herbicides (Mozer et al., 1983).

The structural analysis of a corn gene coding for GST I has been reported

by Shah et al. (1986a). The inbred corn line Missouri 17 contained a single

gene for GST I, whereas the hybrid line 3780A contained two genes (Shah et

al., 1986a). The amino acid sequence of the corn GST I gene showed no apparent sequence homology with the published sequences of animal GSTs

(Shah et al., 1986a).

Table IV presents selected examples of GSTs and other plant enzymes

that have been reported to play a key role in the metabolic detoxification of

several important herbicides. The purification and properties of 0- or

Table IV

Plant Enzymes Known to Metabolize Selected Herbicides



Plant source


Monuron, diuron











Cotton, soybean

Peas, cucumber



Frear et al. (1969)

Makeev et al. (1977)

Corbett and Corbett (1983)

Guddewar and Dauterman

(1 979)

Mozer et at. (1983);

Edwards and Owen (1986)

Frear et 01. (1983a)






Frear and Swanson (1973);

Diesperger and

Sandermann (1979)

Frear et al. (1985)



Fedtke (1983)

Frear et al. (1983b)


Frear and Still (1968)

Gaynor and Still (1983)

Frear (1968)
















Soybean, wheat

Schmitt et at. (1985);

Schmitt et al. (1986)


34 1

N-glucosyl and 0- or N-malonyl transferases from wheat and soybeans

which conjugate the 4-hydroxy derivative of 2,4-D and substituted

chloroanilines, respectively, have been reported recently by Schmitt et al.

(1986). The isolation and characterization of cDNA clones coding for plant

enzymes which detoxify herbicides in higher plants (Table IV) will facilitate

molecular genetic manipulations of these enzymes in the future.

In addition, the potential transfer of bacterial genes which detoxify herbicides into plant genomes, following the modification of such genes for expression in plants, should be exploited for increasing the crop selectivity of

herbicides. A recent example illustrating the potential of this approach is

the isolation of a bacterial gene from Streptomyces sp. which codes for an

enzyme capable of acetylating phosphinothricin, the active ingredient of the

herbicides bialophos and glufosinate (Newmark, 1987). Acetylated phosphilothricin does not inhibit the activity of the enzyme glutamine synthetase. The bacterial gene coding for the enzyme which acetylates phosphinothricin has been engineered into several crop plants, such as tomato,

tobacco (Nicotiana tabacum L.), and potato (Solanum tuberosum L.),

making them tolerant to this herbicide (Newmark, 1987).

3. Modifcation of Herbicide Target Sites

To exert their phytotoxic action most herbicides have to bind to a specific

receptor site, which is usually a protein or an enzyme involved in plant

metabolism. Resistance to such herbicides could develop if proteins or enzymes can be modified to discriminate functionally between the herbicide

and the normal substrate for the binding site. Several examples have been

reported in the literature that illustrate the importance of this mechanism in

the development of resistance to selected herbicides.

a. Plastid-Mediated Resistance to Triazine Herbicides. The most extensively studied example is the development of weed biotypes resistant to the

herbicide atrazine. Following repeated applications of this herbicide in corn

fields of the midwestern United States and of other countries, several weed

biotypes have become tolerant to this herbicide through a plastid-mediated

mechanism of resistance (LeBaron, 1984; LeBaron and Gressel, 1982).

Atrazine is a photosynthesis-inhibiting herbicide known to act on a

plastoquinone-binding membrane protein of 32 kilodalton (kd) termed

“QBprotein” (Arntzen et al., 1983). The QBprotein is the site of herbicide

binding and serves as the second stable electron acceptor of photosystem I1

of the photosynthetic apparatus located in the chloroplast of higher plants

and green algae. Weed biotypes that have developed atrazine resistance at

the chloroplast level share the following common traits:

1. Photosynthetic electron transport in resistant chloroplasts is



insensitive to inhibition by any triazine herbicide.

2. A high-affinity triazine binding site cannot be detected in thylakoid

membranes from resistant chloroplasts.

3. The chloroplast-associated resistance is inherited maternally.

The QB protein is coded for by the psbA gene, which has been sequenced

from several atrazine-sensitive plants such as spinach (Spinacea oleracea L.)

(Zurawski et al., 1982), tobacco (Nicotiana debeneyi) (Zurawski et al.,

1982), and soybean (Spielman and Stutz, 1983) and from atrazine-resistant

weeds such as Amaranthus hybridus (Hirschberg and McIntosh, 1983),

Solanum nigrum (Golubinoff et al., 1984), and Sinapis alba (Link and

Langridge, 1984). In addition, the psbA gene has been sequenced from

atrazine-sensitive and atrazine-resistant green algae and cyanobacteria

(Curtis and Haselkorn, 1984; Erickson et al., 1984; Golden and Sherman,

1984; Golden and Haselkorn, 1985; Karabin et al., 1984; Mets et al., 1986).

The sequences of the psbA gene from several species have demonstrated

that the 32-kd Q B protein is very highly conserved. The resistance of higher

plants, green algae, and cyanobacteria to herbicides acting as inhibitors of

electron transport mediated by photosystem I1 (e.g., triazines, ureas) has

been correlated with a single mutation of the chloroplast psbA gene.

Nucleotide changes resulting in the substitution of selected amino acids

located at positions 219-275 of the psbA gene product are currently viewed

as responsible for the observed plastid-mediated resistance to these herbicides (Fedtke and Trebst, 1987; Arntzen, 1986). In most of the studied

cases, the mutated QB protein is rendered insensitive to triazine herbicides as

a result of a single nucleotide change that replaces serine at position 264 of

the QB protein of susceptible weed biotypes with glycine (Arntzen, 1986;

Mets et al., 1986). In the cyanobacterium Anacystis nidulans, resistant

mutants originated from a change of the serine at 264 to an alanine at the

same position (Golden and Haselkorn, 1985).

The molecular structure of the photosynthetic reaction center of the purple

bacterium Rhodopseudomonas viridis was recently elucidated, using X-ray

crystallography analysis, by Michel’s group at the Max Planck Institute in

West Germany (Deisenhofer et al., 1985). Following this first description of

the high-resolution structure of bacterial membrane protein D,, which corresponds to the QB protein of higher plants, a number of models describing

the herbicide binding domain of the Q B protein and showing the location of

amino acids which have been identified as being altered in herbicide-resistant

membranes have been constructed by Arntzen (1986) and Trebst (1986). Such

models facilitate predictions of mutations which could impart herbicide

resistance not obtained from random mutagenesis.

The psbA gene from resistant weed biotypes or green algae and

cyanobacteria has been cloned and is considered very promising for the



transfer of atrazine resistance to susceptible crop plants such as soybeans

(Gressel, 1985, 1986; Sandermann, 1985). Mets et al. (1986) reported recently

that in genetically mixed chloroplasts, the resistance alleles of the psbA gene

appear to be recessive. They suggested that the exploitation of psbA in the

engineering of plant resistance via gene transfer techniques will most likely require the replacement or inactivation of the endogenous sensitive allele before

useful resistance will be expressed. An alternative approach could involve the

increase of the product of the resistant gene relative to the sensitive protein by

increasing the expression of the resistant gene with an altered promoter.

b. Resistance to Glyphosate. Glyphosate, a nonselective herbicide, is

known to inhibit an enzyme of the shikimic acid pathway, 5-enolpyruvylshikimate-3-phosphate synthase (Amrhein et al., 1983; Steinrucken and

Amrhein, 1980). The enzyme, commonly abbreviated as EPSP synthase, has

been highly purified from Escherichia coli and from pea (Pisum sativum L.)

seedlings (Duncan et al., 1984; Mousdale and Coggins, 1984). The microbial

enzyme is coded for by the aroA gene, which has been cloned from E. coli

(Comai et al., 1983) and Salmonella typhimurium (Rogers et al., 1983). The

mechanism of glyphosate-resistant strains of Salmonella and Aerobacter

aerogenes (Schultz et al., 1984) has been shown to involve a modification of

the target EPSP synthase resulting in a greatly reduced affinity of the enzyme

for glyphosate. Molecular studies with the glyphosate-resistant aroA allele

obtained from mutagenized Salmonella showed that a single base pair change

resulting in a proline-to-serine amino acid substitution at the lOlst amino acid

of the protein was responsible for the development of resistance to this herbicide (Stalker et al., 1985). Attempts to introduce the cloned aroA gene resistant to glyphosate to sensitive crop plants have been reported (Comai et al.,

1985) and they will be discussed in the next section.

c. Resistance to Sulfonylurea and Imidazolinone Herbicides. Although

structurally unrelated, the sulfonyulurea and imidazolinone classes of herbicides have a similar mechanism of phytotoxic activity which involves the inhibition of the enzyme acetolactate synthase (Chaleff and Mauvais, 1984;

Shaner et al., 1984). This is the first specific enzyme for the biosynthesis of

the branched-chain amino acids, valine, leucine, and isoleucine, in higher

plants and microorganisms (Chaleff and Mauvais, 1984; LaRossa and

Schloss, 1984). Resistance to sulfonylureas and imidazolinones is accomplished by a modification of the target enzyme in resistant mutants which renders

it insensitive to these herbicides (LaRossa and Smulski, 1985; Shaner and

Anderson, 1986). Several isozymes of acetolactate synthase (ALS),

designated as ALS I, ALS 11, and ALS 111, are known to exist in

microorganisms (LaRossa and Schloss, 1984). The microbial enzyme from

the enteric bacteria Salmonella typhimurium and E. coli which is mutated to

yield resistance to these herbicides is coded by the ilvB gene (LaRossa and

Smulki, 1985). Tobacco mutants resistant to the sulfonylurea herbicides



chlorsulfuron and sulfometuron-methyl have been isolated recently by direct

selection in tissue culture or by selection following mutagenesis (Chaleff and

Ray, 1984; Chaleff, 1986). The resistance of tobacco to sulfonylurea herbicides

is associated with two loci, and the obtained mutants are commonly designated

as SURA and SURB (Chaleff, 1986). The resistance of yeast to sulfonylurea

herbicides has been characterized by molecular biology precedures, revealing an

altered structural gene for A L S encoding a resistant A L S protein in which a proline amino acid residue in the sensitive A L S is replaced by a serine (Hardy, 1986).

d. Resktance to Dinitroaniline Herbicides. The existence of a biotype of

goosegrass [Eleusine indica (L.) Gaertn.] which developed resistance to the

dinitroaniline herbicide trifluralin following repeated treatments with this herbicide has been reported by Mudge et al. (1984). This biotype was also resistant

to other dinitroaniline herbicides tested (Mudge et al., 1984). Vaughn (1986)

reported recently that an alteration in the target protein involved in the action

of the dinitroaniline herbicides is responsible for the development of resistance

in weed biotypes of goosegrass. Biochemical analysis showed that the

dinitroaniline-resistant biotypes of goosegrass had a tubulin subunit with an

altered mobility as compared to the sensitive biotype (Vaughn, 1986).

4. Increased Production of a Target Enzyme

Increased activity of a target enzyme is another mechanism that may confer

resistance to selected herbicides. Such a mechanism has been demonstrated

empirically with glyphosate-resistant mutants of Aerobacter and cultured

plant cells (Amrhein et al., 1983). In E. coli, overexpression of the enzyme

EPSP synthase was achieved in a controlled way by placing the normal aroA

gene on a multicopy plasmid and then transferring it back into the host

bacteria (Rogers et al., 1983). These bacteria then make nearly 100-fold more

EPSP synthase compared to wild-type strains which are susceptible to the

herbicide glyphosate. Increased EPSP synthase levels have been also achieved

by gene duplication following the use of sequential increases in glyphosate

concentrations in plant tissue culture media. Glyphosate-resistant cultures of

carrot (Daucus carota L.) and of Corydalissemprevirens have been obtained

by this procedure (Amrhein et al., 1983; Widholm, 1984).

As mentioned earlier (Section 111), L-phosphinothricin (the active ingredient of the herbicide’s bialophos and glufosinate) is a mixed competitive

inhibitor of the enzyme glutamine synthetase (GS), which plays a central

role in the nitrogen metabolism of higher plants. Donn et al. (1984) selected

several suspension cell lines of alfalfa that were 20- to 100-fold more resistant to this herbicide. GS enzyme levels were three- to seven-fold elevated in

the variant cell line, suggesting that resistance to L-phosphinothricin was

due to an amplification of a glutamine synthetase gene and overproduction

of the glutamine synthetase enzyme (Donn et al., 1984). The nucleotide



sequence of the amplified alfalfa glutamine synthetase gene was characterized by Tischer et al. (1986). Several regions of homology were observed when

the sequence of this gene was compared to glutamine synthetase sequences

from Anabaena and Chinese hamster (Tischer et al., 1986). The minimum

size of a glutamine synthetase gene amplification unit in the alfalfa tissue

culture line which was resistant to L-phosphinothricin was determined to be

35 kb (Tischer et al., 1986).

Overproduction of acetolactate synthase due to the presence of the IL V2

and ilvG genes, which encode ALS in yeast or E. coli, respectively, has been

reported as an alternative cause of resistance development to the sulfonylurea herbicide sulfometuron-methyl (Falco and Dumas, 1985).

5. Increased Synthesis of Substrates Able to Reverse

the Herbicide-Induced Inhibition

Many of the currently used herbicides resemble structurally normal

cellular substrates and they may function as antimetabolites by mimicking

the structural features or chemical properties of these natural substrates.

The inability of an enzyme or protein to discriminate between the normal

substrate and the herbicide may be responsible for the action of this herbicide (Duessing, 1984). Potentially, a cell could tolerate this type of herbicide action by increasing the intracellular concentration of the natural

substrate. Specific examples demonstrating the involvement of this

mechanism in the development of crop or weed resistance to a particular

herbicide have not yet been reported. The selection, however, of cultured

mutant cell lines of corn (Hibberd et al., 1980), rice (Schaeffler and Sharpe,

1981), tobacco (Widholm, 1976), and carrot (Cella and Iadarola, 1983) that

are resistant to amino acid analogs illustrates the feasibility of this

mechanism. Plant tissue cultures of these resistant mutants have yielded

plants with 10- to 50-fold higher levels of the competing amino acid compared to the susceptible cells (Hibberd et al., 1980; Cella and Iadarola,







Our current knowledge of the mechanisms conferring resistance or

tolerance to all marketed herbicides is far from complete. Recent advances,

however, in our understanding of the biochemical and physiological factors

that render selected plant species resistant to specific herbicides and the

genetic basis of these factors have stimulated considerable interest in the

utilization of genetic manipulations for the improvement of crop tolerance

to given herbicides. Extensive research during the last decade has resulted in



the isolation and characterization of several plant and microbial genes

which regulate the tolerance or resistance of plants to particular herbicides.

Specific examples were presented and discussed in the previous section of

this review.

Uptake, translocation, and compartmentation of selected herbicides in

higher plants are complex physiological processes often determined by

multiple genes. The genetic exploitation of such systems to alter the uptake,

translocation, and compartmentation of selected herbicides and confer crop

tolerance to these herbicides is extremely difficult (Comai and Stalker,


Genetic analyses of crop tolerance to herbicides arising from herbicide

detoxification have shown that this type of resistance is dominant and is

often expressed by a single gene (Comai and Stalker, 1984; Hatzios and

Penner, 1982; Martin, 1985). Identification and characterization of genes

encoding for enzymes involved in herbicide detoxification, as well as advances in our understanding of the processes involved in the regulation of

such genes, offer attractive opportunities for the manipulation of crop

tolerance to herbicides. Selected plant enzymes catalyzing the metabolic

detoxification of specific herbicides are listed in Table IV. In most cases,

however, the genes coding for these enzymes have not been characterized. A

report by Shah et al. (1986a) represents the first successful attempt at cloning and sequencing a plant gene coding for the enzyme GST I involved in

the detoxification of chloroacetanilide herbicides in corn. In a follow-up

study, Wiegand et al. (1986) showed that the expression of this gene can be

regulated chemically with the use of exogenously applied herbicide safeners.

Safeners such as flurazole were shown to act at the transcriptional level, inducing a three- to four-fold increase in the steady state level of the mRNA

encoding the GST I enzyme in corn tissues grown from flurazole-treated

seeds (Wiegand et al., 1986).

Isolation of bacterial genes coding for enzymes detoxifying herbicides

should be also exploited for improvement of crop tolerance to herbicides. A

number of bacterial genes have now been successfully introduced and expressed in plants by using plant promoters, signal sequences, and poly(A) +

signals, etc. (Fraley et al., 1986a). The initial isolation and subsequent in

vitro mutagenesis and selection of resistance may be easier in bacteria than

in higher plants. The aforementioned successful transfer of a degradative

gene from Streptomyces sp., which acetylates phosphinothricin into

solanaceous crops, illustrates the potential of this approach (Newmark,


In situations in which herbicide detoxification results from a chain of

metabolic reactions rather than a single reaction, genetic manipulation of

herbicide tolerance would be more complicated. Genes encoding for all

metabolic enzymes degrading a herbicide and its metabolites would be



needed, resulting in the development of oligogenic resistance systems

(Comai and Stalker, 1984).

Plant resistance to herbicides accomplished by a modification of the herbicide target site or increased production of target enzymes and/or

substrates is often determined by single genes. Such systems are far more

desirable for exploitation because their genetic manipulation would require

the transformation and transfer of a single gene. In fact, the feasibility of

engineering this mechanism of herbicide tolerance is viewed currently as being greater than that of any other agriculturally important trait (Comai and

Stalker, 1984; Hardy, 1986; Netzer, 1984).

Generation of herbicide tolerance or resistance is of immediate scientific

interest because it provides us with a direct selection marker in studying

plant gene expression and regulation. In addition, the introduction of genes

confering herbicide resistance to economic crops could be utilized to improve the agricultural uses of currently registered herbicides.

Crop plants resistant to herbicides can be engineered genetically by four

major approaches including (1) classic plant breeding techniques and mutation breeding; (2) in vitro mutant selection at the cell or tissue level; (3) mutant selection by somatic hybridization; and (4) transfer of cloned genes into

susceptible crop plants.

1. Classic Plant Breeding Techniques and Mutation Breeding

Numerous examples illustrating the genetic variability of the responses of

crops to herbicides have been observed and have been reviewed by Martin

(1985). Success in exploiting the intraspecific variation of plant responses to

herbicides by means of classic breeding approaches has been limited

(Martin, 1985; Beversdorf, 1985a). Standard breeding approaches work only for species that are sexually compatible, a condition that applies to very

few crop plants and weeds (Beversdorf, 1985b; Beversdorf et al., 1980;

Chaleff, 1981). The sources of genes utilized by plant breeders for desirable

characteristics are usually limited by the normal reproductive barriers that

distinguish species.

Classic breeding techniques have been, however, successful for the

development of triazine-resistant crop varieties. The occurrence of a

triazine-resistant biotype of bird’s rape (Brassica campestris L.) has allowed

the use of breeding for the development of commercially useful triazineresistant varieties of canola (Brassica napus L.) (Beversdorf et al., 1980;

Souza-Machado and Bandeen, 1982). Beversdorf et al. (1980) succeeded in

transferring the cytoplasmically inherited resistance to atrazine from bird’s

rape to canola by means of backcrossings between these two cross-fertile

species and selection for chromosome number. Atrazine-resistant canola

(cv. OAC Triton) is presently cultivated in Canada and approximately



200,000acres seeded with atrazine-resistant canola were expected to be produced in 1985 (Netzer, 1984). Under ideal conditions, the yield of both

atrazine-resistant and atrazine-susceptiblecanola crops is equivalent. Under

conditions of high temperature and drought, however, the yield of atrazineresistant canola is reduced by about 20% (Beversdorf, 1985a,b; Netzer,

1984). The poor agronomic performance of the triazine-resistant canola has

been a limiting factor for the further utilization of breeding for atrazine

resistance to other Cole crops and mustards (Beversdorf, 1985a,b).

Mutation breeding, which includes mutagenesis of seeds followed by

screening of seedlings for resistance, offers good promise for manipulating

herbicide resistance at the seedling or whole plant level (Chaleff, 1981). This

approach, however, is likely to require several backcrosses of the isolated,

resistant mutant lines to eliminate undesirable mutations resulting from

random mutagenesis. Early attempts by Pinthus (1972) in utilizing mutation

breeding resulted in the selection of wheat (Triticum aestivum L.) mutants

with increased seedling resistance to the herbicide terbutryne and of tomato

mutants resistant to diphenamid. Populations of these plants grown from

seeds treated with the mutagen ethyl methane sulfonate (EMS) were used in

selecting the aforementioned mutants. More recently, Sommerville et al.

(1985a) reported the successful selection of chlorsulfuron-resistant seedlings

of the small crucifer Arabidopsis thaliana following mutagenesis with EMS.

Similarly, Chaleff (1986) was successful in screening and selecting soybean

mutants with increased tolerance to the sulfonylurea herbicides chlorsulfuron and sulfometuron-methyl by means of mutation breeding.

Tolerance of these mutants to sulfonylureas was conferred by recessive

nuclear mutations which, unlike the mutations recovered in tobacco

(Chaleff and Ray, 1984), did not affect the sensitivity of the enzyme

acetolactate synthase (ALS) to these herbicides.

Although conventional selection techniques and mutation breeding are

laborious and slow processes, these successful examples illustrate the value

of these approaches to investigators attempting to genetically improve crop

tolerance to herbicides. These approaches should not be overlooked.

2. In Vitro Mutant Selection at the Cell or Tissue Level

Attempts to achieve herbicide resistant crops by using cell culture techniques have been made for more than 10 years (Gressel, 1984). Herbicides

that interfere with the metabolic activities of whole plants are expected to

inhibit the growth of cultured cells or tissues as well. In such cases,

herbicide-tolerant mutants can be selected by culturing cells or tissues in the

presence of a herbicide concentration that is toxic to normal cells.

Manipulation of large cell populations, studies on the direct interaction

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