**Herbicide Tolerant Food Legume Crops: Possibilities and Prospects**

N.P. Singh1 and Indu Singh Yadav1,2 *1Indian Institute of Pulses Research, Kanpur 2National Research Centre on Plant Biotechnology, New Delhi, India* 

#### **1. Introduction**

434 Herbicides – Properties, Synthesis and Control of Weeds

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Villarroel, M.J.; Sancho, E.; Ferrando, M.D. & Andreu-Moliner, E. (2003). Acute, chronic and

Walker, C.R. (1971). The toxicological effects of herbicides and weed control on fish and

Walker, C.H.; Hopkin, S.R.; Sibly, R.M. & Peakall, D.B. (2006). *Principles of Ecotoxicology*.

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Wendt-Rasch, L.; Pirzadeh, P. & Woin, P. (2003). Effects of metsulfuron methyl and

Wilson, D.C. & Bond, C.E. (1969). The effects of the herbicide diquat and dichlobenil

environmental chemicals. *Environ Toxicol Chem*, 26, 669-676

(http://water.usgs.gov/nawqa/pnsp/pubs/fs09200/fs09200.pdf).

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(Casoron) on pond invertebrates. Part 1. Acute toxicity. *Trans Am Fish Soc*, 98, 438-

Weeds are one of the major problems in agriculture. Weeds compete with other crops for water and nutrients and, as a result, decrease yields and productivity. Without weed control it is extremely difficult to harvest crops. The advent of mechanization replaced much of the hand labour in the developed world as well as the developing parts of the third world. Mechanical weed control is fraught with high-energy costs, facilitates soil erosion and compaction and has been mostly replaced by chemical weed control using herbicides (Gressel J, 2000). As countries industrialize and develop economically, cheap farm labour becomes unavailable, thus increasing the necessity for cost-effective chemical weed control. In India, weeds cause the highest loss (33%) followed by pathogens (26%), insects (20%), storage pests (7%), rodents (6%) and others (8%). It has been estimated that the potential losses due to weeds in different field crops would be around 180 million tonnes, valued at Rs. 105,0000 millions annually (Anonymous, 2008). Globally, herbicide constitutes 50 percent of the total pesticides sale and in some countries like USA, Germany and Australia; the figure is as high as 60-70 percent. In India, however, the position is different as herbicides form a meager 15 percent of the total pesticide consumption. But still, the consumption has increased rapidly from 4100 metric tons (MT) in 1988-89 to 13,764 MT in 2004 and it is likely to further increase in future (Varshney and Mishra, 2008). Given the harmful economic implications of poor weed management, it is hardly surprising that herbicide production is a main driver of the agrochemical industry. Too often there is no selective chemical that can control a particular weed in a particular crop, as most selectivity between crop and weed are due to catabolic degradation of the herbicide by the crop. Therefore, closely related weeds are to be expected to have similar catabolic pathways as the crop and thus escape the chemical effect. This is one major reason that genetically modified herbicide-resistant crops (GM-HRC) have become so useful, and that biotechnology has been utilized to produce such crops as well as to find new herbicide targets. Selectivity can be enhanced by inserting exogenous resistance genes into the crops or by selecting natural mutations. However, one major concern about transgenic herbicide resistant crops (HRCs) is that the transgene could genetically introgress into related weeds, and make them resistant and therefore, their careful management comes into account.

Herbicide Tolerant Food Legume Crops: Possibilities and Prospects 437

found and marketed, they become research tools of the physiologists and biochemists, first to find a site of action and then as 'anti-metabolites' to further understand and modulate metabolic pathways. Thus the advent of 2,4-D assisted in understanding auxin action, atrazine and diuron (DCMU) in understanding photosystem II, paraquat for photosystem I, dinitroanilines in tubulin to microtubule assembly, dichlobenil for cellulose biosynthesis, etc. Herbicides are the anti-metabolites of choice in dealing with key enzymes such as glutamine synthase [glufosinate (phosphinothricin)], acetolactate synthase (ALS) (many herbicides), acetyl-CoA carboxylase (ACCase) (many herbicides), dihydropteroate synthase (asulam), enolpyruvate-shikimate phosphate synthase (EPSP) (glyphosate) and phytoene desaturase (many herbicides). The genes for most of these enzymes have been isolated and used in transgenic programs. Such research transcended plant biochemistry and agriculture. For example, it was discovered through comparative genomics that plant and trypanosome β-tubulins were similar to each other and different from mammalian β-tubulin. The dinitro-aniline herbicides then proved to be excellent trypanocides (Chan *et al*., 1993; Bell, 1998). The repetitive (mis) use of single herbicides in monoculture over many years predictably leads to the evolution of herbicide-resistant weeds (Gressel & Segel, 1978). The advent of triazine resistance was crucial to the understanding of the role of the *psb*A gene product in the photosystem II binding site, leading to innumerable studies of photosynthesis, biophysics and biochemistry correlated with molecular structure of the gene product. The mutant and natural *psb*A gene products were crystallized and analyzed, leading to new insights into 'drug' (ligand) binding and design (Michel & Deisenhofer, 1988; Deisenhofer & Michel, 1989). Information from herbicide resistance provided the theoretical underpinning for designing transient drought resistant plants. Harvey and Harper (1982) first promoted the idea that paraquat resistance can be similar to oxidative stress tolerance. This was later extrapolated to being similar to transient drought tolerance (Malan *et al*., 1990). This has allowed developing quick pre-tests with paraquat to ascertain the level of transient drought tolerance of transgenic plants bearing genes designed to confer oxidative stress resistance. Genes coding for herbicide resistance developed for agriculture became the selectable markers of choice for generating transgenics, supplanting antibiotic resistance, even when there was no plan for registering the herbicide for use in that crop. The huge corporate investment in HRC and *Bacillus thuringiensis* (*Bt*) toxin containing crops due to perceived market size resulted in the gain of much of our knowledge on promoters, organelle-specific and transit peptides, as well as more recently in organelle transformation. This corporate investment in basic plant molecular biology was manifold greater than the public sector effort, and the spill-over was great. It is important to understand that the transgenic

research is market driven and the market is for weed control.

Table 1.

**5. Recombinant DNA technology used to achieve herbicide resistance** 

alien gene performance; this reflects the ease of transformation in this species.

The techniques used to achieve herbicide tolerance have been reviewed by Cole (1994). Crops which have been transformed to become herbicide tolerant include are shown in

In general, the herbicide tolerance gene is expressed as a determinant which is integrated at a single nuclear locus. Tobacco has often been used as a model crop to study and optimise

#### **2. Chemical weed control**

The controlling of weeds in the growing crops with weedicides increases their yields and ensures the efficient use of irrigation, fertilizers and plant-protection measures, such as the spraying of insecticides and fungicides. The removal of weeds from the growing crops facilitates easy harvesting and gives a high-quality produce without admixture with weed seeds. Chemical weed control can be adopted quite in time and in situations and under conditions, which make manual or mechanical weeding difficult. A great advantage of this method lies in killing weeds in the crop row or in the immediate vicinity of crop plants. The chemical method is easier, less time-consuming and less costly than weeding by hired laborers. However, there are several disadvantages like environment pollution, human and animal health issues related to its use.

## **3. Biological weed control**

Biological weed control is the action of parasites, predators, or pathogens to maintain another organism's population at a lower average density than would occur in their absence. Biological control is usually thought of as intentional introduction of parasites, predators, or pathogens to achieve control, but it is also a natural phenomenon. Biological control will never be the solution to every weed problem. It is employed as one weed management practice among many. Using tools of biotechnology, it is possible to engineer a more potent parasite, predator or mutant which can be deployed to weed control. The biological weed control can be permanent weed management because once an organism is released, it may be self-perpetuating and control will continue without further human intervention. Besides, there are no chemical environmental residues from biological control other than the organism. Bio control may be the best option for management of invasive species. In ideal cases, initial costs are nonrecurring and usually, once the organism is established, no further inputs are needed. There are some situations where biological control is not appropriate. If a plant is a weed in one place and valued in another place, in the same general geographic region, biological control is inappropriate. Spread of a biological control organism, once introduced, cannot be controlled. Biological control is inherently slow, and results are not guaranteed. Some species are geographically local, minor weeds, and development of a biological control for them would be very expensive and not financially wise because of the small-infested area. Release of a biological control organism can induce competitive suppression or extinction of native biological control organisms and other desirable organisms. Biocontrol, particularly in disturbed cropping situations, will not control as many different weeds as other techniques. It won't eradicate weed problems, but most other techniques won't either.

#### **4. Biochemistry and molecular biology of weed control**

The need for developing cost effective chemical weed control systems has led to a vast industrial investment to find and develop selective herbicides and later GM-HRC. Virtually all herbicides marketed are the result of random screening of chemicals. Once success is obtained, further syntheses around the identified chemical are used to find compounds with greater activity and then selectivity. After such compounds have been

The controlling of weeds in the growing crops with weedicides increases their yields and ensures the efficient use of irrigation, fertilizers and plant-protection measures, such as the spraying of insecticides and fungicides. The removal of weeds from the growing crops facilitates easy harvesting and gives a high-quality produce without admixture with weed seeds. Chemical weed control can be adopted quite in time and in situations and under conditions, which make manual or mechanical weeding difficult. A great advantage of this method lies in killing weeds in the crop row or in the immediate vicinity of crop plants. The chemical method is easier, less time-consuming and less costly than weeding by hired laborers. However, there are several disadvantages like environment pollution, human and

Biological weed control is the action of parasites, predators, or pathogens to maintain another organism's population at a lower average density than would occur in their absence. Biological control is usually thought of as intentional introduction of parasites, predators, or pathogens to achieve control, but it is also a natural phenomenon. Biological control will never be the solution to every weed problem. It is employed as one weed management practice among many. Using tools of biotechnology, it is possible to engineer a more potent parasite, predator or mutant which can be deployed to weed control. The biological weed control can be permanent weed management because once an organism is released, it may be self-perpetuating and control will continue without further human intervention. Besides, there are no chemical environmental residues from biological control other than the organism. Bio control may be the best option for management of invasive species. In ideal cases, initial costs are nonrecurring and usually, once the organism is established, no further inputs are needed. There are some situations where biological control is not appropriate. If a plant is a weed in one place and valued in another place, in the same general geographic region, biological control is inappropriate. Spread of a biological control organism, once introduced, cannot be controlled. Biological control is inherently slow, and results are not guaranteed. Some species are geographically local, minor weeds, and development of a biological control for them would be very expensive and not financially wise because of the small-infested area. Release of a biological control organism can induce competitive suppression or extinction of native biological control organisms and other desirable organisms. Biocontrol, particularly in disturbed cropping situations, will not control as many different weeds as other techniques. It won't eradicate weed problems, but

**2. Chemical weed control** 

animal health issues related to its use.

**3. Biological weed control** 

most other techniques won't either.

**4. Biochemistry and molecular biology of weed control** 

The need for developing cost effective chemical weed control systems has led to a vast industrial investment to find and develop selective herbicides and later GM-HRC. Virtually all herbicides marketed are the result of random screening of chemicals. Once success is obtained, further syntheses around the identified chemical are used to find compounds with greater activity and then selectivity. After such compounds have been found and marketed, they become research tools of the physiologists and biochemists, first to find a site of action and then as 'anti-metabolites' to further understand and modulate metabolic pathways. Thus the advent of 2,4-D assisted in understanding auxin action, atrazine and diuron (DCMU) in understanding photosystem II, paraquat for photosystem I, dinitroanilines in tubulin to microtubule assembly, dichlobenil for cellulose biosynthesis, etc. Herbicides are the anti-metabolites of choice in dealing with key enzymes such as glutamine synthase [glufosinate (phosphinothricin)], acetolactate synthase (ALS) (many herbicides), acetyl-CoA carboxylase (ACCase) (many herbicides), dihydropteroate synthase (asulam), enolpyruvate-shikimate phosphate synthase (EPSP) (glyphosate) and phytoene desaturase (many herbicides). The genes for most of these enzymes have been isolated and used in transgenic programs. Such research transcended plant biochemistry and agriculture. For example, it was discovered through comparative genomics that plant and trypanosome β-tubulins were similar to each other and different from mammalian β-tubulin. The dinitro-aniline herbicides then proved to be excellent trypanocides (Chan *et al*., 1993; Bell, 1998). The repetitive (mis) use of single herbicides in monoculture over many years predictably leads to the evolution of herbicide-resistant weeds (Gressel & Segel, 1978). The advent of triazine resistance was crucial to the understanding of the role of the *psb*A gene product in the photosystem II binding site, leading to innumerable studies of photosynthesis, biophysics and biochemistry correlated with molecular structure of the gene product. The mutant and natural *psb*A gene products were crystallized and analyzed, leading to new insights into 'drug' (ligand) binding and design (Michel & Deisenhofer, 1988; Deisenhofer & Michel, 1989). Information from herbicide resistance provided the theoretical underpinning for designing transient drought resistant plants. Harvey and Harper (1982) first promoted the idea that paraquat resistance can be similar to oxidative stress tolerance. This was later extrapolated to being similar to transient drought tolerance (Malan *et al*., 1990). This has allowed developing quick pre-tests with paraquat to ascertain the level of transient drought tolerance of transgenic plants bearing genes designed to confer oxidative stress resistance. Genes coding for herbicide resistance developed for agriculture became the selectable markers of choice for generating transgenics, supplanting antibiotic resistance, even when there was no plan for registering the herbicide for use in that crop. The huge corporate investment in HRC and *Bacillus thuringiensis* (*Bt*) toxin containing crops due to perceived market size resulted in the gain of much of our knowledge on promoters, organelle-specific and transit peptides, as well as more recently in organelle transformation. This corporate investment in basic plant molecular biology was manifold greater than the public sector effort, and the spill-over was great. It is important to understand that the transgenic research is market driven and the market is for weed control.

#### **5. Recombinant DNA technology used to achieve herbicide resistance**

The techniques used to achieve herbicide tolerance have been reviewed by Cole (1994). Crops which have been transformed to become herbicide tolerant include are shown in Table 1.

In general, the herbicide tolerance gene is expressed as a determinant which is integrated at a single nuclear locus. Tobacco has often been used as a model crop to study and optimise alien gene performance; this reflects the ease of transformation in this species.

Herbicide Tolerant Food Legume Crops: Possibilities and Prospects 439

Some microorganisms can detoxify glufosinate by producing an enzyme that causes acetylation of the amino group. The gene encoding the acetylating enzyme has been isolated from *Streptomyces hygroscopicus* (Thompson *et al*., 1987) and from *S. viridochromogenes*  (Wohlleben *et al.,* 1988). It has been referred to as *bar* (for bialaphos resistance) and PAT gene, respectively. The *bar* gene encodes a phosphinothricin acetyl transferase (PAT). In the few countries commercial transgenic crops such sugar beet, canola, soybean, rice and maize

Continuous search for new herbicides that are highly effective and safe for animals and the environment is the need of the hour. A new class of herbicides that fulfils these needs acts by inhibiting specific amino acid biosynthesis pathways in plants (La Rossa 1984). However, most of these herbicides do not distinguish between weeds and crops. Modifying plants to become resistant to such broad-spectrum herbicides would allow their selective use for crop protection. As a consequence, a major effort has been devoted in several laboratories to engineer herbicide-resistant plants. Two approaches have been followed. In the first, a mutant form of the target enzyme is produced which is still active but less sensitive to the herbicide. In this way, mutant plants producing an altered form of the enzyme acetolactate synthase have been selected which are resistant to the sulfonylurea and imidazolinone herbicides (Shaner and Anderson, 1985). In another example, a mutant form of the bacterial aro A gene was expressed in tobacco and conferred tolerance to the herbicide glyphosate (Comai *et al.,* 1985). The second approach involves overproduction of the target enzyme. It has been demonstrated that overexpression of the plant enzyme 5-enol-pyruvylshikimate- 3 phosphate synthase conferred glyphosate tolerance in transgenic petunia plants (Shah *et al*.,

Glyphosate was released by Monsanto Chemical Co. in 1971. Its discovery and release were as revolutionary in weed science as the discovery of 2,4-D. The structure of the amino acid glycine is underlined in following Figure.2 Glyphosate, the Nphosphonomethyl derivative of glycine, is a nonselective, foliar herbicide with limited to no soil activity because of rapid and nearly complete adsorption. It controls perennial grasses and has an advantage over paraquat, because glyphosate translocates. It is the only available herbicide that inhibits EPSP synthase. The enzyme is common in the synthetic pathways leading to the aromatic amino acids phenylalanine, tyrosine, and tryptophan. These amino acids are essential in plants as precursors for cell wall formation, defense against pathogens and insects, and production of hormones (Duke, 1990). The enzyme is not found in animals. Low application volume is more effective than high volume, and small plants are more readily controlled than large ones. In contrast, paraquat, a photosynthetic inhibitor, acts quickly (one or two days) on most plants. Glyphosate activity usually cannot be detected as quickly and may take several days to appear after application. One glyphosate formulation is also used as an aquatic herbicide. Transgenic crops resistant to glyphosate have been developed and marketed. Resistant species include Palmer amaranth, common ragweed, hairy fleabane, goosegrass, Italian ryegrass, rigid ryegrass, and buckhorn plantain. Resistance has been found in Australia,

carrying the *bar* gene has already been released and cultivated commercially.

b. Detoxifying enzyme coding gene

Chile, South Africa, Spain, and in 15 US states.

1986).


Table 1. Transformation of crop species for herbicide tolerance.
