Biotechnology GM technology for Herbicide

Biotechnology

Plant biotechnology and crop improvement

GM technology for Herbicide

Paper No. : 12 Plant biotechnology Biotechnology and crop improvement

Module : 34 GM technology for Herbicide

Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director The Energy and Resources Institute (TERI), New Delhi

Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry Jamia Hamdard University, New Delhi

Paper Coordinator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director The Energy and Resources Institute (TERI), New Delhi

Content Writer: Dr Gulshan Chaudhary, Post Doctorate Fellow, The Energy and Resources Institute (TERI), New Delhi

Content Reviewer: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director

The Energy and Resources Institute (TERI), New Delhi

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Description of Module

Subject Name Biotechnology

Paper Name Plant biotechnology and crop improvement

Module Name/Title GM technology for Herbicide

Module Id 34

Pre-requisites

Objectives

Keywords

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GM TECHNOLOGY FOR HERBICIDES

TABLE OF CONTENTS

1. Learning Objectives

2. Introduction

2.1 Why develop herbicide resistance crops?

3. Glyphosate Resistance

3.1 EPSPSs insensitive to glyphosate

3.2 Detoxification of glyphosate

4. Glufosinate Resistance

4.1 Detoxification of glufosinate molecule

5. Safety aspects of herbicide tolerant technology

5.1 Toxicity and Allergenicity

5.2 Effects on the Plants

5.3 Persistence or invasiveness of crops

6. Advantages of Herbicide Tolerant Crops

7. Disadvantages of transgenic herbicide resistance

8. Current status of herbicide tolerance

9. Summary

10. Figures and Table

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1. LEARNING OBJECTIVES

In the present module, we aim GM crops, Herbicide tolerance crops, its mode of action.

Additionally, we give a preview to understand past, present and future scenario of herbicide

tolerant plants.

2. INTRODUCTION

Weeds cause loss of crop production globally every year. The chemical herbicides which are

used is harmful as, some time it causes harm to plants also. So to overcome chemical

herbicides side effect now days genetic engineering is used to incorporate herbicides

resistance gene in crops know as genetically modified crops (GM).

GM for Herbicide can be done by two type: (1) Target site and, (2) Non target site herbicide

resistance.

1. Target site: In this the target site of herbicide is changed like mutation in amino acid so that

the herbicide does not binds to the site and its effect is lethal to plant.

2. Non target site: In this changes is done in such a way that the amount of herbicide reaching

the target site is very low(non lethal).

2.1 Why develop herbicide resistance crops?

Different herbicides have different Mode of Actions (MOAs) which mean that a target

enzyme no longer functions properly, or at all. This is usually because the molecule of

herbicide has distorted the enzyme molecule. Enzymes are also known to be catalysts

and they provides a platform for specific chemical reaction in which they act directly

or indirectly. The Knocking-out’ of an enzyme with a herbicide shows two main effects

with various consequences:

Chemicals components of the reaction accumulate by damaging directly or indirectly.

Absence of the reaction’s product will restrict growth, either through starvation of

key building blocks or because the reaction makes chemicals which normally protect

the plant.

The enzyme functioning normally Figure 1 (1) while in figure 1(2) molecule of herbicide

stopping biochemically by coming together to form the usual product. As a result the plant

dies due to the damage of building locks. While in figure 1 (3) A mutated enzyme combines

with the herbicide to continue the reaction. Either herbicide is selective to species with this

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type of enzyme, or an individual plant with this mutation could reproduce to establish a

population of resistant weeds.

Examples of gene-based herbicide resistance

Herbicide(s) Effected part/function Mode of development of

herbicide resistance

GM plant

Triazines Photosynthesis

Resistance is due to an

alteration in the psbA

gene, which codes for the

target of this herbicide,

chloroplast protein D-1.

Canola

Sulfonylureas

Inhibitors of AHAS

therefore stopping the

synthesis of branched-

chain amino acids,with

subsequent plant death.

Genes encoding resistant

versions of the enzyme

acetolactate synthetase.

Soybean

,Poplar,

Canola, Flax,

and Rice

Imidazolinones

Inhibitors of AHAS by

stopping synthesis of

the branched-chain

amino acids, with

subsequent plant death.

Strains with resistant

versions of the enzyme

acetolactate synthetase

have been selected in

tissue culture.

Maize, Canola,

Wheat and

Rice.

Aryloxphenoxypropionates,

Cyclohexanediones lipid biosynthesis

These herbicides inhibit

the enzyme acetyl

coenzyme

A carboxylase. Resistance,

selected in tissue culture,

is due either to an altered

enzyme that is not

herbicide sensitive or to

the degradation of the

herbicide.

Maize,

Soyabean and

Cotton.

Glyphosate

inhibition of EPSPS

activity which, disrupts

the shikimate pathway

and inhibits aromatic

amino acid production,

which ultimately

causing the death of

plant.

Resistance is from

overproduction of EPSPS,

the target of this herbicide.

Soybean,

Canola

Cotton, Maize

Bromoxynil

Inhibition of

acetolactate synthase

(ALS)

Resistance to this

photosystem II inhibitor

has

been created with a

bacterial nitrilase gene,

which encodes an enzyme

that degrades this

herbicide.

Tobacco,

Cotton and

Canola

Phenoxycarboxylic acids

(e.g., 2,4-D and 2,4,5-T)

Remain in soil and

toxic to lower mammals

and humans.

Transformation with the

tfdA gene from

Alcaligenes, which

Cotton and

tobacco

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encodes a dioxygenase

that degrades this

herbicide.

Glufosinate

(phosphinothricin)

Inhibits glutamine

Synthetase.

Over 20 different plants

have been transformed

with either the bar gene

from Streptomyces

hygroscopicus or the pat

gene from

S. viridochromogenes. The

phosphinothricin

acetyltransferase that these

genes encode

detoxifies this herbicide.

Canola, Corn,

Cotton, Maize,

Rice and

sugarbeet.

Cyanamide Inhibit catalase,

cytochromoxidase

Cyananide hydratase gene

from the fungus

Myrothecium verrucaria

was introduced. The

enzyme encoded by this

gene converts cyanamide

to urea.

Tobacco,

wheat and

soyabean.

3. Glyphosate Resistance:

It’s a very important herbicides used worldwide. Glyphosate inhibits an enzyme 5-

enolpyruvylshikimate-3-phosphate synthase (EPSPS) of shikimate pathway. An enzyme

enolpyruvylshikimate-3-phosphate synthase catalases the reaction by phosphoenolpyruvate

(PEP) and shikimate-3-phophate (S3P) to form a compound 5-enolpyruvylshikimate-3-

phosphate (EPSP), resulted in to the synthesis of aromatic amino acids. Diagram is showing

the shikimate pathway and glyphosate inhibiting pathway (fig.2).

Work to develop glyphosate resistant crops by genetic engineering are focused on

following strategies (Fig 3):

Overproduction of EPSP synthase.

Introduction of a metabolic detoxification gene.

Introduction of an altered EPSP synthase enzyme with decreased affinity for

glyphosate.

3.1 EPSPSs insensitive to glyphosate:

Since 1985 many researchers have identified many genes from different sources with same or

different mode of action which help plants combat glyphosate (Comai et al. 1985; Stalker et

al. 1985; Kishore et al. 1986; Padgette et al. 1991; Berry et al. 1992; Zhou et al. 1995, 2006;

Ye et al. 2001; Kahrizi et al. 2007). but CP4-EPSPS gene which is isolated from

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Agrobacterium strain. CP4, is best suited for transformation as it is insensitive to glyphosate.

CP4-EPSPS and sensitive EPSPS have identical binding site for substrate glyphosate. CP4-

EPSPS have high affinity for PEP then glyphosate, which allow the shikimate pathway to

function normally, as it ‘bypass’ the endogenous EPSPS (fig.3).

3.2 Detoxification of glyphosate:

Detoxification is another method which can be employed to confer glyphosate. Soil

microorganisms can metabolize glyphosate by two different ways (Fig. 4A): (a) cleavage of

the carbon–phosphorus bond, which results in the formation of phosphate and sarcosine (the

C-P lyase pathway)and (b) oxidative cleavage of the carbon–nitrogen bond on the carboxyl

side, catalyzed by glyphosate oxidoreductase (GOX), which results in the formation of

aminomethylphosphonic acid (AMPA) and glyoxylate (the AMPA pathway).

4. Glufosinate Resistance:

Glufosinate herbicides contain the active ingredient phosphinothricin, which kills plants by

blocking the enzyme responsible for nitrogen metabolism and for detoxifying ammonia, a by-

product of plant metabolism. Glutamine synthase (GS) catalyzes conversion of L-glutamate to

L-glutamine while assimilating ammonia in plants. Phosphinothricin (glufosinate, PPT), is a

herbicide that is analog of glutamate, inhibits glutamine synthase (GS) as L-glufosinate and

L-glutamate are very similar in structure, which results in accumulation of ammonium.

Futher research shows that PPT inhibits part of photorespiration process. Inhibition of GS

result in decrease in glutamate concentration which affect the photorespiration process by

accumulating glyoxylate that inhibits the RuBP-carboxylase that is CO2 formation. Crops

modified to tolerate glufosinate contain a bacterial gene that produces an enzyme that

detoxifies phosphinothricin and prevents it from doing damage.

4.1 Detoxification of glufosinate molecule:

Komoba and Sandermann, 1992 have shown that plants have endogenous metabolism of

glufosinate but its too slow to degrade the herbicide. So, gene from outside need to be

inserted to encode an enzyme that can detoxify L-glufosinate fast and prevent the herbicide

from reaching the target enzyme. Rasche, 1995; Vasil, 1996; Wehrmann et al., 1996 showed

two gene bar from Streptomyces hygroscopicu and pat gene from S. viridochromogenes

encodes Phosphinothricin N-Acetyltransferase (PAT) enzyme. PAT reduces L-glufosinate to

non-phytotoxic metabolite N-acetyl-L-glufosinate (NAG) which will prevent glufosinate to

inhibit GS (Fig.5). So, bar and pat gene are being used to transformation for GM plants

resistant to Glufosinate.

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5. Safety aspects of herbicide tolerant technology

5.1 Toxicity and Allergenicity:

In several countries agencies linked to Government regulatory have ruled that the crops

possessing herbicide tolerant conferring proteins and it do not poses any other environmental

and health risks as compared to non-GM counterparts. Proteins which are introduced are

assessed for the potential toxic and allergenic activity, according to the guidelines developed

by international organizations.

5.2 Effects on the Plants:

The main role of this protein that it expression did not harm the plant growth nor resulted

into the poor agronomic performance compared to parental crops, except for the

expression of an additional enzyme for herbicide tolerance or alteration of an already

existing enzyme, no other metabolic changes occur in the plant.

5.3 Persistence or invasiveness of crops

A major environmental concern associated with herbicide tolerant crops is their potential

to create new weeds through outcrossing with wild relatives or simply by persisting in the

wild themselves. This potential, however, is assessed prior to introduction and is also

monitored after the crop is planted. The current scientific evidence indicates that, in the

absence of herbicide applications, GM herbicide-tolerant crops are no more likely to be

invasive in agricultural fields or in natural habitats than their non-GM counterparts (Dale

et al., 2002).

The herbicide tolerant crops currently in the market show little evidence of enhanced

persistence or invasiveness.

.

6. Advantages of Herbicide Tolerant Crops

It is an excellent weed control, hence higher crop yields

Its flexibility because of its possibility to control the weeds later in the plant’s growth

It also reduces the numbers of sprays in a season therefore reduction in the fuel use

It’s also reduces soil compaction because of less need to go on the land to spray

Because of low toxicity compounds it do not remain active in the soil

It has ability to use conservation-till systems, with has consequent benefits to soil

structure and organisms (Felsot, 2000).

7. Disadvantages of transgenic herbicide resistance

Low toxicity to mammalian

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Its ecotoxicity as have side effects on soil microorganisms and agricultural flora ond

fauna

It develops some herbicide resistant weeds

Have some effect on yield performance

Having single selection pressure and weed resistance

Due to mutation it cause gene escape

It can also responsible for gene flow and contamination of organic crops

8. Current status of herbicide tolerance

Since 1996 to 2016, herbicide tolerat crops consistently occupying the largest planting area of

biotech crops. According to ISAAA in 2016, Herbicide Tolerant crops occupied 86.5 million

hectares or 47% of the 185.1 million hectares of biotech crops planted globally. According to

survey the most common are the glyphosate and glufosinate tolerant varieties. The table 1

shows countries that have approved major Herbicide Tolerant (with single and stacked genes)

crops for food, feed, and/or cultivation

9. Summary:

As we are evolving our techniques are evolving too. Conventional way of removing weed by

use chemical herbicides are not only harmful to weeds but it also effected environment, soil

texture and crop plants too. Herbicides resistance plants is better alternatives to conventional

techniques. When it come to technology then there is always an advantage as well as

disadvantage. So, we should be very careful. From 1996 to 2016, HT crops consistently

occupied the largest planting area of biotech crops. In 2016 alone, HT crops occupied 86.5

million hectares or 47% of the 185.1 million hectares of biotech crops planted globally. The

most common are the glyphosate and glufosinate tolerant varieties.

At present we can get many herbicides resistance plants in market. Still research is go on to

make most of the crop plants herbicides resistance.

Figures

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Figure 1-3Different modes of enzyme reaction

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Figure 2 Shikimate pathway that leads to the biosynthesis of aromatic amino acids and

mode of action of glyphosate on the reaction catalyzed by EPSPS.

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Figure 2 Strategy for the development of glyphosate-resistant

Table 1 Global adoption rate (%) for principal Biotech crops

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Biotechnology GM technology for Herbicide