ADDIS ABABA UNIVERSITYSCHOOL OF GRADUATE STUDIES

DEPARTMENT OF BIOLOGY

GENE FLOW FROM TRANSGENIC CROPS TOTHEIR WILD AND WEEDY RELATIVES

(Seminar Paper)

By Getachew MelakuAdvisor

Teklehaimanot Haileselassie, PhD

November, 2010

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Summary

Gene flow in plants can be defined as the exchange of genes

between different and related populations through pollen transfer

and seed dispersion. Transgene escape from genetically modified

(GM) plants into weedy relatives via gene flow may cause

undesired environmental consequences. Besides, introducing a GM

crop resistant to an agent of selection creates a huge

evolutionary pressure to the wild. This pressure impacts not only

the fitness of the wild but also other organisms. For this

reason, countries like Ethiopia which are believed to be center

of biodiversity, must have a concern for developing reliable

biosafety measures through estimating the performance of crop-

weed hybrids and understanding potential introgression of crop

genes (including trans genes) into weedy populations.

Keywords/Phrases: GMO, gene flow, hybridization, transgene

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1. Introduction

Genetically modified plants are plants whose DNA is modified

using genetic engineering techniques. In most cases, the aim is

to introduce a new trait from any donor individual to the plant

which does not occur naturally in this species (Firbank and

Frank, 2000).

Genetically modified plants have been developed commercially to

improve shelf life, disease resistance, herbicide resistance and

pest resistance (Tripp and Robert, 1999). These plants are also

engineered to tolerate non-biological stresses like drought,

frost and nitrogen starvation or have increased nutritional value

(Beck and Ulrich, 1993). Thus future generations of genetically

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modified (GM) plants are intended to be suitable for harsh

environments, producing increased amounts of nutrients, high

level of pharmaceutical agents and capable on the production of

bioenergy and biofuels as well (Rogers and Parkes 1995). Due to

high regulatory and research costs, the majority of genetically

modified crops in agriculture consist of commodity crops such as,

soybean, maize, cotton and rapeseed (Qaim, 2010).

Since the first commercial release of a transgenic crop in 1994,

the land area planted to these crops has expanded to over 90

million ha worldwide, with approximately 8.5 million farmers in

21 countries (Thies and Devare, 2007). Even if there is a large-

scale commercial release of GMOs both in the public and

scientific domains, they have been suspected for the toxicity or

allergenic effects of transgenes on humans, animals and/or other

beneficial organisms, the persistence of stable transgenes, gene

flow within and between species and alteration of GM crops to

severe weeds (Jorgensen et al., 1996). Besides to these direct

effects, the introduction of non-native plant species and release

of GM crops may induce environmental changes at gene to ecosystem

levels (Bartz et al., 2010). Direct competition between GMO plants

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and wild populations and changes in agricultural practice can

also be considered as impact of GMO on biodiversity (Thompson et

al., 2003). Thus understanding and minimizing the potential

impacts of genetically modified (GM) crops through considering

the relevant genetic, biological, socio-cultural and legal

dimensions is very crucial (Traavik and Lim, 2007).

2. Gene flow

Gene flow in plants can be defined as the exchange of genes

between different and related populations through pollen

transfer. As Pollen flow is a subset of gene flow, we will use a

broad definition including the notion of migration (Legere,

1996). According to Price et al., (1996) seed shedding must also

be considered as another important factor for promoting gene

flow. Especially for crops that have had a relatively short

history of domestication, such as oilseed rape (OSR).

Since the main component of gene flow is the outbreeding rate,

out crossing plants show high level of gene flow than those

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inbreeding forms. But many reports indicate that even in highly

self pollinated crop intraspecies, out crossing takes place to

limited extent (Langevin et al., 1990).

Ellstrand, (2003) suggested that gene flow involves various

factors and varies with species, population, genotypes,

environments, between seasons and within seasons. Thus it is

clear that the efficacy of such process must be tested under a

variety of circumstances.

The consequences of gene flow become apparent through a sequence

of events such as hybridization, introgression, adaptation and

finally dispersion (Jordan, 1999).

2.1 Forms of Gene flow

There are two mechanisms of gene flow, vertical and horizontal.

Hybridization or gene exchange between closely related species

occurs when two different types of plants mate (Stewart et al.,

2003). And the other component of gene flow is a means for the

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co-habitation and co-evolution of prokaryotes (Tamar and Barth,

2005).

2.1.1 Horizontal Gene flow

Stewart et al., (2003) defined horizontal gene flow as the movement

of genes between unrelated species. Like between plants and

microbes. The possibility of horizontal gene flow makes some

people worry that transgenes that code for antibiotic resistance

will move into bacteria to result in new antibiotic resistance

problems for humans. On the other hand, Bertolla and Simonet

(1999) reviewed several published experimental studies on this

burning issue and all of them failed to prove horizontal gene

transfer (HGT) from transgenic plants to bacteria.

Some degree of natural flow of genes, often called horizontal

gene transfer or lateral gene transfer, occurs between plant

species. This phenomenon is facilited by transposons,

retrotransposons, proviruses and other mobile genetic elements

that naturally translocate to new sites in a genome (Monroe,

2006). Similarly, Landis et al., (2000) noticed the common

existence of HGT or the transfer of genetic material from one

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bacterial species (the donor) to another bacterial species (the

recipient) through the involvement of their plasmids or their

transposons.

2.1.2 Vertical Gene flow (Hybredization)

“Hybridization” most commonly refers to mating by heterospecific

individuals but has been applied to mating by individuals of

different subspecies and even among taxonomically

undistinguished but genetically different populations (Rhymer and

Daniel, 1996). Studies suggest that the most likely escape route

for transgenic plants will be through hybridization with wild

plants (National Research Council, 2004). Spontaneous

hybridization with wild relatives appears to be a general feature

of most of the world's important crops. For instance, Ellstrand

and Grant (1981) reported that more than 70% of plant species may

have descended from hybrids. For this reason, domesticated plant

taxa cannot be regarded as evolutionarily discrete from their

wild relatives. Even if gene flow from crop taxa may have a

substantial impact on the evolution of wild populations,

Ellstrand, (2003) stated that as wild species have been the

source of many genes used in crop improvement, there should be8

information about the degree of sexual compatibility among crop

species and their wild relatives. Experimentally measured

hybridization rates between crops and their cross-compatible wild

relatives typically exceed 1%, at a distance of over 100 m or

more (Arias and Rieseberg, 1994). The hundreds of well-studied

cases of natural hybridization used genetically based markers for

demonstrating the entrance and persistence of domesticated

alleles in natural populations (Ellstrand et al., 1999).

Nonetheless, even if hybridization is common, it is not

ubiquitous that the incidence of natural hybridization varies

substantially among plant genera and families (Ellstrand et al.,

1996).

3. Introgression

A natural phenomenon that involves a single hybridization

followed by a series of back-crossing only to one of the parent

for having a sibling which is identical to the back-crossed

parent except having the introgressed gene is known as

introgression (Renno et al., 1997). There are several examples for

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the bidirectional introgression between wild and domesticated

populations (Ellstrand et al., 1999). One pattern of introgression

(from wild to domesticated populations) occurs when farmers use

part of their crop production as seed for the next generation and

the other pattern (from domesticated to wild populations) is

resulted due to expansion of modern agricultural practices (Papa

2005). According to (Wolfe et al., 2001) there is asymmetric

introgression (higher rate of introgression from domesticated to

wild populations) than in the reverse direction. This is probably

because of the much higher population sizes of the domesticated

populations and variability on the types of selection in the

cultivated and wild environments (Papa 2005). Transfer of

adaptive traits between the hybridizing lineages, formation of

hybrid taxa and loss of one of the parental forms through genetic

assimilation by the other are some of the possible outcomes of

introgression (Arnold 1992).

4. Factors determining Gene flow

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Since gene flow between taxa can be viewed as a two step process

called hybridization and introgression, its deterministic factors

should be correlated with these influential components of gene

flow (François et al., 2007). It is widely accepted that

hybridization between a crop and its wiled depends on several key

factors. Like their sympatry, the synchrony of their flowering

periods, the existence of a common vector for the gametes, as

well as their reproductive compatibility and the viability and

fertility of the hybrids (Chapman and Burke 2006). As topography

influences the direction and sped of the wind, a flat land favors

pollen flow over long distances. It is also mentioning that

microtopography seems to have an impact on the behavior of

pollinator insects through hiding or making more visible

potential pollen sources and sinks (Lavigne et al., 1998). Like

hybridization, there are barriers for introgression too; some of

these barriers are incompatibility, genetic instability and

limited hybrid pollen fertility (Rieger et al., 1999). The unique

factor which specifically influences introgression is promising

beneficial or neutral traits to be more preferential than

detrimental genes. For example, silenced genes can be kept in

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recipient genomes, until they are eliminated by genetic drift

(Whitton et al., 1997). Moreover, independent nature of the pollen

vector with the intensity and symmetry of pollen flow determine

the speed of introgression in a sink population (Ellstrand and

Elam, 1993). To fix genes more rapidly, the sink population must

be small in size and should be capable of receiving the pollen

easily (Burges et al., 2005).

5. Ecological consequences

This broad concept called ecological risk is associated with the

release of transgenic crops comprising non-target effects of the

crop itself and the escape of transgenes into wild populations.

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5.1 Non-target effects

5.1.1 Unintended effect on native non-pest species

Alteration on the soil microbial community living under

transgenic plants and the less abundance of non-pest herbivores,

predators, parasitoids and pollinators of the targeted transgenic

crops are the two general categories for the hypothesized non-

target effects of transgenic crops (Diana and Prendeville, 2004).

Though Kourtev et al., (2003) admitted that little is known about

how changes in herbivore abundance or quality might affect the

food web and the efficiency of a relative specific resistant

nature to a particular pest.

A number of studies have investigated the possible impacts of GM

plants and purified recombinant proteins on the honey bee (Apis

mellifera), and it was found that direct toxicity by most widely

grown commercial crops did not show any effect on colony

performance (Malone and Pham_Delegue 2001). However, a year

before Malone et al., (2000) reported that high dosage of serine

protease inhibitors could be able to inhibit bee gut proteases.

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Thus, to minimize the death of adult honey bees and other insects

that feed on pollen or pollen-coated plant tissues, Bogorad,

(2000) found a plastid chromosome-encoded protein which could not

be produced in the pollen as a solution.

5.1.2 Impact on the habitat

According to Diana and Prendeville (2004), alteration on the

habitat requirements of the crop plant itself, cultivation of

crops or persistence of feral plants in previously unsuitable

habitats and persistence of GMO in non agricultural fields in

combination lead to a reduction in the quantity or quality of

native habitat. Besides, when a GM crop cultivar with stacked

genes for multiple resistances to herbicides and other pests is

released into natural ecosystem and remains there, the GM crop

cultivar itself could be a mighty weed that cannot be controlled

by any herbicides available in the farmers' arsenal (Kwon and Kim

2001). Since such GMO plants may invade natural habitats,

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becoming agricultural weeds and cause a change on the type of

biodiversity, they create a large agricultural weed management

burden to the farmers (Conner et al., 2003).

5.2 Transgenic escape to the wild

Ellstrand (2001) suggested that hybridization with wild relatives

has been implicated in the evolution of more aggressive weeds for

seven of the world's 13 most important crops. Thus crop-to-wild

gene flow can create another problem. For instance, transgenes

that increased to high frequency in wild populations might affect

seed production, population size or habitat use in the wild

species. In addition, transgenes for insect resistance that

establish in wild populations could have negative effects on

native herbivores as well as on species with which the native

herbivores interact (Diana and Prendeville, 2004).

On the other hand, Conner et al., (2003) opposed the transgene

escape fears by stating that the development of weedy populations

with herbicide resistance is not a new situation for agriculture

since herbicide-resistant plants have also been developed by

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traditional plant breeding and arise by entirely natural means.

Besides, it is important to recognize that there may be

ecological benefits come up after the release of transgenic

plants through reducing pesticide and herbicide use. (Benbrook,

2003).

6. Gene flow and Evolution

6.1 Gene flow and other evolutionary forces

Hybridization is a frequent and important component of plant

evolution and speciation (Rieseberg and Ellstrand, 1993). For

instance, more than 70% of angiosperm species may have arisen

through the formation of polyploid species through hybridization

(Masterson, 1994). This reality revealed that the rate of

incorporation of foreign alleles under such levels of

hybridization is likely to be orders of magnitude higher than

typical mutation rates and thus, we would expect gene flow in

these systems to be much more important than mutation (Grant,

1975). In other words, gene flow can be considered as a potent

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evolutionary force since a small amount of gene flow is capable

of counteracting the other evolutionary forces of mutation, drift

and selection (Slatkin, 1987). As gene flow within and between

populations has an important role in maintaining population

genetic structures, enabling adaptation to changing environmental

circumstances and reducing vulnerability to evolutionary hazards

such as inbreeding depression and genetic drift are possible

(Campbell, 1991). The process of migration along with the rest of

evolutionary forces is the impetus for the changes in gene

frequencies in natural populations (Cavalli-Sforza, 1966).

The best-known evolutionary consequence of gene flow is its

tendency to homogenize population structure. The conditions for

homogenization will vary depending on whether immigrant alleles

are neutral, detrimental or beneficial in the ecological and

genomic environment of the recipient population (Slatkin, 1987).

But in the absence of gene flow, the evolution of neutral alleles

in a population is regulated by the stochastic process (genetic

drift) which may lead to genetic differentiation among

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populations especially, when the effective population sizes are

small (Wright, 1969).

6.2 Role of transgene flow in evolution

Introduction of a GM crop resistant to a specific herbicide

creates a huge selection pressure to evolve herbicide resistance

in weeds through gene flow and mutation. This evolutionary change

is a very slow and nearly invisible process until it becomes

irreversible and a reality (Kwon et al., 2001). With regard to the

evolution of more aggressive weeds, Crop-to-weed gene flow will

have important practical and economic consequences (Anderson,

1949). Here the evolutionary dynamics of each of these traits

will be determined by the balance between the benefit of the

trait (in the presence of the selective agent) and the cost of

the trait (in the absence of the selective agent) (Simms and

Rausher, 1987). In contrast to costly or neutral characters,

transgenes for characters such as insect or pathogen resistance

and drought tolerance may benefit wild populations and therefore

may increase in frequency in wild populations by natural

selection. For example, damage by herbivores is generally

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detrimental to plant fitness in wild populations and similarly

viruses and fungal pathogens also commonly reduce fitness of wild

plants. This scenario suggests that transgenic herbivore and

pathogen resistance would be favored by natural selection in many

wild relatives of crop plants (Maskell et al., 1999).

6.2.1 Wild extinction due to gene flow

Under the appropriate conditions, hybridization between a common

species and a rare one can send the rare species to extinction in

a few generations (Ellstrand, 2001). For instance, hybridization

with domesticated species has been implicated in the extinction

or increased risk of extinction of two wild species of the

world's 13 most important crops. A risk of extinction by

hybridization may also occur when a previously allopatric rare

taxon becomes sympatric or peripatric with a recently introduced

crop and this risk will get higher as hybridization and

introgression proceed over generations (Ellstrand and Elam,

1993). This is because, introduced species (subspecies) can bring

about a genetic change. In most of the cases, habitat

modifications can breakdown reproductive isolation between native

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species which is followed by subsequent mixing of gene pools and

potential loss of genotypically distinct populations (Rhymer and

Daniel, 1996). Besides, even if introgression occurs, it can be

limited in various ways. For example, it can be unidirectional.

Either of the hybrids can fail to mate with individuals of one

parental taxon or such mattings can be sterile. Further,

hybridization itself can be unidirectional which means males of

one species breeding with females of the other species, but not

the opposite cross (Rhymer and Daniel, 1996). So the problem of

extinction by hybridization is not only dependent on the relative

fitness but also on the patterns of mating (Ellstrand and Elam,

1993). On the basis of hybrid fitness, Ellstrand, (1992) noted

that, the fitness costs associated with hybridization may be

severe enough to select for the evolution of secondary isolating

mechanisms even if, a rare species will often lack the genetic

variation necessary for such evolution.

6.2.2 Gene flow and wild fitness increment

Hybridization can allow for rapid evolutionary change by

producing novel gene combinations (Barton and Hewitt, 1989). For

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this reason, botanists recognize that hybridization may increase

genetic variation at both the genic and genotypic levels. This

condition in turn leads to an increased fitness and adaptation to

new environments in the existing taxa (Ellstrand, 1992). Hybrid

vigor or heterosis resulting through hybridization is also a

well-known phenomenon and has been documented more frequently

than outbreeding depression has (Hartl and Clark, 1989). In

addition, gene flow within and between populations can reduce

vulnerability to evolutionary hazards such as inbreeding

depression, genetic drift and maintain population genetic

structures (Ellstrand, 1992).

6.2.3 Transgenic wild plants and their impact on the evolution of

others

The evolution of enhanced weediness of transgenic wild plants

will depend on whether hybridization occurs or the hybrids can

reproduce in the wild or one or more alleles from the

domesticated plant can confer an advantage on the wild (Ellstrand

and Elam, 1993). Thus when transgenes for resistance to

herbivores or pathogens increase to high frequency in wild

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populations, then there will be immediate and negative effects on

the targeted organisms. By contrast, if these species have

additional host plants, the effect of the transgene will depend

on how important the transgenic-wild plant is in the diet of the

herbivore or pathogen (Charlet et al., 1997). In contrast, as

these affected species may evolve counter-resistance, the change

will not be static. This is because of the speed for counter-

resistance development which depends on the strength of selection

over the transgenic host, the presence of alternative hosts and

pre-existing variation for resistance (Diana and Prendeville,

2004). Even if the evolution of new insect biotypes due to the

introduction of an insect resistant GMO plants is the major area

of concern, experiences from the commercial host plant resistance

breeding has shown that there is no direct relationship between

the deployment of insect resistant cultivars and the evolution of

new insect biotypes (Sharma and Ortiz, 2000). But according to

FAO (2004), most scientists agree that extensive long-term use of

Bt crops with glyphosate, gluphosinate and herbicides associated

with herbicide tolerant (HT) crops can promote the development of

resistant insect pests and weeds. Likewise, plant breeders

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incorporated new resistance genes which simultaneously made

insects and pathogens to evolve mechanisms for overcoming their

survival (Sharma and Ortiz, 2000).

Eventhough, some efforts have already been made to introduce

multiple traits in a single crop cultivar, Gressel (1980)

proposed that one way to delay the evolution of herbicide

resistance in weeds is to stack two herbicide resistance genes in

a GM crop and to use a mixture of herbicides with different modes

of action.

7. Risk assessment

In the first years following commercialization of genetically

modified organisms, the primary risk assessment researches were

focused on developing detection and monitoring systems to account

for unwanted GM DNA in foodstuffs and crops (Traavik and Lim,

2007). Through time, researchers turned their attention to

environmental risk assessment focused on describing numerous

environmental effects that can be caused by GM crops and

transgene escape (Chandler et al., 2008). This is the reason for

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considering the past 10 years as time for researchers to carry

out experimentation on measuring gene flow and alteration of GMOs

to series weeds (Darmency et al., 2007).

7.1 Risk assessment on transgene flow to the wild

species

It is known that impact of gene flow is dependent on its

magnitude which is idiosyncratic, varying among species,

populations, individuals and even years Surprisingly, when

compatible species with similar flowering phenologies grow in

spatial proximity, levels of gene flow may vary (Ellstrand,

1992). For example, differences in the relative sizes of the

source and sink populations may result in different rates of

hybridization and gene flow (Devaux et al., 2007). Even if there

are multitude factors that can impose the task of quantifying

probability of gene flow, Heard et al., (2003) highlighted the

impact of variability on weed density in a GMO field as an agent

for transgenic escape. Even if such factors complicate the task

of measuring gene flow, Ellstrand and Elam (1993) could

measured it by testing the progeny of the weed from a large sized

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population through techniques free from hand pollination and

embryo rescue.

As consequences of gene flow will be irreversible and may only be

detected when the impact is already beyond the manageable range,

(Kwon et al., 2001). Having a decision on the probability of gene

flow is the one among the various risk analysis measures. Hence,

Hails and Timms-Wilson (2007) used the mathematical expression:

 

7.2 Risk assessment for the weediness of GMO

The appropriate measure of invasion risk for a plant is its

finite rate of increase (λ). Under stable age distribution and

absence of density-dependent constraints especially for crops

with discrete non-overlapping generations, (Crawley et al., 2001)

formulated λ in its simplest form as:

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Here St is the number of seeds in generation t and St+1 the number

of seeds in the subsequent generation. This method is more

valuable than estimates of a single demographic process

(performance at any particular life history stage) which provide

no information about invasiveness when used in isolation (Parker

and Kareiva 1996).

(Kwon et al., 2001) advised governments of countries that do not

cultivate but importing bulk unprocessed GM crop commodities to

have consideration and systematic approach employing accurate

assessment of gene flow on practical and useful basis for

effective and appropriate management of gene flow from GM to non-

GM crops

8. Management and Genetic methods to minimize

gene

flow

8.1 Ecological methods

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Daniell, (2002) emphasized on the system of surrounding the

transgenic crop with barren zones or barrier crops to reduce

pollen flow. In addition to applying buffer zones as a means of

prevention, other strategies like removal of flowers from the

transgenic plants, removal of sexually compatible species,

adjustment of flowering time and isolating genetically modified

crops by distance have also been used (Dale, 1992). Quist and

Chapela (2001) recommended avoiding plantation of transgenic

crops in their center of biodiversity or in the habitat of their

conventional varieties and wild relatives as a prevention

measure. Hardy, (2009) declared that farmers may be able to

reduce the spread of rogue transgenic plants by separating their

transgenic crops from their traditional crops or through

protecting their traditional seed banks not to be contaminated

with transgenic seeds. Moreover, governments can also play a role

by monitoring the cultivation of GM crops. Kwon et al., (2001)

tried to finalize that the principal mitigation measures would be

an appropriate selection of biotechnologically available targeted

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species with low out-crossing rate or minimum sexually compatible

wild relatives.

8.2 Genetic method of controlling gene flow

Various genetic methods like plastid transformation, genetic

alteration, recoverable block of function (RBF), RNA-i based post

transcriptional gene silencing methods and other means of

biotechnological applications have a significant role on

preventing transgene transmission (Daniell, 2002).

Hills et al., (2007) reviewed the two Genetic Use Restriction

Technologies (GURTs) called T-GURT (trait-GURT) and V-GURT

(varietal-GURT). Here the former technique allows seed formation

with inhibiting expression of the transgene and the latter

prevents transmission of the gene in the seed. Like T-GURT, Singh

et al., (2007) announced the concept of gene containment based on

epigenetic imprinting and RNAi-based post-transcriptional gene

silencing method of preventing pollen-mediated gene flow through

allowing seed set to occur normally. Victor et al., (2001)

described RBF having a blocking sequence linked to the gene of

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interest and a recovering sequence that can pick up the blocked

function of the host plant through changes on the specific

chemical or physical treatment for resulting phenotypic change or

death of the host plant. Like RBF use of ‘tandem constructs’ will

result in inviability, sterility and death of the transgenic

wiled hybrid (Gressel 1999).

As plastid transformation has 10–50 times expression levels over

nuclear transformation, molecular biotechnologists emphasize on

its advantages (Kwon et al., 2001). Absence of gene flow was

recorded even from the pollen produced by a plant with

metabolically active maternal plastids due to loss of the plastid

at the state of pollen maturity (Nagata et al 1999). And

inheritance of plastid genes in a strictly maternal fashion on

most of crop plants (Smith, 1989) are the other advantages of

this method. On the contrary side, expressing a transgene to a

very high level or stacking a variety of transgenes in the

chloroplast may mean that reduced quantity of amino acids used

for the synthesis of ribulose-1,5-bisphosphate carboxylase (Kwon

et al 2001).

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To compensate the limitation of plastid transformation Mariani et

al., (1990) designed localized expression of specific genes, such

as RNases, to prevent pollen formation and the production of

male-sterile plants.

The genetic modification may also be used to inhibit flower

formation which is only a feasible approach for non-seed products

(Lemmetyinen et al., 2001).

8.3 Cliestogamy

Cleistogamy is quite a common phenomenon in cultivated plants

which is believed to be found in 29 families and in about 70

genera of the kingdom plantae (Lord, 1981). Plants producing

cleistogamic flowers need not to open at all to complete

fertilization and are characterized by a reduction in number and

size of floral parts such as stamens and by modifications of the

perianth (Chhabra and Sethi, 1991).

9. Recommendation

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Even if genetic engineering plays a magnificent role especially

for ensuring food security in the world, debates on the benefits

and costs of GM crops and transgene escape continue among

scientists. But the problem is that, none of these debates can

critically assess the ability of science and scientists on

providing evidence necessary for rational decisions. This is

because most of model-based risk assessments used qualitative

data that might fail to provide relatively unambiguous,

transparent and internally consistent analysis. Besides, the

suggested ecological and evolutionary changes which are

considered as adverse effects can be lowered or prevented by

applying aggregates of prevention measures simultaneously.

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