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Plants genetically modified (GM) for insect resistance (IR) have to be tested and compared to their non-GM counterparts with respect to several aspects. This thesis summarizes the effects of three transgenic lines of oilseed rape (Brassica napus) expressing pea (Pisum sativum) seed lectin (PSL) in the pollen on a target pest: the pollen beetle (Meligethes aeneus), and a non-target insect: the honey bee (Apis mellifera). The competitive ability of the transgenic plants was tested to evaluate potential invasive characters. Finally, attitudes towards GM crops among Swedish farmers were surveyed. Pollen beetle adults and larvae were exposed to three PSL expressing plant lines and two control lines without any PSL. Fourteen life history parameters were studied and significant differences between transgenes and controls were found for egg size and larval mortality. This means that the modification does not prevent direct damage to the attacked crop but the effects could, together with the action of natural enemies, lead to a reduced pollen beetle population. The sensitivity of honey bee larvae to PSL containing pollen was tested by feeding them diets with high levels of transgenic or control pollen. The addition of pollen had a positive effect on developmental time and larval weight but no differences were detected between transgenic and non-transgenic pollen. Competitive ability was tested by growing transgenic plants either in monoculture or mixed with control plants, with or without pollen beetles, and with or without pollinators (bumblebees). Plant characters related to plant fitness were measured but transgenic plants did not benefit from the transformation regarding pest damage. However, yield was higher on transgenic plants when grown mixed with control plants than when grown in monoculture, and the opposite was true for control plants. A majority of the surveyed farmers were negative to GM crops and considered consumers' unwillingness to buy GM products as the largest drawback, while higher yield was considered the largest potential benefit from growing such crops.
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Oilseed Rape Transformed with a Pea
Lectin Gene
Target and Non-target Insects, Plant Competition, and
Farmer Attitudes
Faculty of Natural Resources and Agricultural Sciences
Department of Ecology
Doctoral thesis
Swedish University of Agricultural Sciences
Uppsala 2007
Acta Universitatis Agriculturae Sueciae
2007: 95
ISSN 1652-6880
ISBN 978-91-576-7394-7
© 2007 Anna Lehrman, Uppsala
Tryck: SLU Service/Repro, Uppsala 2007
Lehrman, A. 2007. Oilseed rape transformed with a pea lectin gene. Target and non-target
insects, plant competition, and farmer attitudes. Doctor’s dissertation.
ISSN 1652-6880, ISBN 978-91-576-7394-7
Plants genetically modified (GM) for insect resistance (IR) have to be tested and compared
to their non-GM counterparts with respect to several aspects. This thesis summarizes the
effects of three transgenic lines of oilseed rape (Brassica napus) expressing pea (Pisum
sativum) seed lectin (PSL) in the pollen on a target pest: the pollen beetle (Meligethes
aeneus), and a non-target insect: the honey bee (Apis mellifera). The competitive ability of
the transgenic plants was tested to evaluate potential invasive characters. Finally, attitudes
towards GM crops among Swedish farmers were surveyed.
Pollen beetle adults and larvae were exposed to three PSL expressing plant lines and two
control lines without any PSL. Fourteen life history parameters were studied and significant
differences between transgenes and controls were found for egg size and larval mortality.
This means that the modification does not prevent direct damage to the attacked crop but
the effects could, together with the action of natural enemies, lead to a reduced pollen
beetle population.
The sensitivity of honey bee larvae to PSL containing pollen was tested by feeding them
diets with high levels of transgenic or control pollen. The addition of pollen had a positive
effect on developmental time and larval weight but no differences were detected between
transgenic and non-transgenic pollen.
Competitive ability was tested by growing transgenic plants either in monoculture or
mixed with control plants, with or without pollen beetles, and with or without pollinators
(bumblebees). Plant characters related to plant fitness were measured but transgenic plants
did not benefit from the transformation regarding pest damage. However, yield was higher
on transgenic plants when grown mixed with control plants than when grown in
monoculture, and the opposite was true for control plants.
A majority of the surveyed farmers were negative to GM crops and considered
consumers’ unwillingness to buy GM products as the largest drawback, while higher yield
was considered the largest potential benefit from growing such crops.
Keywords: Brassica napus, Meligethes aeneus, pea lectin, Pisum sativum, Apis mellifera,
transgene, performance, attitude, GMO.
Author’s address: Anna Lehrman, Department of Ecology, Swedish University of
Agricultural Sciences, SE-750 07 Uppsala, Sweden
Raps modifierad med ärtlektin
Skadegörare, nyttodjur, växtkonkurrens och
attityder bland lantbrukare
Växter genmodifierade (GM) för insektsresistens (IR) behöver testas och
jämföras med motsvarande icke-GM gröda för flera egenskaper. Denna
avhandling summerar effekten av tre GM-linjer av raps (Brassica napus),
som utrycker ärtlektin (Pisum sativum) (PSL) i pollenet, på dess
skadegörare, rapsbagge (Meligethes aeneus), och en nyttoinsekt, honungsbi
(Apis mellifera). De transgena plantorna testades även för att utreda om
modifieringen inneburit ökad konkurrenskraft. Slutligen undersöktes
attityderna till GM-grödor bland Sveriges lantbrukare.
Både larver och vuxna rapsbaggar fick äta på någon av de tre transgena
linjerna eller på någon av två kontrollinjer som inte innehöll något
ärtlektin. Fjorton olika parametrar testades men det var bara äggstorlek och
larvdödlighet som skiljde sig signifikant mellan transgena och icke
transgena plantor. GM-plantorna skulle därmed inte vara direkt skyddade
mot skadegöraren, men om denna effekt skulle bestå under naturliga
förhållanden skulle den transgena rapsen, tillsammans med rapsbaggens
naturliga fiender, kunna begränsa en av de svåraste skadegörare på raps i
norra Europa.
Möjliga negativa effekter av PSL-pollenet på bilarver testades genom att
mata larverna med höga nivåer av antingen pollen från den transgena
rapsen, eller pollen från kontrollplantor. Pollentillskottet i dieten hade
positiv effekt på utvecklingstid och vikt men inga skillnader kunde påvisas
mellan transgent och icke-transgent pollen.
Plantornas konkurrenskraft undersöktes genom att odla transgena plantor
för sig eller i blandning med icke-transgena, med eller utan rapsbaggar och
pollinatörer (humlor). Flertalet växtkaraktärer relaterade till fitness mättes
men den transgena rapsen blev inte mindre skadad jämfört med kontroll
plantorna. Däremot gav PSL-rapsen lägre avkastning när den odlades
separat jämfört med den blandade odlingen medan det motsatta gällde för
Majoriteten av lantbrukarna var negativa till GM-grödor.
Konsumenternas negativa attityd sågs som den största nackdelen och en
högre avkastning som den största fördelen om de skulle odlas en gröda
modifierad för insektsresistens.
Introduction, 7
Breeding for insect resistance, 8
Domestication of plants, 8
Plant defence, 9
Improving insect resistance in plants using biotechnology, 9
Global status of transgenic crops, 10
Risk/ benefit assessments, 10
Toxicity to non-target organisms, 11
Gene introgression, 11
Resistance development, 12
Additional effects of gene transfer, 12
Environmental benefits, 12
The plant and its pests, 13
Brassica history, 13
Rapeseed breeding and cultivation, 13
The major pest; the pollen beetle, 14
The making of a transgene, 16
Finding the gene, 16
Introduction of the gene and plant line production, 16
Transgene assays, 17
Lectin quantification, 17
Pollen beetle assays, 17
Honey bee assay, 20
Plant invasiveness, 22
The grower, 22
Economic benefits, 23
Farmer survey, 23
Conclusions, 24
References, 25
Acknowledgement – Tack, 30
Papers I-V
The present thesis is based on the following papers, which will be referred to by
their Roman numerals:
I. Lehrman, A., Åhman I & Ekbom, B. 2007. Influence of pea lectin
expressed transgenically in oilseed rape (Brassica napus) on adult pollen
beetle (Meligethes aeneus). Journal of Applied Entomology 131, 319-
II. Lehrman, A., Åhman I & Ekbom, B. Effect of pea lectin expressed
transgenically in oilseed rape on pollen beetle life history parameters.
III. Lehrman, A. 2007. Does pea lectin expressed transgenically in oilseed
rape (Brassica napus) influence honey bee (Apis mellifera) larvae?
Environmental Biosafety Research 6 (in press).
IV. Åhman, I., Lehrman, A. & Ekbom, B. Competitive ability of oilseed rape
transformed for pollen beetle resistance. (Manuscript)
V. Lehrman, A. & Johnson, K. Swedish farmers attitudes, expectations and
fears in relation to growing genetically modified crops. (Manuscript)
Paper I is reproduced by kind permission of Blackwell Publishing. Paper III is
reproduced by kind permission of EDP Sciences.
The ability to introduce novel traits into plants by gene transformation has
provided plant breeders with new opportunities to breed for higher yields,
improved insect and disease resistance, tolerance to abiotic stress and herbicides,
new qualities of plant products etc. The potential environmental impacts
connected to the technology are in many ways similar to that of conventional
crops, although the novelty of some transgenic crops may involve new challenges
(Dale, Clarke & Fontes, 2002). More than 10 years have passed since the first
commercial transgenic crop was introduced in agriculture (James, 2006) but this
plant breeding technology is still not universally accepted. The potential risks of
negative environmental effects have resulted in extensive pre-release tests of
transgenic varieties where every new trait and crop combination and new
environment into which the transgenic crop is introduced have to be assessed (Nap
et al., 2003). However, benefits need to be acknowledged and weighed against the
risks, and in the case of transgenic insect resistance the result can be beneficial
both for the environment and the growers. Pest control today often relies on
pesticide application, an alternative that often is harmful to organisms besides the
targeted insect. At farm level the pesticides also expose the farmer to toxic
chemicals and require economic input in purchase, equipment, and labour.
In this thesis I have evaluated some of the anticipated benefits and risks with a
transgenic oilseed rape that express a protein from pea (Pisum sativum), pea seed
lectin (PSL). This plant material was the first product of an effort to develop
transgenic oilseed rape cultivars with resistance to an important insect pest in
northern Europe, the pollen beetle (Meligethes aeneus Fab.). However, the
commercial aspect was abandoned and the plant material is here used as model for
testing issues related to a transgenic insect resistant crop.
I will begin by describing the domestication and breeding of plants that resulted
in the crops we have today, continuing with the benefit and risk approach to
transgenic plants. I also present the history of the Brassica crop, pollen beetle
ecology, crop damage, and current control of the insect pest. The introduction ends
with a description of how the transgenic plants were developed and the initial
studies of their resistance to pollen beetles (Melander et al., 2003). The benefits
from the introduced trait with respect to negative effects on the pollen beetle had
previously only been studied to a limited extent. The damage from the pollen
beetle is mainly attributed to the hibernated adults and therefore it was necessary
to determine the effect of the transgenes on this life stage (paper I), but to be able
to draw any conclusions about the effects on the population level the entire life
cycle had to be considered (paper I and II) and in order to evaluate the potential
effects at population level several life history parameters had to be studied. In this
thesis I summarize results from pollen beetle assays with PSL oilseed rape (paper I
and II).
In the following section I give a short introduction to one of the most important
beneficial insects, the honey bee (Apis mellifera (L.)). Because the foreign protein
is only expressed in the pollen of the plant, the number of species that would be
exposed to the transgenic protein is rather limited. However, honey bees also feed
on pollen and any negative impact on this pollinator would be devastating. Here I
present the effects found on bee larvae feeding on pollen from the transgenic
oilseed rape (paper III).
The next section deals with the plant material and its potential increase in
competitive ability from the traits introduced by transformation. Insect resistance
reducing the damage to the resistant plant could give it a competitive benefit in
contrast to plants not carrying the resistance trait, which in turn could result in
increased invasiveness. Several wild relatives to oilseed rape can be found in the
vicinity of the crop fields in Europe, and some of these can most likely hybridise
with the transgenic crop. Feral populations of plants originating from Brassica
oilseed cultivation can also be found in and outside the fields. Therefore it was
important to test the competitive ability of the transgenic plants in comparison
with their conventional counterpart (paper IV).
Finally, application of plant biotechnology at farm level will depend on attitudes
among the producers. Surveys on attitudes often ask what position a person takes,
but often not why. Therefore I performed a survey among Swedish farmers on
their perception of potential benefits and drawbacks connected to insect resistant
transgenic crops (paper V).
Breeding for insect resistance
Domestication of plants
Evolution among plants, as all organisms, is driven by the selection for individuals
with the highest fitness; the ability to produce as many fertile offspring as possible
in that specific environment. Plants are forced to cope with UV-radiation, draught,
flooding, herbivores, and diseases in competition with other plant species as well
as their relatives. Evolution would not be possible without reoccurring genomic
changes; altered gene sequences, expressions, functions, or chromosome numbers,
creating new traits that give the plant an advantage over its competitors.
Depending on if the mode of reproduction is crossbreeding, self-fertilization or
vegetative, the trait may or may not be transferred to other plants and combined
with the genes of the receiving plants.
The domestication of plants started long before Darwin and Mendel enlightened
us about evolution and inheritance. As soon as humans started collecting seeds to
be cultivated more than 10 000 years ago (Balter, 2007), the man-made evolution
of crops began. Preparation of the soil, removal of weeds, draining, irrigation etc.,
have changed the conditions for natural selection and today, many domesticated
are plants more or less dependent on humans for their survival (Hyams, 1971).
Uniform ripening and shatter resistance are traits that facilitate harvest. In
addition, reduced toxicity for consumption and resistance to pests and pathogens
are preferable in crops and have been more or less consciously selected for during
the history of plant breeding. Breeding against toxicity to humans and livestock
and at the same time for resistance to pests can cause conflicts because many of
the substances toxic or repellent to pests, such as alkaloids, have the same effect
on humans. Selection against such traits often results in a crop more sensitive to
pests and an increased need for pesticide application.
Plant defence
Resistance to insects can be defined as inherited plant characteristics that reduce
the effect of insect attack compared to susceptible plants that are more severely
damaged (Smith, 1989). The less damaged plant might be avoided by the insect
(antixenosis) or be less suitable as a host (antibiosis). A third way to withstand
pest attacks is tolerance. An ideal resistance is a combination of all three
mechanisms where tolerance is most desirable since no pressure is put on the
insect to adapt (Smith, 1989). For crops in modern agriculture, antixenosis may be
insufficient since this may depend on what other choices the insects have, and
several hectares of monoculture may force the insect to choose the less preferred
host for feeding or oviposition. Large areas of susceptible host crops also increase
the population growth of the pest if it is not hampered by natural enemies or other
pest control methods.
Plants are protected against insect pests by both physiological and chemical
traits. Thick tissues and epidermis, spines, thorns, and hairs, in some cases with
glands, can prevent herbivory by insects and secondary metabolites,
allelochemicals, can be toxic, deterrent or repellent to the herbivore (Dethier,
1970) and/or attract natural enemies to the pest (Dicke, van Poecke & de Boer,
2003). Both chemical and physical defence can be constitutively expressed or
induced by herbivory, in the whole plant or restricted to specific plant parts.
Improving insect resistance in plants using biotechnology
It is often difficult to find resistance to insects in cultivars. Often one has to use
wild relatives, and if the resistance is a quantitative trait (affected by many genes)
crossings and back crossings to transfer the desired genes is very resource
demanding. Less desirable genes may also be linked with the gene of interest and
therefore difficult to get rid of by crossings and selections. Development in plant
biotechnology has enabled not only selection of single genes but also the
possibility to transfer genes from unrelated plants, other organisms, or even
synthetic genes, to the genome of a crop plant. For a gene to be translated, a
promoter is needed that is recognised by the transformed crop plant’s RNA
polymerases. The selection of promoter enables gene expression to be restricted to
specific tissues of the plant. The gene construct also needs to contain a selectable
marker that makes it possible to select successfully transformed plants. There are
two major techniques to make the transfer, one biological using the bacterium
Agrobacterium tumefaciens as a vector (Gheysen et al., 1985). Another, physical,
is to use a “genegun” where micro particles coated with the gene construct are shot
into living cells (Klein et al., 1987).
The first mission in the transformation procedure is to find a potential resistance
factor and to isolate the gene(s) coding for it. Several plant proteins have been
tested as potential resistance factors to be used against insect pests; for example
proteinase inhibitors (PIs) and lectins (Carlini & Grossi-de-Sá, 2002). But the only
insect resistant (IR) transgene crops grown commercially today are plant varieties
of maize and cotton resistant to insect pests by the transformation of genes coding
for δ-endotoxin from the bacterium Bacillus thuringiensis. There are several
hundreds of strains of B. thuringiensis coding for different δ-endotoxins, which
are highly specific toxins to certain insect groups but harmless to mammals (Betz,
Hammond & Fuchs, 2000).
Global status of transgenic crops
From the first commercialization in 1996 the global area of biotech/genetically
modified (GM) crops has increased to 102 million hectares in 2006 of which 32%
were IR GM crops (James, 2006). 19% of the IR GM crops contained “stacked”
IR and herbicide tolerance genes, making the plant resistant both to insect pests
and an herbicide. USA accounts for more than half of the acreage of transgenic
crops (54 million ha), followed by Argentina (18 million ha), Brazil (11.5 million
ha), Canada (6.1 million ha), India (3.8 million ha), and China (3.5 million ha)
(James, 2006). The first commercial planting of GM crops in Europe took place in
1998 in Spain, which still is the major grower in the EU. However, the 65 000 ha
planted with GM crops in Europe in 2006 (Brookes, 2007) make up less than 0.1%
of the global area planted with transgenes.
Risk/ benefit assessments
A risk can be defined as the likelihood of an event multiplied by (negative) impact
of that event (Conner, Glare & Nap, 2003). When addressing the introduction of a
transgenic crop, risks and benefits have to include both human health and
environmental effects. The countries that grow GM crops have different
frameworks regulating the release, growing and monitoring of transgenic crops
emphasising potential risks for humans and environment. However, the
philosophy behind the regulation can differ; New Zealand and EU demand
extensive information, focusing on the plant development process. This is in
contrast to, for example, USA and Canada that focus more on the product
(Conner, Glare & Nap, 2003).
The transgenic plant needs to be characterized, and the features by which the
transgene differs from its corresponding conventional variety determined. A
distinction has to be made between traits that occur in native plants and novel
ones, or traits from exotic plants or other organisms. Although a trait is found in a
native plant, expressing it in another plant species might expose new organisms to
the novel trait. Also, the level of the potential toxin might be higher in the
transgenic plant and expressed in other tissue than in the original plant species.
Different transformation events might lead to different levels of gene expression
or affect the expression of other genes. Therefore all transgenic crops have to be
approved on a case by case basis (EU, 2001). Environmental as well as
agricultural benefits have to also be addressed, the latter will be dealt with in the
section concerning the grower.
Toxicity to non-target organisms
Transgenes that are to be used for human and livestock food obviously have to be
evaluated for potential toxicity to the same, but also the toxicity to other organisms
that might come in contact with the plant, directly or indirectly, has to be
evaluated. Risk of direct effects on non-target organisms depends on the level of
exposure to the novel gene product i.e. if the organism feeds on the plant tissue
where the transgene is expressed, and the potential hazard or toxicity of the same.
The level of gene expression is also important for potential toxicity. The toxin can
also be passed on in the food web to predators of the plant herbivores (Birch et al.,
1999) and parasitoids (Romeis, Babendreier & Wäckers, 2003). The toxin might
also end up in the environment if it persists in the soil after plant material or
insects that ingested the toxin decay (Groot & Dicke, 2002). Plant proteins might
also end up in the soil through root exudates which can affect soil bacteria,
protozoa, nematodes, or earthworms (Groot & Dicke, 2002).
In the transgenic oilseed rape tested in my study, the expression of the pea lectin
was restricted to the anthers and pollen and thereby only organisms feeding on
those plant parts would be directly exposed to the novel protein. Oil from the
seeds is the only part of the plant that is used for human consumption today and
does not contain much protein, so even if the lectin were to be expressed in the
whole plant the level of lectin in the oil would be very low. The by-product from
oil extraction is, however, fed to livestock, which then would be exposed to the
transgenic protein. However, the protein comes from a plant used as both human
and animal feed, the garden pea, where it occurs at a concentration of about 2% of
seed protein (Edwards et al., 1991) which is more than twice the levels found in
the pollen of the studied oilseed rape (paper I). The only way that the pea lectin
will end up in the environment is through the rapeseed pollen, but a much more
important contribution to the environment should be from seeds of the pea plant,
the origin of the transferred lectin.
Gene introgression
The advantage that a transgenic plant would have over other plants by the new
added trait has also been widely debated. Concern has been expressed for the
creation of new weedy plants, the risk of the transformed plants crossing with wild
relatives, and that plants with the transformed trait could out-compete native
populations (Ellstrand, Prentice & Hancock, 1999). Crop plants in general are
rather poor at competing outside the fields; actually they are not strong
competitors in the field either, which is the reason for the use of herbicides and
tillage. But weedy plants that are the product of natural hybridization between a
crop and their wild relatives do exist (Ellstrand, Prentice & Hancock, 1999) and
this problem is not exclusive for transgenic plants.
For outcrossing to occur, the plants must be at least partially cross pollinated,
flower at the same time, and be related in such way that they produce fertile
offspring (Conner, Glare & Nap, 2003). For a wild plant to gain increased fitness
from crossing with the crop plant, traits that will improve the offspring’s total
fitness outside the field are required, or the gene has to be closely situated to other
beneficial genes in the genome (Chapman & Burke, 2006). However, most of the
crops traits will lower the fitness of the first hybrid generation. If conditions are
good though, the offspring might survive to reproduce, this time back crossing
with the wild plants and thereby regaining some of the traits needed to succeed in
the wild. Selection pressure will then favour individuals that have the most
favourable traits, of which one might be the transformed one. If this is at all
probable, one has to address what the consequences would be. Plants with the
gene would only benefit if the targeted herbivores are restricting the plant
population, or if other organisms on that plant are affected by the trait.
Concerns have been raised regarding the risk of horizontal gene transfer (i.e.
outside the reproductive process, across species barriers) of transgenic DNA or
marker genes from the vector (Kleter, Peijnenburg & Aarts, 2005; Weaver &
Morris, 2005). Whether or not transgenic DNA is more (or less) likely to be
horizontally transferred than non-transgenic DNA has not been determined.
Resistance development
The agricultural success of Bt is unquestionable but irrespective of GM or
conventionally bred resistance, the reliance on just one resistance factor makes it
likely that resistance will be of short duration. Eventually, somewhere, there will
be individuals that overcome the resistance. Precautions are taken to prevent pests
overcoming plant resistance, for example by gene stacking, susceptible crop
refuges and rotations of crops and insecticides.
Additional effects of gene transfer
The transformation process, tissue culturing, and gene inserts can have effects on
other genes in the plant genome (Latham, Wilson & Steinbrecher, 2006). Changes
in plant phenotype due to the transformation might affect the attractiveness and
nutritional value of the plant which is why the transgene is screened for nutritional
and known secondary compounds for comparison with the non-transgenic
counterpart (EU, 2003). The transformed gene might also behave differently in its
new environment (Prescott et al., 2005), such alteration could, however, also occur
by natural mutations. When a gene is expressed in the new genome it may affect
the expression of other genes, interact with those genes or function differently due
to post-translational processes compared to when expressed in the organism of
origin (Prescott et al., 2005).
Environmental benefits
Potential benefits must be identified in order to weigh them against the risks
posed. The major environmental benefit from insect resistant crops is the reduction
in insecticide application. Many insecticides persist in the environment affecting
many other organisms and sometimes also end up in our food and water.
Reduction in insect diversity and natural enemies of the insect pest makes pest
control even more dependent on insecticides. A positive side effect of reduced
insecticide application is reduction in CO2 emission from less machinery use
(Brookes & Barfoot, 2006). In some areas the growers experienced less fungal
disease in the Bt crops which may also lead to fewer fungicide applications
(Brookes, 2007).
The plant and its pests
Brassica history
The oldest and most widespread Brassica crop is turnip rape (Brassica rapa syn.
B. campestris), which grows both wild and cultivated in Europe and Asia. Both
south-east Asia and south-west Europe have been suggested as sites of origin for
the genus (Baranyk & Fábry, 1999). Seeds dating back to the Bronze Age have
been found (Baranyk & Fábry, 1999), and rapeseed (B. rapa and B. napus) was
cultivated in ancient Rome, mainly as green fodder (Fussel, 1955). During the 19th
century rapeseed production increased in Sweden and peaked in 1866 when 3000
tons were produced (Andersson & Granhall, 1954). Cultivation then declined,
probably due to access to imported mineral oil that could be used for lamps
(Baranyk & Fábry, 1999) and other vegetable oils for margarine and soap
production (Andersson & Granhall, 1954). The shortage of edible oils in Europe
after World War II caused the production of oilseed crops to increase again
(Appelqvist & Ohlson, 1972). Rapeseed cultivation was also adopted in Canada
and later on in USA and Australia (Baranyk & Fábry, 1999).
Rapeseed breeding and cultivation
Breeding of oilseed crops has, apart from an increased yield, focused on
glucosinolate and erucic acid content, resistance against pathogens and pests, frost
resistance (Baranyk & Fábry, 1999; Meyer, 1997), and lately also on transgenic
herbicide tolerance (Mazur & Falco, 1989). Today oilseed rape is grown for the oil
which is used in food production, and for technical applications such as fuel and
lubricants. A by-product is a protein rich meal which is used in fodder. Cultivation
of rapeseed reached over 25 million ha globally in 2006 with India as the largest
grower (7.3 million ha), followed by China (6.7 million ha), Canada (5.3 million
ha) and Europe (4.7 million ha) (FAOSTAT, 2007). In 2006 transgenic rapeseed
was grown on 4.8 million ha, but the only commercially available varieties are
transformed for herbicide tolerance and are restricted to Canada and USA (GMO
Compass, 2007). In Sweden rapeseed cultivation has shown an increase during
recent years (SJV, 2006) and was, in 2006, grown on 90 760 ha (FAOSTAT,
2007), which accounts for about 3 % of the arable land (not including farms with
less than 2 hectares). In contrast to the general decline in herbicide and fungicide
use in Sweden, insecticide use increased by over 20% (active substance) from
1998 to 2006. The increased insecticide use was mostly due to increased use in
peas and oilseed rape (SCB, 2007).
Brassica pests
A large number of insect pests cause damage to oilseed rape. For example: the
seed weevil Ceutorhynchus assimilis (Payk.) (Free & Williams, 1978; Gould,
1975), the pod-infesting gall midge Dasineura brassicae Winn. (Gould, 1975), the
aphid Brevicoryne brassicae L. and flea beetles of the genera Phyllotreta and
Psylliodes. But the most severe pest in oilseed rape in northern Europe is the
pollen beetle (Meligethes aeneus F.) (Alford, Nilsson & Ulber, 2003) which can
cause up to 70% yield loss (Nilsson, 1987) and accounts for the main insecticide
use in rapeseed in Sweden.
No insect resistant oilseed rape variety has been marketed although efforts have
been made to breed for resistance against the aphid Lipaphis erysimi Kalt. (Sekhon
& Åhman, 1992) and the flea beetle Phyllotreta spp. (Lamb 1989). In a search for
resistance sources, more than 90 populations of spring oilseed rape were screened
for insect resistance in south Sweden. Even though a couple of the most promising
populations gave higher yields than standard varieties when no insecticide was
applied, the highest yielding population only gave 36% of the yield compared to
when insecticides were applied (Åhman, 1993).
The major pest; the pollen beetle
The dominate pollen beetle species in oilseed rape in Sweden is M. aeneus
followed by M. viridescens (Karltorp & Nilsson, 1981). Among the 489 beetles
that were examined in my study, not a single M. viridescens was found. Neither in
the studies by Nilsson, was there any species other than M. aeneus detected in the
same geographic area (Nilsson, 1994). Meligethes aeneus will hereafter be
referred to as the pollen beetle.
Life cycle
The pollen beetle is univoltine (one generation per year) and hibernates as an adult
(Fig. 1). As the name implies, the pollen beetles mainly feed on pollen, in the fall
and early spring in a variety of plants. Later in spring they move to Brassica plants
to feed and reproduce, and the eggs are laid in small clusters preferably in 2-3 mm
large buds through beetle-made holes (Ekbom & Borg, 1996). Egg production can
last up to 2 months (Fritzsche, 1957), or as long as plants suitable for oviposition
are present (Hopkins & Ekbom, 1996), which can result in a production of several
hundreds of eggs per female (Fritzsche, 1957).
Plant damage
The most severe damage to the crop occurs before the flowers are open when the
beetles feed on the small buds, and if many eggs are laid in the same bud the
larvae might also damage the ovaries of the flower. The plant can compensate for
bud damage (Gould, 1975), or even overcompensate, by the production of side
branches (Tatchell, 1983). But this makes harvest difficult because seeds on side
branches ripen later than those on the main shoot. Pollen beetles do not normally
Fig. 1. The pollen beetle life cycle: The adults emerge in spring and feed on spring flowers
before emigrating to Brassica plants to feed, mate and oviposit. Eggs are laid in the buds
and the 1st instar larvae feed in the developing flower. The 2nd instar larvae continue to feed
in the open flowers before dropping to the ground for pupation in the soil. The eclosing
adults feed on a variety of flowers before overwintering in the top soil or leaf litter, usually
in a forest nearby. (Graphics by UppsalAnimation©)
fly when the temperature is below 14°C (Fritzsche, 1957) which usually means
that winter rape already has begun to flower before the beetles move to the crop,
but spring rape flowers later and can suffer severely when the beetles move to
those fields.
Control history
In the absence of rapeseed crops, the pollen beetle is restricted to its natural host
plants; cruciferous weeds; and reproductive success is dramatically increased as
soon as cultivation of Brassica is intensified (Hokkanen, 2000). During the 19th
century damage was occasionally so severe in Sweden that the crop could only be
used as green fodder or had to be ploughed under (Lampa, 1893 in Nilsson, 1994).
The first efficient pest control became available after the Second World War in the
form of DDT, which was replaced by organic phosphates (OP) in the 1960s, and at
the same time control thresholds were adopted to minimize insecticide use
(Nilsson, 1994). In the early 1980s the less noxious pyrethroids replaced the OP’s.
However, the extensive use of pyrethroids has resulted in development of
resistance in pollen beetles in several countries (Derron et al., 2004; Ekbom &
Kuusk, 2001; Hansen, 2003). In Sweden, farmers in such areas are granted
permission to use OP’s. From 2007 Swedish farmers are also allowed to spray
with neonicotinides, which are harmless for mammals but the recommendation is
instar larva
instar larva
to alternate between these substances to prevent or delay further development of
resistance. The economic threshold for spraying is close to the most favourable
density for the beetles to maximize their offspring, and thereby an unwanted side
effect of chemical protection is a high pest population in the following year
(Hokkanen, 2000; Veromann et al., 2007).
The making of a transgene
Finding the gene
In order to find a resistance factor that could be transformed into oilseed rape
several lectins, proteinase inhibitors (PIs) and patatin were tested in a feeding
assay using pollen beetle larvae and two lectins also on adults (Åhman &
Melander, 2003). The Concanavalin A (Con A) lectin from jackbean (Canavalia
ensiformis L.) caused the highest larval mortality of the tested plant proteins. The
problem with Con A is that it can be toxic to mammals, which makes the protein
unsuitable for transformation of crops used for food and fodder. Lectins are a wide
and heterogenic group of proteins with the common characteristic that they bind
reversibly to carbohydrates (Van Damme et al., 1998). In plants the lectins are
suggested to function as storage proteins, defence against herbivory and/or
nodulation of root hairs (Kijne, 1996). Function and toxicity of lectins towards
insects is highly variable and seems to be rather specific depending on the lectin
and insect species combination (Murdock & Shade, 2002), even within the same
insect order (Sétamou et al., 2002). Con A binds to mannose and glucose a feature
shared with the pea (Pisum sativum) lectin (PSL) (Strosberg et al., 1986), however
PSL is harmless to mammals (Grant, 1989). The posttranslational process of Con
A is also more complex than that of the PSL, which might lead to changed protein
structure when expressed in a different organism, and therefore the PSL was
chosen for further testing (Melander et al., 2003). When pollen beetle larvae were
fed oilseed rape anthers soaked in 1% solution of PSL, survival was reduced by
84% and the body mass by 79%. These results encouraged development of
transgenic oilseed rape expressing the PSL (Melander et al., 2003).
Introduction of the gene and plant line production
The pollen specific promoter of the Sta44-4 gene from B. napus was combined
with the PSL gene isolated from pea. This promoter was chosen in order to restrict
the expression of the lectin gene to the developing pollen in anthers, which should
minimize the impact of the transgene on both the plant and non-target organisms.
The Sta44-4-pea lectin gene complex was then ligated with the NPTII marker gene
(kanamycin resistance) to enable selection of transformed plants. The final
construct was transferred to the spring rape cultivar Westar using A. tumefaciens
as a carrier (Melander et al., 2003).
Self pollination of transformed plants (T0) and their offspring (T1) gave a second
generation of inbred plants (T2) and their anthers were analyzed for PSL content.
Twenty of the primary transformants were tested in a feeding assay on pollen
beetle larvae; a slight reduction in survival and up to 46% weight reduction was
recorded (Melander et al., 2003). A negative correlation between lectin
concentration in anthers and larval weight was detected. In order to reduce the risk
of segregation for transgenity in following generations, doubled haploid (DH)
plants were generated from microspores (from T3 plants) (Åhman et al., 2006).
Plants from five transformation events were selected as microspore donors, of
which two did not contain any PSL. Those zero plants were expected to be non-
transgenic, since their ancestors were negative for the NPTII gene construct when
tested in generations T1 and T2. The T2 generation of the transgenic plant lines had
been tested in a feeding assay with pollen beetle larvae that resulted in a reduction
in larval growth by 13-24%. (Melander et al., 2003). Among the 171 DH lines
produced, five plant lines (one from each transformation event) were selected for
further testing (Åhman et al., 2006).
Transgene assays
Lectin quantification
Anthers from each of the five DH lines, designated T-17, T-83, T-132 (transgenes)
and C-101 and C-112 (controls), used in my studies were sampled in 2004 for PSL
quantification. The pollen specific promoter used in the construct results in gene
expression late in the pollen development (Hong et al., 1997). Therefore both buds
and recently opened flowers were selected to determine if there was any difference
in PSL expression in those two stages of developing anthers, but no such
differences were found (paper I). The PSL concentration in the anthers was 0.2,
0.6 and 0.7% (of total soluble protein) in T-83, T-132 and T-17 respectively. No
lectin could be detected in the anthers of the control lines (paper I).
Pollen beetle assays
The initial test of PSL-expressing oilseed rape on pollen beetle larvae showed a
negative effect; a reduction in body mass (Melander et al., 2003). A low adult
weight, which could be the result of low larval weight, has been shown to reduce
the beetle’s winter survival rate (Hokkanen, 1993). The adult stage is also the most
damaging to the rapeseed crop and therefore the effect of PSL on adults was of
particular interest. Additionally, even though a substance is not acutely toxic,
small effects on various life history parameters can lead to a decline of the
population (Larsson, Ekbom & Björkman, 2000; Sétamou et al., 2003). To be able
to calculate the potential effects of the transgene on pollen beetles at the
population level, effects of the five selected plant lines, were tested on all life
stages of the pollen beetle.
Beetles were collected as soon as they emerged after hibernation and stored cold
until used in the different tests. The effect on adult beetles when fed only anthers
(where the PSL was expressed), was tested by controlling the weight change and
feeding for individual beetles every third day over a three week period (paper I).
No significant difference in feeding, weight or survival could be detected between
the plant lines.
Oviposition was tested by first isolating groups of beetles in cages with one of
the five plant lines to allow them to feed and mate (paper I). The beetles were then
weighed and individually isolated on racemes, from the same plant as the pre-
treatment, for 10 days. Every second day the beetles were moved to a new raceme
and the number and length of the eggs laid in the buds recorded. No difference in
insect mortality between the plant lines was found, neither could any difference in
number of eggs laid be detected. However, the egg sizes tended to increase for
beetles feeding on the control lines but were unchanged or decreasing for the
transgenic lines. Egg size is known to be reduced on suboptimal hosts (Ekbom &
Popov, 2004) and it would have been interesting to continue to follow the females
until they ceased to oviposit to see if egg size continued to diverge. From a
resistance point of view egg size was of less interest since no correlation between
egg size and survival, weight or development time was found in the following
study on larval development (paper II). Females lost more weight than males
during the raceme test. This is probably due to oviposition reducing the weight,
which was supported by the fact that no difference in weight change could be
detected in the anther study where the females had no opportunity to lay eggs.
However, there was no correlation between weight loss and number of eggs laid.
To obtain eggs for tests of larval development and survival (paper II) adults were
isolated on the different plant lines in cages in order for them to feed and mate and
then isolated smaller groups on individual plants for 24 h. Eggs were then
dissected from the buds and the developing larvae were continuously fed anthers
from the test plants. Hatching, development time until adult, larval weight and
survival were recorded. The only difference in larval weight was found between
C-112 and T-132; larvae feeding on C-112 weighed significantly more. The main
effect was found on survival until pupation where larvae on all the transgenic lines
had significantly higher mortality compared to the control lines (paper II).
Adult offspring
Groups of beetles from the previously described pre-treatment cages were isolated
on several plants for 24 h. The emerging larvae fed on the same plant before
pupating in the pot soil, and eclosing beetles were collected and weighed. This test
was performed both in 2004 and 2005 and gave somewhat contradictory results;
beetles developed on T-132 had the lowest mean weight in 2004 but the highest in
2005 (paper II). There was no consistent difference in weight between beetles
from the group of transgenic and the group of control lines, instead a significant
difference was found between the two control lines in 2005. The result implies
that there might be some other plant quality affecting offspring weights, which
will be discussed below.
I also wished to follow beetles developed on the transgenes throughout the
following winter and during the winters 2003-2006 I tested winter survival.
Initially (2003-2004) I tested survival in groups divided by weight among beetles
collected in the autumn. The proportion of survivors was higher in the groups with
heavier beetles and the average weight among the hibernated beetles only
increased among the small beetles. These results are in accordance with the results
from the hibernation study by Hokkanen (1993) where the mean average weight
among the pollen beetles increased from autumn to spring.
The following two years beetles produced in the test for adult weights were
overwintered, but it was only in the second year any beetles survived to the
following spring (paper II). The less successful hibernations during those last two
years might be due to colder winters, but the 2.4% survivors collected in the
spring in 2006 is within the range of the 2-15% survival recorded by Hokkanen
(1993). The fact that no surviving beetles were found the previous spring might be
due to the lower number of beetles tested that year (paper II). One might argue that
the low number of surviving test beetles depends on the more or less artificial
rearing, compared to beetles collected in the field, but in both years survival of the
reared beetles was compared with survival of beetles collected outdoors a couple
of weeks before the pots were placed in the ground, and mortality was equally
Population effects
Melander et al. (2003) suggested that there is a dose response to PSL in pollen
beetle larvae, something I did not find clear evidence for in my tests. The lectin
content in T-83 was an intermediate between control plants and the high PSL lines
but not the results, something that would be expected if the effect was dependent
on the level of PSL. Even though open pollinated rapeseed varieties are produced
to be as uniform as possible to start with, they are not clones and also, during
subsequent propagation steps individual plants may become more and more
different from one another via crossings. Unfortunately, I had no control over the
genetic uniformity of the seed lot of Westar used for the transformations.
Furthermore, the transformation and tissue culturing might also have altered or
disturbed the expression of other genes in the plants (Latham, Wilson &
Steinbrecher, 2006), affecting the pollen beetle in unpredictable ways.
All parameters measured are summarized in table 1, and the results from the five
plant lines are ranked according to beetle performance. When summarizing the
effects shown in the table, certain patterns for transgenic and non-transgenic lines
emerge, but far from all measured parameters differ significantly and therefore the
ranking should be interpreted with caution. However, even if only considering the
parameters in which the plant lines differed significantly, the effects of the PSL
expressing oilseed rape would reduce the pollen beetle population by half
compared to the control plants (paper II).
Table 1. Summary of parameters measured on the pollen beetle when fed the five plant lines
(transgenes T-17, T-83, T-132, and control lines C-101 and C-112). The ranking of plant
lines corresponds to the beetle performance; highest feeding, weight gain, and survival,
highest number of eggs laid, largest eggs, and least weight loss, highest larval weight,
shortest development time, and highest percentage of beetles surviving the winter are given
rank one (lowest rank = five). Different letters indicate significant difference between plant
lines (P < 0.05 for specific tests se paper I and II). Mean ranking of plant lines when
considering all parameters measured
Adults - Anther assay
Adults - Raceme assay
feeding weight
change1 survival ovi-
position egg size weight
changem survival
T-83a C-101 C-101* T-132 C-101a T-83 C-101 C-112
C-112ab C-112 C-112* C-101 C-112ab C-112 T-132 T-83
T-17ab T-132 T-17* C-112 T-17ab C-101 T-17 T-17
C-101ab T-83 T-83* T-17 T-83ab T-17 T-83 C-101
T-132b T-17 T-132 T-83 T-132b T-132 C-112 T-132
Adult weight
weight dev. time
to adult 2004 2005 2005-2006
C-112a T-83a C-112a C-112a T-132a C-101 C-112 2.3
T-17ab C-101ab C-101a T-17ab C-112a T-83 C-101 2.5
C-101ab C-112b T-132ab T-83ab T-17ab T-132 T-83 3.0
T-83ab T-17b T-83ab C-101ab T-83b T-17 T-17 3.3
T-132b T-132b T-17b T-132b C-101b C-112 T-132 3.9
1 both sexes, f females, m males, * same value
Honey bee assay
Which insects besides the targeted pollen beetle might be affected by the
transformed plants? Because the expression of the PSL gene is restricted to the
anthers and pollen in the plant, other pollen feeders would be the major organisms
directly exposed to the protein by feeding. The honey bee is one of the most
important beneficial insects in agriculture and many crop plants over the world are
more or less dependent on bees for reproduction (Klein et al., 2007). Moreover,
oilseed rape yield and oil content in seed have shown to to be higher when the
apiary is close to the field (Fries & Stark, 1983). With wild bee species declining
insect pollinated plants are even more dependent on the honey bee (Kearns,
Inouye & Waser, 1998; Westerkamp & Gottsberger, 2000) and in the pre-release
tests of transgenic crops, the honey bee is one of the first insects to be tested.
Bees are social insects and the worker honey bees collect pollen and nectar which
is brought to the hive. If the novel gene is expressed in the whole plant the bees
could be exposed to the transformed protein through the nectar, but the amino acid
(protein) levels in nectar are very low and it is through the pollen the bees would
come into contact with the transgenic protein. The nursing bees in the hive feed on
pollen, not only to provide themselves with protein but also to produce royal jelly,
which is fed to the larvae (Crailsheim et al., 1992; Haydak, 1963) during the first
3-4 days in larval development (Crailsheim, 1990). Later on the brood food
contains less protein and more sugar from nectar and also some pollen
(Babendreier et al., 2004; Crailsheim, 1990). The exact composition of the food
depends on whether the larvae are raised to become a drone, worker or a queen. It
has been suggested, but so far not proven, that proteins could be transferred to the
larvae through the royal jelly produced in the hypopharyngeal glands. Many
lectins are stable proteins, insensitive to insect proteases and variations in pH
(Peumans & Van Damme, 1995), and could hypothetically pass through bee
digestion without degradation. In the young bee larvae the peritrophic membrane
that lines the midgut epithelium is not fully developed (Davidson, 1970), which
probably makes them more sensitive to harmful or toxic substances in the food.
Selection of test plants and protein quantification
Three plant lines from the initial tests on pollen beetles were selected using results
from the pollen beetle assays; plant line T-132 had the most negative effect on the
beetles while the control C-112 had the most positive effect. Additionally line T-
17 was also tested because this plant line has a similar level of PSL as T-132
(paper I). The PSL quantification in the dried pollen showed higher PSL content
compared to the freshly frozen anthers, which was probably due to lower total
protein content because of protein degradation (paper III). No lectin could be
detected in the control lines.
Feeding assay
I wanted to create a worst case scenario where the larvae would receive as much
pollen as possible during the early stages of their development (paper III). To test
a potential toxic protein such as PSL on bee larvae under natural and, at the same
time, controlled conditions is difficult because sick and dead larvae would be
removed by nurse bees several days before being detected by the human eye
(Brødsgaard, Ritter & Hansen, 1998). Methods have therefore been developed to
culture bee larvae in the lab. In my study newly hatched larvae were collected
from a comb and gently placed on the surface of the food, which was a mix of
sugar solution, royal jelly and pollen. The amount of pollen fed to the larvae in the
hive changes during development and the exact amount eaten by the larvae may
vary with the protein content of the pollen. Before testing the transgenic pollen I
therefore examined how much oilseed rape pollen could be fed to the larvae
without increasing their mortality (paper III). The optimal test would have
included a gradual increase in pollen as larvae developed, since the second instar
might cope with higher pollen levels. However, the levels at which the larval
mortality increased was approximately the same when tested from day 4 and
onward (Brødsgaard pers. comm.).
Diet without pollen was used as an extra control for possible negative effect of
pollen in general. Significant differences were found between treatments.
However those differences were not between control and transgenic plants but
between diet with and without pollen. Larvae fed diets containing pollen
developed faster and weighed more than larvae fed the control diet, although the
control diet is supposed to contain the nutrition needed for the larvae. The
mortality was rather high in all diets compared to other studies on bee larvae
(Aupinel et al., 2005; Genersch, Ashiralieva & Fries, 2005), which was probably
due to handling of the larvae, but there was no difference in mortality between
diets until pupation. Although only tested on larvae and with dried pollen, my
results indicate that PSL at 1.2% of total soluble protein poses no hazard to the
honey bee. Adult bees, especially the nursing bees, would eat more pollen
compared to the larvae who receive the pollen in the brood food mix. Thus if a
PSL containing crop variety were to be considered for commercial cultivation
adult bees would also have to be tested.
Plant invasiveness
The invasiveness of a plant, its ability to spread to, establish and expand at new
locations, depends on many factors such as mode of pollination, seed dispersal,
competitive ability, and environment (Chapman & Burke, 2006; Conner, Glare &
Nap, 2003). If wild relatives to the plant are growing in the range of pollen
dispersal hybrids might form that could out-compete the native population.
In order to test if the oilseed rape had gained a higher competitive ability from
the transgenic character in our case, a greenhouse study was performed where the
transgenic plant lines were grown alone and in mixtures with the control lines,
with and without bumblebees (Bumbus terrestris (L.)), and with and without
pollen beetles (paper IV). When the transgenic plants were grown in mixture with
control plants, in the experiment with pollinators but without pollen beetles, they
produced more viable seeds than when grown alone. There was an opposite trend
for the control plants, which implies that there might be lower viability in the
pollen from the transgenic plants. This might be due to the expression of the
foreign protein affecting the pollen or that the transformation caused
rearrangements in the genome (Forsbach et al., 2003). The PSL did not seem to
give the plants any competitive benefit with respect to reduced pollen beetle
damage. In fact, in the absence of bumblebees the pollen beetle had a positive
effect on the plant yield, presumably due to increased self-pollination of the plant
by the beetle.
The grower
Apart from the potential changes in plant ecology of the transgene there might also
be changes in farming practices that have to be included in a risk/benefit
assessment. There have been several benefits at farm level from growing IR GM
crops. Reports from the first 10 years of commercial use of IR GM crops show
economic benefits from decreased insecticide and machinery use, as well as
reduced labour costs including scouting. Reduced pesticide use means less damage
to other organisms in the field and reduced exposure to the farmer who handles it.
At farm level the risk of GM crops crossing with non-GM crops can be
important for both the GM farmer and the non-GM farmer. In the EU, products
that contain more than 0.9% GM (unintentional mix) have to be labelled (EU,
2003). A non-GM farmer may receive a lower price if transgenes are mixed in his
harvest at higher levels than 0.9% and thereby suffer economic loss.
Economic benefits
Of the 10.3 million farmers growing GM crops in 2006, 9.3 million were small,
resource poor farmers in developing countries (James, 2006). Non governmental
organisations (NGOs) often state that the only beneficiaries from GM-crops are
the large seed companies. However, calculations show that, besides the seed
companies, both consumers and growers have benefited economically from the
application of the technology (Fernadez-Cornejo & Caswell, 2006). Among the
growers in developing countries not only economic benefits are important. Due to
poor application techniques and equipment they are often highly exposed to
pesticides and thereby have much to gain in improved health from growing IR
The expenses for GM seeds and technological fees need to be weighed up by
increased yield or higher quality products and/or reduced production costs in order
to be beneficial for the grower. Additionally, there must be retailers willing to buy
the product. Other benefits such as less exposure to insecticides and reduced
impact on beneficial insects and environment may also motivate a grower to select
a GM crop.
No GM crops are grown for commercial use in Sweden so far, and the IR GM
crop approved in the EU; Bt maize resistant to the European corn borer (Ostrinia
nubilalis) and the Mediterranean stemborer (Sesamia nonagroides), is currently of
no interest to Swedish farmers. Maize is, however, increasing as a crop in the
south of Sweden and with a warmer climate one can expect an increase of pests on
maize. With GM products beginning to enter the Swedish market and with the
approval of GM soy-meal as feed in Swedish meat production, farmers already
have to make choices about GM technology, despite the fact that they do not yet
grow them in their fields.
Farmer survey
What do the Swedish farmers believe are the potential benefits and drawbacks if
they were to grow IR GM crops? In a survey in 2005, Swedish farmers were asked
first about their attitude towards GM crops in general, and then asked two
questions where they selected statements about potential benefits and drawbacks
of an IR GM crop (paper V). This meant that we could connect their general
attitude to their perception of benefits and risks, and also get an idea about how
selections of specific benefits were connected to perceived risks. A majority, 57%,
was negative to GM crops but as many as 30% were neither positive nor negative.
A higher number of growers with large farms were surveyed than what is
representative for Swedish farmers, and the response frequency was also higher
among owners of large farms. Owners of large farms were more positive towards
GM crops in general, which means that the actual percentage of farmers in
Sweden that are negative to GM crops probably is higher than that in the survey
(paper V). The difference in attitude between owners of small and large farms
might be due to the fact that larger farms generally use more insecticides and
therefore have more to gain from growing an IR GM crop. Furthermore, owners of
large farms more often had higher education in agriculture which was correlated to
a more positive attitude towards GM crops.
The major concern among all farmers in the survey was scepticism among
consumers, which may result in an unwillingness to buy the product. Only 20% of
Swedish consumers are willing to buy GM foods. However, more than twice as
many would buy such food if the crop was grown in a more environmentally
friendly way compared to conventional crops (Fjæstad, Olofsson & Öhman,
2003), something that the farmers might not be aware of.
The relation between attitude and selected statements reveal four groups of
farmers with different concerns and expectations (paper V). A general positive
attitude to GM crops was connected to potential for reduced damage to other
organisms, reduced insecticide costs, and reduced health risk for the grower as
well as concerns about more expensive seeds. The contrasting group saw no
benefits from growing IR GM crops and believed that the crop could be dangerous
for both humans and other organisms. A third group saw higher yields as the most
probable benefit but were, at the same time, concerned about consumer attitudes to
GM products. The fourth group (contrasting the third) was concerned about genes
spreading from the GM crop to both wild relatives to the plant and to conventional
crops. The farmers could also point out additional potential benefits and
drawbacks that they found important in connection with the questions. The most
frequently mentioned concern was the lack of knowledge about the technology.
This might reflect why as many as 30% claimed to be neither positive nor negative
to GM crops. The selection of statements by “neutral” farmers often coincided
with the selection by positive farmers, which may be interpreted as many farmers
acknowledging the benefits but at the same time experiencing high uncertainty
about the potential risks.
I have developed a system for testing the effects of Brassica host characters on
performance of pollen beetles and found that PSL oilseed rape can reduce the rate
of population increase. The transgenic plants did not gain any competitive benefit
by their negative effect on the pollen beetle; neither did the feeding assay with the
honey bee larvae indicate any negative effects. All my assays were conducted
under greenhouse and/or lab conditions. Although such studies provide essential
knowledge, it is not until field trials are performed that the full impact can be
Furthermore, my studies show the importance of testing more than one
transgenic and one control line to be able to draw conclusions about the effects of
the transgene. It may be important to screen for alterations in the genome
introduced by the transformation, doubled haploid production, or tissue culturing,
and to control for possible genetic heterogeneity in the starting material for
transformations. Also negative effects of the transgene on the plant, such as the
reduced pollen quality need more attention.
The question is if we can find another toxin, as effective and precise as Bt, for
the control of pests. We may have to accept some infestations in the field to make
crop production as environmentally friendly as possible. Although pollen beetle
larvae showed an increased mortality, the PSL oilseed rape by itself is probably
not the single solution to the pollen beetle problem. If PSL oilseed rape were to be
grown at larger scales it might, together with natural enemies, which under current
pest control are hampered by insecticides (Veromann, Luik & Kevväi, 2006;
Veromann et al., 2006), prevent rapid population growth of pollen beetles. This is,
of course, contingent on there being no negative effects of the GM crop on the
natural enemies. It should also be kept in mind that regardless of method used to
control the pest, if the pest population is reduced, it will affect the biological
community (Shelton, Zhao & Roush, 2002).
The goal for future agriculture should be to use farm practices that have as little
environmental impact as possible for the attained yield. A sustainable agricultural
practice should ideally implement all the available knowledge, and this might
include the usage of transgenic crops. When decisions are to be made regarding
new crop varieties, improved by gene transformation or through other plant
breeding techniques, both risks and benefits need to be addressed, and be based on
scientific knowledge.
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Acknowledgements – Tack!
För en som är så självcentrerad som jag, så passar det väl bra att börja
acknowledgements med att prata om mig själv. Jag är kanske inte lat, men väldigt
bekväm. Så Barbara och Inger, jag vet inte hur ni burit er åt men nu är jag alltså i
mål. Barbara, du har all min respekt och det är väl lika bra att jag erkänner att jag
var lite rädd för dig i början. Du har en fantastisk förmåga att känna när jag behövt
uppmuntran, eller när jag behöver en knuff, eller ja en spark framåt. Trots många
andra uppdrag har du alltid tid för dina studenter. Inger! Om det inte varit för dig
så hade jag kanske inte doktorerat överhuvudtaget. Och att kalla dig biträdande
handledare känns inte helt rätt då du varit delaktig i mer eller mindre allt jag
företagit mig under åren. Jag hoppas att jag smittats av din noggrannhet, även om
jag inte alltid verkat helt nöjd när mina manus kommit tillbaka med mer röd än
svart text… Alla goda ting lär ju vara tre och så även i detta fall. Johan, mitt
molekylärbiologiska skyddsnät! Även om vi inte jobbat ihop någon längre tid har
det varit väldigt skönt att du funnits till hands och jag uppskattar att du tagit dig tid
när frågorna hopat sig.
Det finns fler som bidragit till att det finns några artiklar och manus i
avhandlingen. Plötsligt på vårkanten kom en låda från Danmark och jag kastades
in i en värld av hypop..hypofaryngeala körtlar, grafting tools och drottninggelé,
som förresten INTE är gott (borde vetat bättre än att fråga en rättsentomolog?).
Som tur var fick jag guidning av ett gäng med koll på bin. Ingemar, till slut blev
det rätt i alla fall! Tusen tack för att du tog dig tid. Eva, utan dig hade isoleringen i
källaren i 30 graders värme nästan känts tråkig. Tack för all hjälp & lycka till med
avhandlingen! Och Anders, hur många strängar har du egentligen på din lyra (har
han någon lyra överhuvudtaget? Kanske en tung bas?)? Jag saknar B-korridoren
med Anders trummande i skrivbordet & Ingemars svärande över dåliga manus
(men det här med Mac…).
One of the papers would definitely be of questionable quality without the efforts
of England’s most lovely girl Katy, clever and annoyingly young! Those 11 days
in the Boldrewood bunker was without doubt efficient, but never underestimate
the scientific value of shopping! Just so you know, I see it as my mission to hook
you up with a certain football player..
Per ”formel1” Hydbring! Ibland har man tur. Om jag inte varit sådär trött på
doktorandrådsmötet hade jag antagligen inte fått tillfälle att på kort tid och med
bästa tänkbara hjälp, göra det molekylära jobb som krävdes för att mina artiklar
skulle bli publicerbara. Tur också att jag hann utnyttja LG’s grupp innan ni
försvann till KI! Se nu till att lösa cancergåtan så att jag kan skryta med att jag
jobbat med dig!
Under de första tre åren spenderade jag somrarna i Svalöv och där blev ni,
Helena, Yvonne och Lillemor min andra familj. Stort tack till övriga på
oljeväxtavdelningen på SW som gjorde att jag alltid kände mig välkommen.
I am very grateful for being part of the department of Entomology, to me you
guys was everything a new PhD student could ask for. A big thank you to ALL the
people in the house, who contribute to the positive atmosphere. Ingen nämnd,
ingen glömd brukar det ju heta men det finns speciell person på institutionen som
var väldigt viktig för mig som ny och förvirrad student, Hjördis! Nu har du ju fått
sällskap av ett par som inte heller är så tokiga men din hjälp och omtänksamhet
har betytt otroligt mycket! Tack till alla sköna doktorander på institutionen!
Sandra, du var den första jag träffade på entomologen och du har liksom Kakan
blivit en av mina bästa vänner i huset. Du är den mest omtänksamma och
osjälviska person jag träffat, men lite mer ego har du kanske ändå blivit i mitt
sällskap? Kakan, min roommate. Det har hänt en hel del under de här åren eller
hur? Nu har ju jag varken gift mig eller blivit gravid men ändå. Något som inte
förändrats är prasslet av kak- & godispapper framåt eftermiddagen och att jag
fortfarande tror på allt du säger!? Nu får du ju dras med mig ett tag till men passar
på att tacka för sällskapet och att du hållit kvar mig på marken när jag varit på väg
upp i det blå. Ska nog ta och köpa den där Kalle Jularbo-skivan ändå.
Thank’s to the chemical ecology group in Alnarp, especially Bill and Marcus.
You were the ones who introduced me to research and made me believe that this
might be something for me!
Jag har även haft ett liv utanför arbetstid (!) och där finns det en så lång rad av
människor så det nästan skulle krävas en separat avhandling för att tacka alla!
Mina vänner sedan barndomen, Anna & Helena, och Linda som, trots att vi bor en
bit ifrån varandra alltid finns där. Det fantastiska Lundagänget (utan inbördes
ordning!); Sussi, Matilda, Astrid, Manne, Micke, Siv, Johan, Åsa, Nina m fl. Tack
Micke för att du vågade sammanföra dina två mest envisa & bestämda vänner! Det
har lett till både det ena & andra. Nyår i Hassela x 5, charter i Halmstad med mina
partners in crime Tina (det är inte aktuellt) & Nina (jag sitter på bussen). På köpet
fick jag också lära känna Norrlands Nations antagligen galnaste gäng, bl. a. Marita
(var är min väska?), jag hoppas att du flyttar (eller jag kanske?) lite närmare för nu
ses vi på tok för sällan.
Tack till Tannér-Persson’s, min extra familj i Skåne, som jag alltid lyckades
hälsa på när det vankades mat! Men är det inte lite skumt att jag fått två bilar
lagade otaliga gånger men aldrig sett mekanikern? Misstänker att du Jan är som
Stålmannen, fast istället lagar du bilar i hemlighet..
Lugnet infinner sig alltid när jag får chans att besöka mitt barndomshem
Bäckäng. Karin, Christian & Fredrik, tack för att ni tar så bra hand om vårt gamla
hus! Ser fram emot att fira Lille-Jul med er i minst 18 år till!
Tack till min underbara familj. Mamma, den bästa som finns. Vad jag än tar mig
för så är du alltid på min sida, även om det inte varit lätt att hänga med i svängarna
(läs humör). Pappa! Det här trodde du väl inte (och inte jag heller) när jag hoppade
av gymnasiet efter 2 månader? Vi är ju lika du & jag, vilket tog tid att erkänna,
och du förstår mig så väl. Marianne, tack för att du från dag ett fått mig att känna
mig välkommen! På köpet fick jag Lundahlbröder x 3! Tack också till min bror
Mats med familj som jag är otroligt stolt över men träffar allt för sällan.
Sist, killen som bidragit till att göra det sista året till en roligare resa och som
envisats med att jag kunde klara av det här. Tack Daniel, jag tror på dig också.
... Sources: AgroAtlas, 2009;Bromand, 1990; Bartlet et al., 1996;Hokkanen and Wearing, 1996;Ruther and Thiemann, 1997;Girard et al., 1998;Kift et al., 2000;Ulmer, 2002;Ester et al., 2003;Du Toit, 2007;Kazana et al., 2007;Khattab, 2007;Lehrman, 2007;Valantin-Morison et al., 2007;Smallegange et al., 2007;Knodel and Ganehiarachchi, 2008;Cartea et al., 2009. ...
... Of the 10 Meligethes species reported from brassicaceous plants in Europe, M. aeneus is by far the most common on cultivated brassicas (Blight and Smart, 1999). Pollen beetles are considered important pests on oilseed brassicas and cause serious yield losses to oilseed rape crops, with yield reductions of more than 80% reported (Lamb, 1989;Ekbom, 1995;Ekbom and Borg, 1996;Ruther and Thiemann, 1997;Ekbom and Ferdinand, 2003;Bartlet et al., 2004;Hansen, 2004;Williams, 2006;Kazachkova, 2007;Lehrman, 2007). Although pollen beetles are important pests, particularly for oilseed rape in Northern Europe (Jönsson and Anderson, 2007), and turnip rape, they do not oviposit on all Brassicacea plants (Bartlet et al., 2004). ...
... Sources: AgroAtlas, 2009;Bromand, 1990; Bartlet et al., 1996;Hokkanen and Wearing, 1996;Ruther and Thiemann, 1997;Girard et al., 1998;Kift et al., 2000;Ulmer, 2002;Ester et al., 2003;Du Toit, 2007;Kazana et al., 2007;Khattab, 2007;Lehrman, 2007;Valantin-Morison et al., 2007;Smallegange et al., 2007;Knodel and Ganehiarachchi, 2008;Cartea et al., 2009. ...
... Of the 10 Meligethes species reported from brassicaceous plants in Europe, M. aeneus is by far the most common on cultivated brassicas (Blight and Smart, 1999). Pollen beetles are considered important pests on oilseed brassicas and cause serious yield losses to oilseed rape crops, with yield reductions of more than 80% reported (Lamb, 1989;Ekbom, 1995;Ekbom and Borg, 1996;Ruther and Thiemann, 1997;Ekbom and Ferdinand, 2003;Bartlet et al., 2004;Hansen, 2004;Williams, 2006;Kazachkova, 2007;Lehrman, 2007). Although pollen beetles are important pests, particularly for oilseed rape in Northern Europe (Jönsson and Anderson, 2007), and turnip rape, they do not oviposit on all Brassicacea plants (Bartlet et al., 2004). ...
... These insect pests are the major un-equalisers of growth and crop yield of brassicas and their importance varies by geographical location (Hokkanen and Wearing, 1996; Kanrar et al., 2002). Some of the major insect pests that attack Brassicacea crops worldwide are listed in Table II and presented inFigure 3. Sources: AgroAtlas, 2009; Bromand, 1990; Bartlet et al., 1996; Hokkanen and Wearing, 1996; Ruther and Thiemann, 1997; Girard et al., 1998; Kift et al., 2000; Ulmer, 2002; Ester et al., 2003; Du Toit, 2007; Kazana et al., 2007; Khattab, 2007; Lehrman, 2007; Valantin-Morison et al., 2007; Smallegange et al., 2007; Knodel and Ganehiarachchi, 2008; Cartea et al., 2009. ...
... Of the 10 Meligethes species reported from brassicaceous plants in Europe, M. aeneus is by far the most common on cultivated brassicas (Blight and Smart, 1999). Pollen beetles are considered important pests on oilseed brassicas and cause serious yield losses to oilseed rape crops, with yield reductions of more than 80% reported (Lamb, 1989; Ekbom, 1995; Ekbom and Borg, 1996; Ruther and Thiemann, 1997; Ekbom and Ferdinand, 2003; Bartlet et al., 2004; Hansen, 2004; Williams, 2006; Kazachkova, 2007; Lehrman, 2007). Although pollen beetles are important pests, particularly for oilseed rape in Northern Europe (Jönsson and Anderson, 2007), and turnip rape, they do not oviposit on all Brassicacea plants (Bartlet et al., 2004). ...
... These insect pests are the major un-equalisers of growth and crop yield of brassicas and their importance varies by geographical location (Hokkanen and Wearing, 1996; Kanrar et al., 2002). Some of the major insect pests that attack Brassicacea crops worldwide are listed in Table II and presented inFigure 3. Sources: AgroAtlas, 2009; Bromand, 1990; Bartlet et al., 1996; Hokkanen and Wearing, 1996; Ruther and Thiemann, 1997; Girard et al., 1998; Kift et al., 2000; Ulmer, 2002; Ester et al., 2003; Du Toit, 2007; Kazana et al., 2007; Khattab, 2007; Lehrman, 2007; Valantin-Morison et al., 2007; Smallegange et al., 2007; Knodel and Ganehiarachchi, 2008; Cartea et al., 2009. ...
... Of the 10 Meligethes species reported from brassicaceous plants in Europe, M. aeneus is by far the most common on cultivated brassicas (Blight and Smart, 1999). Pollen beetles are considered important pests on oilseed brassicas and cause serious yield losses to oilseed rape crops, with yield reductions of more than 80% reported (Lamb, 1989; Ekbom, 1995; Ekbom and Borg, 1996; Ruther and Thiemann, 1997; Ekbom and Ferdinand, 2003; Bartlet et al., 2004; Hansen, 2004; Williams, 2006; Kazachkova, 2007; Lehrman, 2007). Although pollen beetles are important pests, particularly for oilseed rape in Northern Europe (Jönsson and Anderson, 2007), and turnip rape, they do not oviposit on all Brassicacea plants (Bartlet et al., 2004). ...
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Brassica crops are grown worldwide for oil, food and feed purposes, and constitute a significant economic value due to their nutritional, medicinal, bioindustrial, biocontrol and crop rotation properties. Insect pests cause enormous yield and economic losses in Brassica crop production every year, and are a threat to global agriculture. In order to overcome these insect pests, Brassica species themselves use multiple defence mechanisms, which can be constitutive, inducible, induced, direct or indirect depending upon the insect or the degree of insect attack. Firstly, we give an overview of different Brassica species with the main focus on cultivated brassicas. Secondly, we describe insect pests that attack brassicas. Thirdly, we address multiple defence mechanisms, with the main focus on phytoalexins, sulphur, glucosinolates, the glucosinolate-myrosinase system and their breakdown products. In order to develop pest control strategies, it is important to study the chemical ecology, and insect behaviour. We review studies on oviposition regulation, multitrophic interactions involving feeding and host selection behaviour of parasitoids and predators of herbivores on brassicas. Regarding oviposition and trophic interactions, we outline insect oviposition behaviour, the importance of chemical stimulation, oviposition-deterring pheromones, glucosinolates, isothiocyanates, nitriles, and phytoalexins and their importance towards pest management. Finally, we review brassicas as cover and trap crops, and as biocontrol, biofumigant and biocidal agents against insects and pathogens. Again, we emphasise glucosinolates, their breakdown products, and plant volatile compounds as key components in these processes, which have been considered beneficial in the past and hold great prospects for the future with respect to an integrated pest management.
... The impact of these unspecific methods on C. pallidactylus has not been evaluated, so far. Additionally, a new method had been established in the laboratory for controlling pollen beetle by transforming oilseed rape with a pea lectin gene (Lehrmann 2007). The usage of transgenic insect-resistant Brassica crops as an instrument of IPM is not possible in Europe because of low public acceptance and legal guidelines (Davis 2006). ...
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The search for plant resistance to insect pests in oilseed rape (Brassica napus L. var. oleifera Metzg.) is still in an early stage of development. In this study, a wide spectrum of Brassicaceae representing a broad genetic variability (B. napus and B. rapa cultivars, breeding lines, resynthesized rapeseed lines, other Brassica spp.) was evaluated for resistance characteristics to cabbage stem weevil (Ceutorhynchus pallidactylus) for the first time. Two new methods were developed for the screening of host plant quality of a large assortment under controlled conditions in climate chambers to allow a rapid and reliable selection of genotypes with partial or full resistance. For the quantification of leaf feeding by adult beetles, 106 genotypes were tested in no-choice screening tests for their susceptibility. In comparison to the standard cultivar ‘Express’, the average leaf area consumed by C. pallidactylus was reduced significantly by more than 40% on ten oilseed rape cultivars, four resynthesized rapeseed lines and five other accessions. In dual-choice screening tests for the evaluation of oviposition preferences, female C. pallidactylus laid significantly fewer eggs into plants of two oilseed rape cultivars, five resynthesized rapeseed lines and eight accessions of B. oleracea and B. fruticulosa, respectively, than into plants of the standard cultivar ‘Express’. Within three years, field experiments were carried out to evaluate the susceptibility of 42 Brassica genotypes to infestation by C. pallidactylus under field conditions. A significant correlation was found between the results obtained by laboratory screening tests and by field experiments, thereby confirming the reliability of the new laboratory tests for predicting the susceptibility of Brassica genotypes to C. pallidactylus under field conditions. In a semi-field trial, no significant relationship was found between the total leaf glucosinolate content and the intensity of attack by C. pallidactylus in 12 genotypes tested. However, the stem injury coefficient was significantly correlated with single glucosinolate compounds, particularly aromatic glucosinolate gluconasturtiin and the indolic glucosinolates glucobrassicin, 4-methoxybrassicin and 4-hydroxybrassicin. The larval parasitism of C. pallidactylus and C. napi by Tersilochus obscurator and T. fulvipes (Hym.: Ichneumonidae), respectively, was found to be significantly different between host plant genotypes. In three cultivars of turnip rape, B. rapa, the level of parasitism of C. napi by T. fulvipes was significantly reduced compared to cultivars of winter oilseed rape, B. napus. The level of parasitism was also significantly affected by host plant architecture. In summary, the results present a new method useful in studying essential fundamentals for the development of oilseed rape cultivars with partial resistance to cabbage stem weevil and provide evidence for the impact of host plant genotype on multitrophic interactions between stem-boring insect pests and specialised parasitoids.
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Transgenic crops genetically engineered for enhanced insect resistance should be compatible with other components of IPM for the pest resistance to be durable and effective. An experimental potato line was genetically engineered to express an anti-aphid plant protein (snowdrop lectin, GNA), and assessed for possible interactions of the insect resistance gene with a beneficial pest predator. These extended laboratory studies are the first to demonstrate adverse tri-trophic interactions involving a lectin- expressing transgenic crop, a target pest aphid and a beneficial aphidophagous predator. When adult 2-spot ladybirds (Adalia bipunctata[L.]) were fed for 12 days on peach-potato aphids (Myzus persicae Sulzer) colonising transgenic potatoes expressing GNA in leaves, ladybird fecundity, egg viability and longevity significantly decreased over the following 2–3 weeks. No acute toxicity due to the transgenic plants was observed, although female ladybird longevity was reduced by up to 51%. Adverse effects on ladybird reproduction, caused by eating peach-potato aphids from transgenic potatoes, were reversed after switching ladybirds to feeding on pea aphids from non-transgenic bean plants. These results demonstrate that expression of a lectin gene for insect resistance in a transgenic potato line can cause adverse effects to a predatory ladybird via aphids in its food chain. The significance of these potential ecological risks under field conditions need to be further evaluated.
A method of measuring the effect of honeybees on a field of rapeseed (Brassica campestris) was tested. The density of honeybees in the field was significantly related to distance from an apiary. Other gradients in the field were investigated with cages that excluded honeybees, the cages being used to calculate correction factors to compensate for variations in yield due to parameters other than insect density. The results showed that the design was practicable, and indicated that higher densities increased both seed yield and oil content of seed. The method needs to be tested where a more pronounced density gradient can be obtained.
Small colonies of emerged bees were kept in confinement, and fed fresh pollen, or pollen two years old supplemented with: nothing, pantothenic acid, 20% dried brewers' yeast, 20% vitamin-free casein, casein plus pantothenic acid. The number of sealed brood cells in these colonies (in the above order) was 8·9, 1·0, 2·8, 4·4, 5·6, and 5·8 times the number in the colonies fed old pollen alone. Deterioration of proteins and of pantothenic acid is thus partially responsible for the inferior value of stored pollen for brood rearing.
This chapter discusses the gene flow and introgression from domesticated plants into their wild relatives. Domesticated plants cannot be regarded as evolutionarily discrete from their wild relatives. Most domesticated plants mate with wild relatives somewhere in the world, and gene flow from crops may have a substantial impact on the evolution of wild populations. Planting, growing, and harvesting plants for food, fiber, fodder, pharmaceuticals, and many other uses have profound effects on the ecology and evolution of organisms in the surrounding habitats. Some of these effects may be direct and obvious. The chapter uses population genetic theory to predict the evolutionary consequences of gene flow from crops to wild plants and discusses two applied consequences of crop-to-wild gene flow—the evolution of aggressive weeds and the extinction of rare species. It suggests the ways of assessing the likelihood of hybridization, introgression, and the potential for undesirable gene flow from crops into weeds or rare species.
Cruciferous oilseeds are attacked by a large number of insect pests (Lamb 1989). Blossom beetles, Meligethes spp., and other species of Coleoptera like Psylliodes chrysocephala and Ceutorhynchus spp. are severe pests in Europe. Chrysomelid beetles, Phyllotreta spp. and Entomoscelis americana, are the important pests in Canada. The Indian mustard aphid, Lipaphis erysimi (Fig. 1) is by far the most devastating insect on Brassica oilseeds in India. The aphid is reported to cause an average loss of about 50% in seed yield (Bakhetia 1983). Bagrada cruciferarum and Athalia proxima on germinating and young crop, and Agrotis sp. on rainfed crop, are other sporadic pests in India (Sekhon, unpubl.). Out of the 38 insect pests attacking rapeseed and mustard crops in India, ten are of particular economic significance (Bakhetia 1987). The cabbage aphid, Brevicoryne brassicae,a pest on Brassica vegetables in temperate and warm temperate parts of the world, attacks Brassica oilseeds only rarely (Blackman and Eastop, 1985). The detailed list of the important pests of brassicas is presented in Table 1.
A 647-bp 5'-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to theß-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.