Abiotic stress and transgenics: Implications for reproductive success and crop-to-wild gene flow in Brassicas

Abstract

Various abiotic and biotic stressors affect crop and weed plant performance in agroecosystems. Ozone (O3) tolerance in plants is partly regulated by the genotype and phenotypical properties, and it varies greatly in related species of wild and crop backgrounds. Thus, a continuous increase in atmospheric O3 concentration could change population dynamics of sexually compatible crop and weed species, and consequently affect crop-to-wild gene flow in the future. One way to build resistance against a biotic stressor, in this case insect-mediated herbivory, in crop plants is transgene-mediated insecticidal toxin production. In this study we aimed to describe how the physiological and phenological responses in a crop Brassica and its weedy relatives functioned to affect their comparative O3 tolerance. Furthermore, we studied how harbouring a transgene affects these responses in B. napus and B. rapa × transgenic B. napus BC2F2 backcross hybrid plants to reveal any within-plant trade-offs among toxin production, growth and O3 tolerance. We found a higher number of O3 symptoms but more effective compensatory assimilate allocation directed to reproduction for wild B. rapa compared to crop B. napus under elevated O3. This result suggested that the invasion-orientated strategy of producing a high number of seeds when vegetative growth is limited might improve the performance of weedy species under elevated O3. The probabilities for crop-to-wild transgene flow could be increased through higher seed production in hybrids under elevated O3, but the germination of hybrid seeds in particular was hampered by O3. The presence of transgenes did not perturb fecundity, within-plant biomass allocation or O3 tolerance of B. napus.ZusammenfassungVerschiedene abiotische und biotische Faktoren beeinflussen die Performanz der Nutz- und Unkrautpflanzen in Agrarökosystemen. Die Ozon-(O3)-Toleranz der Pflanzen wird teilweise durch den Genotyp und durch phänotypische Eigenschaften reguliert und variiert in großem Maße bei verwandten Pflanzen mit einem wilden bzw. Nutzpflanzen-Hintergrund. Deshalb könnte eine kontinuierlich steigende O3-Konzentration in der Atmosphäre die Populationsdynamik von sexuell kompatiblen Nutz- und Unkrautarten verändern und in der Zukunft als Konsequenz den Genfluss von Nutz- zu Unkrautarten beeinflussen. Ein Weg um eine Resistenz gegenüber einem biotischen Stressor, in diesem Fall die Herbivorie durch Insekten, bei einer Nutzpflanze aufzubauen, ist die transgen vermittelte Produktion von insektiziden Toxinen. In dieser Untersuchung war es unser Ziel, zu beschreiben, wie die physiologischen und phänologischen Reaktionen bei der Nutzpflanze Brassica und ihren verwandten Unkräutern funktionierten, um ihre O3-Toleranz im Vergleich zu beeinflussen. Darüber hinaus untersuchten wir, wie die Anwesenheit eines Transgens diese Reaktionen bei B. napus und transgenen B. rapa x B. napus BC2F2 Rückkreuzungshybriden beeinflusst, um irgendwelche “trade offs” zwischen der Toxinproduktion, dem Wachstum und der O3-Toleranz innerhalb der Pflanzen festzustellen. Im Vergleich zu genutztem B. napus fanden wir unter erhöhtem O3-Gehalt eine größere Anzahl von O3-Symptomen aber auch eine effektivere, kompensatorische Assimilate-Allokation in Richtung auf die Reproduktion bei wildem B. rapa. Dieses Ergebnis lässt vermuten, dass, wenn das vegetative Wachstum limitiert ist, die invasionsorientierte Strategie durch die Produktion einer großen Anzahl von Samen die Performanz der unkrautartigen Arten bei erhöhtem O3-Gehalt verbessern könnte. Die Wahrscheinlichkeit für den Genfluss von transgenen Nutz- zu Wildpflanzen könnte durch die höhere Samenproduktion bei Hybriden bei erhöhtem O3-Gehalt erhöht sein, wenn auch die Keimung der Hybridsamen bei erhöhtem O3-Gehalt behindert wurde. Die Anwesenheit von Transgenen störte bei B. napus weder die Fruchtbarkeit, noch die Biomassenallokation innerhalb der Pflanze oder die O3-Toleranz.

Full text

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Basic and Applied Ecology 11 (2010) 513–521
Abiotic stress and transgenics: Implications for reproductive success
and crop-to-wild gene flow in Brassicas
Sari J. Himanena,b,∗, Anne-Marja Nergb, Guy M. Poppyc, C. Neal Stewart Jr.d,
Jarmo K. Holopainenb
aMTT Agrifood Research Finland, Plant Production Research, Lönnrotinkatu 5, FIN-50100 Mikkeli, Finland
bDepartment of Environmental Science, University of Eastern Finland, Kuopio Campus, P.O. Box 1627, FIN-70211 Kuopio, Finland
cSchool of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK
dDepartment of Plant Sciences, University of Tennessee, 2431 Joe Johnson Drive, Knoxville, TN 37996-4561, USA
Received 15 September 2009; accepted 27 June 2010
Abstract
Various abiotic and biotic stressors affect crop and weed plant performance in agroecosystems. Ozone (O3) tolerance in
plants is partly regulated by the genotype and phenotypical properties, and it varies greatly in related species of wild and crop
backgrounds. Thus, a continuous increase in atmospheric O3concentration could change population dynamics of sexually
compatible crop and weed species, and consequently affect crop-to-wild gene flow in the future. One way to build resistance
againstabioticstressor,inthiscaseinsect-mediatedherbivory,incropplantsistransgene-mediatedinsecticidaltoxinproduction.
In this study we aimed to describe how the physiological and phenological responses in a crop Brassica and its weedy relatives
functionedtoaffecttheircomparativeO3tolerance.Furthermore,westudiedhowharbouringatransgeneaffectstheseresponses
in B. napus and B. rapa×transgenic B. napus BC2F2backcross hybrid plants to reveal any within-plant trade-offs among toxin
production, growth and O3tolerance. We found a higher number of O3symptoms but more effective compensatory assimilate
allocation directed to reproduction for wild B. rapa compared to crop B. napus under elevated O3. This result suggested that
the invasion-orientated strategy of producing a high number of seeds when vegetative growth is limited might improve the
performance of weedy species under elevated O3. The probabilities for crop-to-wild transgene flow could be increased through
higher seed production in hybrids under elevated O3, but the germination of hybrid seeds in particular was hampered by O3.
The presence of transgenes did not perturb fecundity, within-plant biomass allocation or O3tolerance of B. napus.
Zusammenfassung
Verschiedene abiotische und biotische Faktoren beeinflussen die Performanz der Nutz- und Unkrautpflanzen in Agrarökosys-
temen.DieOzon-(O3)-ToleranzderPflanzenwirdteilweisedurchdenGenotypunddurchphänotypischeEigenschaftenreguliert
und variiert in großem Maße bei verwandten Pflanzen mit einem wilden bzw. Nutzpflanzen-Hintergrund. Deshalb könnte eine
kontinuierlich steigende O3-Konzentration in der Atmosphäre die Populationsdynamik von sexuell kompatiblen Nutz- und
Unkrautarten verändern und in der Zukunft als Konsequenz den Genfluss von Nutz- zu Unkrautarten beeinflussen. Ein Weg
um eine Resistenz gegenüber einem biotischen Stressor, in diesem Fall die Herbivorie durch Insekten, bei einer Nutzpflanze
∗Corresponding author at: MTT Agrifood Research, Plant Production Research, Lönnrotinkatu 5, FIN-50100 Mikkeli, Finland. Tel.: +358 40 738 9873;
fax: +358 15 226 578.
E-mail address: Sari.Himanen@mtt.fi (S.J. Himanen).
1439-1791/$ – see front matter © 2010 Gesellschaft für Ökologie. Published by Elsevier GmbH. All rights reserved.
doi:10.1016/j.baae.2010.06.007

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514S.J. Himanen et al. / Basic and Applied Ecology 11 (2010) 513–521
aufzubauen, ist die transgen vermittelte Produktion von insektiziden Toxinen. In dieser Untersuchung war es unser Ziel, zu
beschreiben, wie die physiologischen und phänologischen Reaktionen bei der Nutzpflanze Brassica und ihren verwandten
Unkräutern funktionierten, um ihre O3-Toleranz im Vergleich zu beeinflussen. Darüber hinaus untersuchten wir, wie die Anwe-
senheit eines Transgens diese Reaktionen bei B. napus und transgenen B. rapa x B. napus BC2F2 Rückkreuzungshybriden
beeinflusst, um irgendwelche “trade offs” zwischen der Toxinproduktion, dem Wachstum und der O3-Toleranz innerhalb der
Pflanzen festzustellen. Im Vergleich zu genutztem B. napus fanden wir unter erhöhtem O3-Gehalt eine größere Anzahl von
O3-Symptomen aber auch eine effektivere, kompensatorische Assimilate-Allokation in Richtung auf die Reproduktion bei
wildem B. rapa. Dieses Ergebnis lässt vermuten, dass, wenn das vegetative Wachstum limitiert ist, die invasionsorientierte
Strategie durch die Produktion einer großen Anzahl von Samen die Performanz der unkrautartigen Arten bei erhöhtem O3-
Gehalt verbessern könnte. Die Wahrscheinlichkeit für den Genfluss von transgenen Nutz- zu Wildpflanzen könnte durch die
höhere Samenproduktion bei Hybriden bei erhöhtem O3-Gehalt erhöht sein, wenn auch die Keimung der Hybridsamen bei
erhöhtem O3-Gehalt behindert wurde. Die Anwesenheit von Transgenen störte bei B. napus weder die Fruchtbarkeit, noch die
Biomassenallokation innerhalb der Pflanze oder die O3-Toleranz.
© 2010 Gesellschaft für Ökologie. Published by Elsevier GmbH. All rights reserved.
Keywords: Brassica napus ssp. oleifera (oilseed rape; canola); Brassica rapa; Ozone tolerance; Transgene introgression; Crop–weed
population dynamics
Introduction
Diurnal background concentration of tropospheric ozone
(O3) already exceeds 40ppb in many regions and trends
towards increasing levels are predicted to continue for the
coming decades (Sitch, Cox, Collins, & Huntingford 2007).
This can make O3an important contributor on plant compet-
itive dynamics. Oxidative stress by elevated O3is a severe
environmental challenge for plants: phytotoxic symptoms
arise and this affects photosynthetic processes, phenologi-
cal development and yield (Booker et al. 2009). Most wild
plant species are classified as susceptible to O3compared
with crop species, which have been selected through breed-
ing to be more robust under variable environments (Pleijel
& Danielson 1997; Davison & Barnes 1998; Biswas et al.
2008). Genetically regulated O3 tolerance (Biswas et al.
2008), but also differences in phenology (e.g. maturation
age), life-histories (Pleijel & Danielson 1997) and pheno-
types (Overmyer et al. 2008) affect O3responses of plants
andcouldalterthecompetitivedynamicsofco-speciesunder
elevated O3.
The introduction of transgenic crops has initiated wide
ecological research of potential gene escape into native
species (Stewart, Halfhill, & Warwick 2003). The probabil-
ities for transgene flow have been extensively studied on
Brassicas (e.g. Warwick et al. 2003; Halfhill et al. 2004;
Kelly, Bowler, Breden, Fenner, & Poppy 2005; Warwick,
Légère, Simard, & James 2008), since wild Brassica species
occur worldwide and exist commonly in agroecosystems.
They are capable of hybridizing with cultivated Brassicas,
which enables segregation of common genomic material
among these plants (Wilkinson, Elliott, et al. 2003; Halfhill
et al. 2004). Unwanted introgression of transgenes into wild
speciescouldhavesevereecologicalconsequences,although
the pathway required for a transgene to be fully introgressed
into a wild-plant genotype is a complicated one in which
the success of the hybrids are crucial (Wilkinson, Sweet,
& Poppy 2003). Environmental stresses, including elevated
O3, are candidates for affecting the performance and com-
petitiveness of introgressed plant individuals and thus are
critical in assessing the environmental risk of a transgene
introgressing into the genome of a crop’s wild relatives. Our
study is the first to compare the performance of nontrans-
genic and transgenic crops, wild relatives and introgressed
transgene-carrying back-cross hybrid plants under elevated
O3.
Bacillusthuringiensis(Bt)Cry1Ac-transgenicoilseedrape
(Brassica napus ssp. oleifera), insecticidal against numer-
ous key Lepidopteran Brassica pests, is a model plant used
widely in ecological risk assessment studies for crop-to-wild
gene flow (e.g. Halfhill, Richards, Mabon, & Stewart 2001;
Warwicketal.2003).Previously,wehaveshownthatBttoxin
concentration in Bt-producing B. napus is not compromised
by, but increased under high atmospheric O3(Himanen et
al. 2009), and the plants exhibit similar responses to chronic
andacuteO3elevationduringvegetativegrowthastheirnon-
transgenicparentplants(Himanenetal.2008).Here,ourfirst
aim was to test for a trade-off, as a result of intrinsic costs of
constitutiveBttoxinproduction,amongreproductionandO3
tolerance in Bt B. napus. Secondly, we assessed whether ele-
vatedO3affectscertainphysiologicalcharacteristics(growth,
allocationtoreproduction,seedsize)andthereforetheperfor-
manceofsexuallycompatiblewildB.rapaandcropB.napus
plants, representing different ecological life-history strate-
gies (Moles and Westoby 2006), in a different way. Finally,
we evaluated the O3responses and within-plant allocation
patterns of introgressed Bt-transgene-carrying B. rapa×B.
napus BC2F2back-crossed hybrid plants to reveal whether
elevated O3could affect the probability of transgene escape
through altered performance or reproduction of hybrids. Our
results could reveal important aspects for assessing compet-
itive advantage of introgressed transgene-carrying hybrids,
wild relatives and crop plants in future O3-enriched atmo-
spheres.

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S.J. Himanen et al. / Basic and Applied Ecology 11 (2010) 513–521515
Materials and methods
Plants used in the experiments were: 1) non-transgenic
Brassica napus ssp. oleifera (oilseed rape) cv. Westar (par-
ent line), 2) its Bt-transgenic line GT1 F4 (containing a
synthetic Bt Cry1Ac gene and a green fluorescent protein
(gfp) marker gene under CaMV 35S promoters, as described
by Halfhill et al. 2001), 3) Brassica rapa wild accession
2974 (Milby, Québec, Canada; Halfhill et al. 2005) and 4)
wildB.rapa(describedabove)×crop(Bt-transgenicGT1B.
napus) BC2F2hybrid carrying the transgene and having B.
rapa ploidy level and phenotype (hybridized as described in
Halfhill, Millwood, Weissinger, Warwick, & Stewart 2003).
Plants were grown in four identical environment-
controlled growth chambers (2.6m3, Bioklim 2600T, Kryo-
Service Oy, Helsinki, Finland) under 16:8h photoperiod,
20/16◦C thermoperiod and minimum 250?molm−2s−2
PAR irradiance. One hundred and four seeds from each
genotype were individually sown into 0.66l pots in
2:1:1 fertilized compost (Kekkilä, Finland, NPK: 100-30-
200mgl−1): B2 Sphagnum peat (Kekkilä, Finland, NPK:
110–40–220mgl−1): sand (0.5–1.2mm) mixture and placed
in a randomised block design into each of the four chambers
(26 plants of each genotype per chamber). Chronic O3fumi-
gation (supplied as in Himanen et al. 2009) was started after
emergence (7 days after sowing) with two chambers hav-
ing close to 0ppb O3(filtered air) and two having 100ppb
elevated O3treatment (8h daily, 8.30 to 16.30h). Elevated
O3concentration of 100ppb was used, as it caused severe
but not detrimental phytotoxic challenge to the plants in ear-
lier screening experiments (Himanen et al., unpublished). In
these screenings, filtered air (0ppb) and 50ppb (high ambi-
ent concentration) were also compared and close to identical
responses in the plants warranted the use of no O3elevation
(filtered air) as the control treatment. To avoid any effects of
chamber-specific growth conditions, the plants inside cham-
bersandthetreatmentsamongchamberswererotatedweekly
(as in, e.g. Black, Stewart, Roberts, & Black 2007, except
during flowering to prevent pollen contamination between
treatments). The plants were transplanted into 1.4l pots at
22 days after sowing. During flowering, flowers were hand-
pollinated,usingafinebrush,attwo-dayintervalswithineach
O3treatment.
For biomass determination, the dry weight (DW) of leaves
(dried at 60◦C) was measured individually for six plants
per genotype and O3treatment (three plants per chamber)
at 22 days and 33 days after sowing. Net photosynthesis was
measured for the second growth leaf of six plants per geno-
type and O3treatment (three plants per chamber) 22, 28 and
35 days after sowing with a CI-510 Portable Photosynthe-
sis System (Cid Inc., Vancouver, WA, USA) under saturating
lightintensityof1800?molm−2s−2PAR.TheCO2inputfor
the cuvette was taken from the growth chambers. O3dam-
age (percentage of total leaf area damaged, assessed from
photographs of the plants analyzed using Adobe Photoshop
software) was determined for the same plants at days 28 and
35. The abscission of leaves for each genotype in each cham-
ber was determined by collecting and determining DW of
senesced leaves at two- to four-day intervals.
Theplantswereharvestedwhenover75%ofallpodswere
dry (yellow in colour), but none had started to open, which
occurredat11–12weeksaftersowing.Atharvest,stemheight
was measured and the number of branches was counted. The
DW of stem, roots, pods and seeds in main rachis, and pods
andseedsinbranchesforeachplantweremeasuredafterdry-
ing. Harvest index was calculated as the ratio of seed DW to
total plant DW. For presentation of the results (Table 4), har-
vest index and root:shoot ratio were multiplied by 100. The
1000-seed weight was calculated after weighing all or 100
seeds per plant, for main rachis and branch seeds separately.
Germination of the seeds (collected from main rachis) was
tested in 20 replicate Petri dishes (diameter 100mm) lined
with moisturised filter paper and placed in a growth chamber
at 20/16◦C. Seeds from all eight treatments were randomly
assignedtoeightsectorsineachdish,withfiveseedspersec-
tor. Final germination percentage was determined after 14
days.
Before statistical analysis, all data were checked for nor-
mality and equality of residual error variances and then
appropriatelytransformed(logorsquareroot)ifnecessaryto
gain normality. Percentage values were arcsin-transformed
prior to analysis. Results were analyzed using REML lin-
ear mixed models with plant genotype and O3 treatment
as fixed effects and chamber as a random factor. Post hoc
tests based on estimated marginal means with Bonferroni
correction were conducted both among O3treatments and
genotypes,separately.Leafsenescenceandseedgermination
results were tested with general linear models with genotype
and O3as fixed effects. Correlation between root+stem DW
and seed DW was tested using Pearson correlation tests sep-
arately for each genotype and O3treatment. All data were
analyzed using SPSS for Windows 14.0 statistical package
(SPSS Inc., Chicago, IL, USA).
Results
Vegetative growth stage responses
At 22 days after sowing, transgene-harbouring BC2F2
hybrid and wild B. rapa plants had higher leaf DW than the
B. napus genotypes (Tables 1 and 2). By day 33, O3reduced
leaf DW in B. rapa (PO3< 0.05), whereas there was no dif-
ference in leaf DW between genotypes at this time-point.
Photosynthesis rates were similar in all plant genotypes and
at both O3levels at day 22 (Tables 1 and 2). By days 28 and
35, the photosynthesis rate was lower in hybrid and B. rapa
plantsthaninB.napusgenotypeplantsgrownunderelevated
O3.
Percent damage to the total leaf area caused by O3was
higher in the first (at 28 days) and second (at 28 and 35
days) growth leaves of hybrid and B. rapa plants than in

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516S.J. Himanen et al. / Basic and Applied Ecology 11 (2010) 513–521
Table 1. Leaf dry weight and net photosynthetic rate (A) during vegetative growth (mean±SEM) for non-transgenic and Bt-transgenic B.
napus, B. rapa×transgenic B. napus BC2F2hybrid and wild B. rapa plants grown under control conditions (filtered air) or elevated (100ppb
8hday−1) O3.
ControlElevated O3
B. napus
Bt B. napus
BC2F2hybrid B. rapaB. napus
Bt B. napus
BC2F2hybrid
B. rapa
Leaf dry weight (g)
22 days 0.18 ± 0.02a
33 days 1.11 ± 0.21
A (?molm−2s−1)
22 days 22.5 ± 1.38
28 days 22.5 ± 2.08a
35 days 17.0 ± 0.99
Net photosynthesis was measured for 2nd growth leaves. Different letters indicate statistically significant (P<0.05) difference between genotypes among
control and elevated O3treatments.
0.12 ± 0.01a
0.91 ± 0.13
0.36 ± 0.05b 0.42 ± 0.03b
1.01 ± 0.18
0.17 ± 0.02a
1.01 ± 0.14
0.15 ± 0.02a
0.87 ± 0.06
0.33 ± 0.07ab
0.67 ± 0.12
0.44 ± 0.05b
0.61 ± 0.141.17 ± 0.26
21.0 ± 1.40
22.3 ± 0.72ab
22.4 ± 2.39
21.2 ± 1.26
16.1 ± 1.65b 18.2 ± 0.62ab
17.8 ± 1.59
22.1 ± 0.96 23.1 ± 0.43
21.1 ± 0.50a
19.2 ± 1.22a
21.5 ± 0.68
22.2 ± 0.42a
21.1 ± 0.67a
23.0 ± 0.53
7.80 ± 1.61b
6.67 ± 3.08b
22.7 ± 0.63
9.95 ± 1.30b
10.3 ± 1.99b15.3 ± 1.79
Table 2. Mixed model results for main effects of genotype (non-transgenic B. napus, Bt-transgenic B. napus, B. rapa×transgenic B. napus
BC2F2hybrid and wild B. rapa) and O3treatment (control or 100ppb elevated O3) and their interaction on vegetative and reproductive growth
stage parameters and within-plant allocation (as percentage of total plant DW) at harvest.
Genotype F3,151
O3F1,151
Gt×O3F3,151
Vegetative stage parameters:
Leaf DW 22 days
Leaf DW 33 days
A 22 days
A 28 days
A 35 days
29.0***
0.74
0.87
28.2***
12.5***
0.02
5.31*
1.73
22.8***
6.69*
0.41
1.12
0.21
7.25***
4.48***
Reproductive stage parameters:
Plant height
Branch number
Stem DW
Root DW
Root+stem DW
Total pod DW
Total plant DW
Main rachis seed DW
Branch seed DW
Total seed DW
Seed germination
1000-seed weight
In main rachis
In branches
42.2***
13.1***
6.96***
16.4***
9.18***
9.51***
12.5***
83.6***
0.71
19.0***
114.1***
0.07
0.41
27.2***
64.0***
36.3***
15.3***
33.5***
17.9***
1.36
9.13**
4.99*
4.25**
1.35
3.44*
2.45
3.61*
1.34
0.12
4.09**
3.06*
3.80*
1.98
120.5***
51.8***
0.22
0.94
1.17
2.66
Within-plant allocation:
Root: shoot ratio
Harvest index
Roots
Stem
Pods in main rachis
Seeds in main rachis
Pods in branches
Seeds in branches
11.7***
5.14**
9.53***
0.72
12.3***
35.8***
26.0***
6.19***
44.3***
1.99
39.9***
0.05
0.17
0.01
0.14
3.24
1.32
5.75**
5.53***
4.16**
1.47
2.07
0.75
3.41*
Statistical significance in bold (***P<0.001,**P<0.01,*P<0.05).

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S.J. Himanen et al. / Basic and Applied Ecology 11 (2010) 513–521517
Table 3. Foliar O3damage (% of total leaf area, mean±SEM) 28 and 35 days after sowing in non-transgenic and Bt-transgenic B. napus, B.
rapa×transgenic B. napus BC2F2hybrid and wild B. rapa plants grown under elevated (100ppb 8h day−1) O3. 1st growth leaf denotes the
oldest leaf.
Growth leaf
B. napus
Bt B. napus
BC2F2hybrid
B. rapaF3,39genotype
28 days
1
2
3
4
60.0±3.3a
10.0±6.0a
0
0
65.8±2.5a
12.5±0.8a
0
0
100.0±0.0b
95.0±5.0b
39.2±9.2
9.7±5.3
99.2±0.8b
94.2±0.8b
35.8±7.5
11.3±10.3
100.6***
148.5***
0.079
0.021
35 days
1
2
3
4
53.8±38.8
7.5±7.5a
0
0
76.3±13.8
20.0±0.0a
3.8±3.8
0
100.0±0.0
91.3±6.3b
68.8±18.8
37.5±25.0
100.0±0.0
96.3±3.8b
55.0±12.5
5.0±5.0
1.166
79.12***
6.746
1.625
Statistical significance in bold (***P<0.001). Different letters indicate statistically significant (P<0.05) difference between genotypes.
B. napus genotypes (Table 3). Hybrid and B. rapa plants
senesced earlier in the control treatment than the B. napus
genotypes (Fig. 1A). Leaf senescence occurred earlier in all
genotypes under elevated O3(by approximately one week)
(Fig. 1B).
Reproductive growth stage responses
Plant height was lower in BC2F2 hybrid plants in the
control treatment, and in B. rapa and hybrid plants under
elevated O3than in the B. napus genotypes (Table 4). Height
was reduced by elevated O3only in B. rapa plants (PO3<
0.05, Table 2, interaction gt×O3). Hybrid plants had more
branches than the other genotypes (Table 4).
In the control treatment, hybrid plants had lower stem DW
than B. rapa, lower root DW than the B. napus genotypes,
lower root+stem DW than non-Bt B. napus and B. rapa and
lowertotalDWthannon-BtB.napus(Table4).Intheelevated
O3treatment, stem, root, stem+root and total DWs were
lowerinB.rapaandhybridplantsthaninB.napusgenotypes.
Elevated O3reduced root DW in all genotypes, but stem and
root+stem were significantly reduced by O3only in B. rapa
and hybrid plants (PO3< 0.05, Table 2, interaction gt×O3).
TotalpodandseedDWforB.napusgenotypeswerehigher
thanforhybridandB.rapaundercontrolconditions,whereas
seed DW under elevated O3differed only between non-Bt B.
napus and hybrid plants (Table 4). Elevated O3reduced pod
DW in all genotypes and seed DW in B. napus genotypes
(Table 2, interaction gt×O3).
Seed dry weights for the main rachis were lower in hybrid
and B. rapa plants than in B. napus genotypes in the con-
trol treatment (Table 4). Non-Bt B. napus had also a higher
seed DW in the main rachis than Bt B. napus under elevated
O3. Elevated O3reduced seed DW in the main rachis of B.
napus genotypes (PO3< 0.05), whereas those of hybrid and
B.rapaplantswereunaffected(Table2,interactiongt×O3).
On branches, O3reduced seed DW only for non-Bt B. napus
plants (PO3< 0.05, Table 2, interaction gt×O3).
Fig. 1. Leaf senescence patterns (mean±SEM) for non-transgenic
and Bt-transgenic B. napus, BC2F2B. rapa×transgenic B. napus
hybrid and wild B. rapa plants grown under (A) control con-
ditions (filtered air) and (B) elevated (100ppb 8hday−1) O3.
Statistically significant main effects for genotype (gt) (F3,7), O3
(F1,7) and their interactions (F3,7) are shown (*P<0.05,**P<0.01,
***P<0.001). Different letters above bars indicate statistically sig-
nificant(P<0.05)differencebetweengenotypesamongcontroland
elevated O3treatments.

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S.J. Himanen et al. / Basic and Applied Ecology 11 (2010) 513–521
Table 4. Variables measured at harvest (mean±SEM) for non-transgenic and Bt-transgenic B. napus, B. rapa×transgenic B. napus BC2F2hybrid and wild B. rapa plants grown under
control conditions (filtered air) or elevated (100ppb 8h day−1) O3.
ControlElevated O3
B. napus
Bt B. napus
BC2F2hybrid
B. rapa B. napus
Bt B. napus
BC2F2hybrid
B. rapa
Plant height (cm)
Branch number
Stem DW (g)
Root DW (g)
Root+stem DW (g)
Total pod DW (g)
Total plant DW (g)
118.5 ± 2.0a
3.1 ± 0.2a
4.61 ± 0.25ab
0.83 ± 0.06a
5.44 ± 0.29a
6.77 ± 0.37a
12.3 ± 0.66a
119.1 ± 2.6a
3.2 ± 0.2ab
4.39 ± 0.22ab
0.84 ± 0.04a
5.23 ± 0.26ab
6.52 ± 0.39a
11.8 ± 0.63ab
97.6 ± 3.3b
4.3 ± 0.1c
3.85 ± 0.18b
0.61 ± 0.04b
4.46 ± 0.22b
5.36 ± 0.26b
9.81 ± 0.32b
111.1 ± 3.4a
3.8 ± 0.2bc
4.73 ± 0.33a
0.78 ± 0.07ab
5.51 ± 0.36a
5.05 ± 0.29b
10.6 ± 0.40ab
122.8 ± 3.0a
3.5 ± 0.2ab
4.17 ± 0.21a
0.65 ± 0.05a
4.82 ± 0.25a
5.52 ± 0.24
10.3 ± 0.46a
126.6 ± 2.0a
3.4 ± 0.2a
4.02 ± 0.15a
0.65 ± 0.03a
4.67 ± 0.18a
5.47 ± 0.29
10.1 ± 0.45a
97.7 ± 2.4b
4.0 ± 0.2b
3.10 ± 0.17b
0.33 ± 0.03b
3.43 ± 0.19b
4.57 ± 0.36
8.00 ± 0.36b
101.0 ± 1.8b
3.8 ± 0.1ab
3.14 ± 0.14b
0.38 ± 0.03b
3.52 ± 0.17b
4.90 ± 0.21
8.42 ± 0.29b
Seed DW (g)
In main rachis
In branches
1.93 ± 0.09a
1.39 ± 0.15
3.32 ± 0.16a
84.7 ± 3.6a
1.85 ± 0.08a
1.20 ± 0.10
2.99 ± 0.17a
87.5 ± 4.0a
0.74 ± 0.07b
1.33 ± 0.15
2.07 ± 0.15b
40.0 ± 7.9b
0.95 ± 0.11b
1.04 ± 0.11
1.99 ± 0.18b
10.6 ± 4.6c
1.64 ± 0.09a
0.92 ± 0.07
2.56 ± 0.13a
81.2 ± 6.3a
1.31 ± 0.05b
1.12 ± 0.11
2.43 ± 0.14ab
85.6 ± 4.2a
0.69 ± 0.08c
1.26 ± 0.15
1.95 ± 0.19b
16.3 ± 5.8b
0.88 ± 0.05c
1.27 ± 0.11
2.15 ± 0.11ab
7.1 ± 3.4b
Total seed DW (g)
Germination (%)
1000-seed DW (g)
In main rachis
In branches
4.21 ± 0.11a
4.04 ± 0.14a
18.0 ± 8.0ab
27.4 ± 5.0a
4.13 ± 0.13a
3.69 ± 0.23a
19.1 ± 5.0a
25.4 ± 4.0a
2.25 ± 0.22b
2.39 ± 0.11b
15.9 ± 9.0b
21.1 ± 1.3b
2.25 ± 0.13b
2.42 ± 0.14b
16.9 ± 1.4ab
19.3 ± 1.8b
3.98 ± 0.16a
3.62 ± 0.03a
15.4 ± 8.0a
25.1 ± 10.0
4.33 ± 0.12a
4.20 ± 0.28a
16.2 ± 5.0a
23.8 ± 5.0
2.33 ± 0.17b
2.09 ± 0.27b
10.7 ± 6.0b
23.6 ± 1.9
2.01 ± 0.14b
2.12 ± 0.13b
12.0 ± 8.0b
25.5 ± 0.9
Root: shoot ratio
Harvest index
Different letters indicate statistically significant (P<0.05) difference between genotypes among control and elevated O3treatments.

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S.J. Himanen et al. / Basic and Applied Ecology 11 (2010) 513–521519
Fig. 2. Within-plant distribution of DW at harvest (mean % of total plant DW), for non-transgenic and Bt-transgenic B. napus, BC2F2B.
rapa×transgenic B. napus hybrid and wild B. rapa plants grown under control conditions (filtered air) or under elevated (100ppb 8hday−1)
O3.Differentlettersinbarsindicatestatisticallysignificant(P<0.05)differencebetweengenotypesamongcontrolandelevatedO3treatments.
B. napus genotypes had a higher germination rate than
hybrid and B. rapa, irrespective of O3treatment (Table 4).
Hybrid plants had also higher germination than B. rapa in
the control treatment. Elevated O3reduced the germination
percentage for hybrid plant seeds only (PO3< 0.05). The
1000-seed weights were higher in the B. napus genotypes
than in B. rapa and hybrid plants in main rachis and branch
seeds, and the weights were not affected by elevated O3
(Table 2).
Within-plant allocation responses
In the control treatment, hybrid plants had the lowest
root:shoot (R:S) ratio, and Bt B. napus the highest, this dif-
ference being the only statistically significant one (Table 4).
The R:S ratio was reduced by elevated O3in all genotypes
(Table 2) and B. napus genotypes had higher R:S ratios than
hybrid and B. rapa plants under elevated O3. Harvest index
(HI) was higher in B. napus genotypes than in hybrid and B.
rapa plants in the control treatment. Under elevated O3, HI
was equal in all genotypes. Elevated O3increased HI solely
for B. rapa (PO3< 0.05, Table 2, interaction gt×O3).
The allocation of DW to roots was higher under elevated
O3forB.napusgenotypesthanforBC2F2hybridandB.rapa
(Fig. 2). Elevated O3reduced percentage DW in roots in all
other genotypes except non-transgenic B. napus (Table 2,
interaction gt×O3). More biomass was allocated to stem
DW in B. rapa than other genotypes in the control treatment.
Elevated O3increased percentage allocation to stem for the
B. napus genotypes, but reduced it for B. rapa (interaction
gt×O3). The allocation to DW of pods was higher towards
branchesinhybridandB.rapathanB.napusgenotypesunder
elevatedO3.Inthecontroltreatment,B.napusgenotypeshad
a higher percentage allocation to seeds in main rachis than
hybrid and B. rapa, whereas hybrid plants had the lowest
allocation to main rachis seeds under elevated O3. Percent
biomass of seeds in branches was lower for non-Bt B. napus
thanforhybridandB.rapaplantsunderelevatedO3.Elevated
O3increased percentage allocation to branch seeds only in
B. rapa (PO3< 0.05, interaction gt×O3).
Root+stem DW correlated positively with total seed DW
fornon-BtandBtB.napusgenotypesinthecontroltreatment
(r=0.879, P<0.001 and r=0.897, P<0.001, respectively),
and for Bt plants the correlation was significant also under
elevated O3 (r=0.777, P<0.001). In contrast, seed DW
was negatively correlated to stem+root DW for B. rapa
plants in the control treatment (r=−0.556, P=0.011) and
a marginally statistically significant negative correlation was
foundforhybridplantsgrownunderelevatedO3(r=−0.432,
P=0.057).
Discussion
Ozone tolerance of wild and cultivated Brassicas:
implications for crop–weed population dynamics
The high percentage of O3 lesions and reduced pho-
tosynthesis rates in wild B. rapa and transgene-carrying
back-crossed hybrid plants suggested high O3 sensitivity,
which is typical for native species (Davison & Barnes 1998).
B. rapa plants had earlier senescence under elevated O3
than crop B. napus plants, and O3-induced differences in
phenology might reduce overlap of their flowering periods
and, hence, cross-fertilization. Important from the aspect of
reproduction was also the increased harvest index and higher
percentage of total DW allocated to branch seeds, resem-

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520S.J. Himanen et al. / Basic and Applied Ecology 11 (2010) 513–521
bling indeterminate growth, in B. rapa under elevated O3.
Interestingly, seed DW correlated positively with root+stem
DW in the B. napus genotypes indicating better reproduc-
tion with higher vegetative biomass, whereas these were
inversely related in B. rapa. Wild B. rapa seems to have
a stronger assimilate sink in seeds that leads to a more
pronouncedwithin-planttrade-offandallocationtoseedpro-
duction than that in B. napus. This might benefit B. rapa
population development under abiotic stresses such as ele-
vated O3. The transgenic back-crossed hybrid plants lacked
such a high allocation as the parent B. rapa to reproduction,
which might be attributable to interference with the B. napus
genome (Halfhill et al. 2003).
Previously, Black et al. (2007) also found compensation
for O3damage during the vegetative stage in B. campestris
(syn. B. rapa); as a result, O3-exposed plants produced
a mature seed yield comparable to control plants. Simi-
larly, Sutherland, Justinova, and Poppy (2006) reported a
highercompensationforherbivoredefoliationinwildB.rapa
(increase in biomass) than in B. rapa×B. napus F1hybrid
or B. napus plants. In nature, it is beneficial for a wild weedy
species such as B. rapa to rely on a fast growth cycle, high
seed number and relatively small seed size in a bet-hedging
strategy to maximize possibilities of finding suitable habi-
tats to invade and establish a seedbank in the soil (Moles
& Westoby 2006). In contrast, human-bred B. napus pro-
duces a lower number of high-quality seeds (Zia et al. 2008),
whichcanrestricttheircompensatorypotential.Basedonour
results, the crop B. napus showed less foliar O3damage and
thereforewouldhaveahigherchanceofsurvivalafterhighO3
episodes, yet wild B. rapa had higher compensatory alloca-
tiontoreproduction,whichcanenhanceitspersistenceunder
O3. This emphasizes the need for follow-up field studies on
the ecological fitness and competitive dynamics (Monaghan,
Metcalfe,&Torres2009)ofBrassicaspeciesfromweedyori-
gins and agricultural backgrounds in a natural O3-enriched
agroecosystem.
We also found reduced germination percentage for the
back-crossed hybrid seeds under elevated O3, which could
beimportantforcompetitiveabilitiesandinvasiveness.Small
seeds of weeds have been earlier suggested to be more vul-
nerabletoenvironmentalstresses(Harbur&Owen2004)and
hybrid plants typically have lower seed size than their parent
types(Wei&Darmency2008).The1000-seedweightforthe
B. napus cultivar used here was approximately double that of
B. rapa and hybrid seeds. Field tests to ascertain whether O3
affects hybrid seed dormancy or actual viability should be
performed.
Bt transgene interaction and abiotic stress
effects on transgene flow
There were no observed trade-offs between transgenicity
and reproductive ability in B. napus, as was reported ear-
lier (Mason, Braun, Warwick, Zhu, & Stewart 2003). Lower
main rachis seed DW in Bt plants compared to the non-Bt
genotype under elevated O3did, however, suggest that there
waseitheratemporalphenologicalorallocationaldifference,
which was then balanced for in total seed DW. The growth
and reproductive responses to elevated O3were comparable
in Bt-transgenic and non-transgenic B. napus genotypes.
The fecundity of the Bt-transgenic B. rapa back-crossed
hybrids was equal to B. rapa and lower than those of the
B. napus genotypes in control conditions. The similarity of
O3responses in wild B. rapa and the transgene-harbouring
back-crosses revealed that the impact of phenotype proper-
tiesexceededthepotentialeffectsconferredbythisparticular
transgeneonO3responses.However,fromanecologicalper-
spectiveitcouldbemeaningfulthatthereproductiveoutcome
of the back-crossed hybrids was less affected by elevated O3
thanthatoftheB.napusparentplants.Thus,itisimportantto
continue assessments for potential changes in transgene flow
dynamicsbyelevatedO3.Assessmentofwhetherdifferential
O3sensitivity will render competitive advantage that might
affect crop–weed population dynamics or the probability of
unwanted exchange of genetic material between crop and
wildplantswouldbenefitfrommeasuringcomparativepollen
releaseandsynchronizationoffloweringinO3-enrichedfield
environments. Another important aspect to consider is the
roleoflowgerminationrateoftheback-crossedhybridseeds
(Wei & Darmency 2008) under O3stress that we observed
here, which could serve to restrict transgene flow.
Acknowledgments
We thank Timo Oksanen for technical help and Virpi
Tiihonen, Jaana Rissanen, Juuso Heinonen and Maria Saas-
tamoinen for assistance in experiments. This work was
supported by the Academy of Finland (grant no. 105209)
(S.J.H., A-M.N. and J.K.H.), the Finnish Cultural Founda-
tion (S.J.H.), ISONET (MRTN-CT-2003-504720) (J.K.H.),
USDA Biotechnology Risk Assessment Program (C.N.S.),
BBSRC and NERC (G.M.P.).
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