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―
Chinese Science Bulletin 2006 Vol. 51 No. 13 1578—1585
DOI: 10.1007/s11434-006-2021-4
Transgene directionally inte-
grated into C-genome of Bras-
sica napus
LI Jun, FANG Xiaoping, WANG Zhuan, LI Jun,
LUO Lixia & HU Qiong
Key Laboratory of Oil Crops Genetic Improvement of the Ministry of
Agriculture, Institute of Oil Crops Research, Chinese Academy of Agri-
cultural Sciences, Wuhan 430062, China
Correspondence should be addressed to Fang Xiaoping (email:
xpfang@public.wh.hb.cn)
Received March 14, 2006; accepted April 17, 2006
Abstract Integration of a transgene into a
C-genome chromosome plays an important role in
reducing ecological risk of transgenic Brassica napus.
To obtain C-genome transgenic B. napus, herbi-
cide-resistant bar gene was firstly transferred into B.
oleracea var. alboglabra mediated by Agrobacterium
tumefaciens strain LBA4404. Then using the trans-
genic B. oleracea as paternal plants and 8 non-
transgenic varieties of B. rapa as maternal plants, C-
genome transgenic B. napus with bar gene was arti-
ficially resynthesized by means of ovary culture and
chromosome doubling. Among 67 lines of the resyn-
thesized B. napus, 31 were positive, and 36 were
negative according to PCR test for bar gene. At least
2 plants from each line were kept for PPT spray con-
firmation. The result was in consistence with the PCR
test. Genomic Southern blotting of three randomly
chosen lines also showed that bar gene had been
integrated into the genome of resynthesized B. napus
lines.
Keywords: Bar gene, C-genome, directional transgene, resynthesis of
Brassica napus.
Transgenic Brassica napus has been widely planted
in Canada, the United States, and some other countries.
In China, although the policy for genetically modified
foods has not yet opened, genetically modified rape-
seed oil as raw material for biodiesel offers particularly
good prospects. A major concern for the release of
transgenic B. napus is that the transgenes may diffuse
into other wild relatives to form super weeds. A lot of
studies confirmed the unavoidable genetic drift and
introgression of B. napus to its relative weeds, espe-
cially B. rapa (2n=20, AA) and B. juncea (2n=36,
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AABB)[1 4]. For example, herbicide resistant transgene
has been found in wild B. rapa population in Canada[5].
To prevent transgenic B. napus from bringing ecologi-
cal environment risk after natural interspecific hybridi-
zation is becoming a main subject of researches[6].
B. rapa and B. juncea are common field weeds in
China. The AC-genome B. napus is easily hybridized
with A-genome B. rapa[7,8] or AB-genome B. juncea[9].
The hybridization of AC-genome B. napus with
C-genome B. oleracea, however, is extremely diffi-
cult[10,11]. Thus to eliminate ecological risk of transgenic
B. napus, reduction of transmission probability of ex-
ogenous genes to offsprings of the hybrid between
transgenic B. napus and A- or AB-genome species is of
significant importance.
Mikkelsen et al.[12] proposed the hypothesis that
there might be safe sites for gene integration in B.
napus, i.e. there are chromosome regions with a low
probability of gene transmission to backcross genera-
tions with B. rapa as the recurrent parent via homolo-
gous recombination. Specifically, the presence of
chromosomes of either the A-genome or C-genome
determines the transmission frequency of a resistance
gene in subsequent backcross generations. Metz et al.[8]
and Zhu et al.[13] found that transgene in some lines
disappeared faster than others in backcross generations
with B. rapa when studying the inheritance of trans-
genes in crosses and backcrosses of transgenic B. napus
with B. rapa. They suggested that the ecological risk of
gene diffusion is small when transgenes are inserted
into the C-genome of B. napus. On the basis of the dis-
tribution probability of different chromosomes in hy-
brids and backcross generations of B. napus with B.
rapa, Lu et al.[14] built three mathematical models to
analyze transmission rate of transgene in different
backcross generations after the diffusing of the trans-
gene from A-genome or C-genome, with or without
selection pressure. The result supports the conclusion
obtained by Metz et al.[8] that transgene integrated on a
C-chromosome is safer than that on an A-chromosome.
Two problems exist in the previous studies on the
risk evaluation of C-genome transgeneic B. napus[8,12,14].
(1) There is no direct experimental evidence for the
assumption that the backcross populations with faster
transgene lost derived from a transgenic B. napus line
whose transgene was located on C-genome. (2) Strate-
gies for the development of C-genome transgenic B.
napus with low ecological risk were not pointed out.
Since the chromosomes of B. napus are tiny, and the A-
Chinese Science Bulletin Vol. 51 No. 13 July 2006

and C-genomes are highly homologous in sequence and
hardly distinguishable[15], it is not possible to ensure the
transfer of target gene into C-genome using B. napus as
the experimental material. In our experiments, we first
transferred bar gene into the genome of B. oleracea var.
alboglabra (CC), then hybridized the transgenic B. ol-
eracea var. alboglabra (CC, 2n=18) with non-trans-
genic B. rapa, and finally obtained resynthesized B.
napus with transgene on C-genome, thus directionally
integrated the target bar gene into C-genome of B.
napus. These C-genome transgenic B. napus lines pro-
vide a good material basis for investigation of the exis-
tence of transgene in alien species and offspring popu-
lations, and for further confirmation of the ecological
risk of transgene diffusion in C-genome transgene of B.
napus with experimental evidence, as well as for the
investigation of exchange frequency between A-ge-
nome and C-genome of B. napus.
1 Material and methods
1.1 Plant material
Hong-Kong Zhonghua Jielan (B. oleracea var. al-
boglabra, CC, 2n=18) was used in genetic transforma-
tion. Eight B. rapa (AA, 2n=20) accessions: Heiyebai
(No. QF01), Rongyou Aikangqing (No. QF04), Tezao
50 Baicaitai (No. QF05), Huangjindi Xiaobaicai (No.
QF06), Huangjin Xiaobaicai (No. QF08), Jiuyuexian
Hongcaitai (No. QF10), Chaoshan Tianbaicai (No.
QF11), and a double-low B. rapa cultivar (No.QF15).
Except the double-low B. rapa introduced from Canada,
all other experimental materials come from China.
1.2 Agrobacterium strain and plasmid
Agrobacterium tumefaciens strain is LBA4404 and
its plasmid is pCAMBIA3300. NPTII gene was used
for bacteria screen and bar gene was used for plant
screen (Fig. 1).
1.3 Genetic transformation of B. oleracea
Agrobacterium strain LBA4404 with pCAM-
BIA3300 plasmid was inoculated in a YEB plate with
50 mg/L Kanamycin and cultured at 28℃ for 2 d. A
single colony was inoculated in 5 mL liquid medium
(with 50 mg/L Kanamycin), shaken on an orbital shaker
at 200 rpm, and cultured at 28℃ overnight. The bacte-
ria liquid culture was inoculated in a YEB plate with 50
mg/L Kanamycin and cultured at 28℃ for 2 d. After
colonies grew up, the petri dish was put in a 4℃ refrig-
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erator for later use. In each experiment, a single colony
was inoculated in a YEB plate with 50 mg/L Kanamy-
cin, cultured at 28℃ for 1―2 d. Thalli was washed off
with 1/2MS liquid medium (pH 5.4), and diluted to A600
＝0.08, then used for infection.
The seeds of B. oleracea were sterilized using 70%
alcohol for 5 ― 10 s, 1% Dichloroisocyanuric acid
(DICA), for 8―15 min and washed with sterile water
for 3―4 times, implanted in 1/2MS medium and grown
for 4―5 d at 25℃, 16 h/8 h day/night). Then cotyle-
dons with 1―2 mm long petiole and 0.5―0.7 cm hy-
pocotyl sections were cut off as cotyledon and hypo-
cotyl explants, respectively.
Fig. 1. Plasmid map of pCAMBIA3300.
The explants were incubated in Agrobacterium liq-
uid. Infection was carried out at 22℃ for 5―10 min by
gentle shaking. The explants were moved to MS+1
mg/L 2.4-D+0.2 mg/L 6-BA medium, cultured in dark
at 22℃ for 2―3 d, put in MS+4.5 mg/L BA+5 mg/L
AgNO3+500 mg/L Cb for 5―7 d, then moved to selec-
tive medium MS+4.5 mg/L BA+5 mg/L AgNO3+500
mg/L Cb+10 mg/L PPT and finally cultured for 3―4
weeks. Green seedlings were cut off and implanted in
rooting medium MS+0.2 mg/L NAA+10 mg/L PPT.
After complete plants were established, they were
transplanted into pots and cultured with proper shade
and moisture.
1.4 PCR detection of T1 transgenic B. oleracea
In order to avoid false positives and Agrobacterium
contamination of T0 transgenic plant, T1 seeds were
obtained by bud pollination of T0 plants that were posi-
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tive in PPT screening. T1 seeds were sown in an iso-
lated chamber in Spring, 2004, and sprayed with 13.5%
Basta® at 1:200 dilution at seedling stage. Leaves of
resistant transgenic B. oleracea and non-transgenic B.
oleracea were used for genomic DNA[16] extraction by
CTAB method. PCR was performed with 20 μL reac-
tion system, with negative control (total DNA of
non-transgenic B. oleracea plants as template) and
positive control (plasmid DNA as template). Reaction
conditions were: 95℃, 2 min; 94℃, 30 s, 50℃, 30 s,
72℃, 1 min, 35cycles; 72℃, 5 min. PCR products was
checked by 1% agarose gel electrophoresis.
For bar gene detection, the forward primer was
5′-GAT CTC GGT GAC GGG CAG GA-3′ and the
reverse primer was 5′-GGC GGT CTG CAC CAT CGT
CAA-3′, synthesized by Shanghai Bioasia Biotechnol-
ogy Co. Ltd.
1.5 Genetic analysis of the transgenic B. oleracea
T1 seeds were sown in an isolated chamber in Spring
2004, and sprayed with 13.5% Basta® at 1:200 dilution.
The number of alive and dead plants was recorded and
PCR was performed to detect bar gene in live plants,
the segregation ratio of bar gene was calculated and χ2
test was performed.
1.6 Resynthesis of B. napus
In the test field of Qinghai Academy of Agricultural
and Forestry Sciences (Xining) in Spring 2004, eight B.
rapa species as female parents and T1 transgenic B.
oleracea with bar gene as male parent were crossed. 7
d after pollination, ovaries were cultured on MS me-
dium with 500 mg/L hydrolyzed casein (with 0.8% agar
and 3.0% sucrose) after sterilization (70% alcohol for
5―10 s, 1% DICA for 8―10 min, washed with sterile
water for 3―4 times). After ovaries were cultured for
nearly 35―40 d, seeds were peeled out of the ovaries,
inoculated into MS medium till they grew into normal
seedlings. The seedlings were cut off, treated in MS
medium with 0.01% colchicine for 7―10 d for chro-
mosome doubling. Then they were planted in rooting
medium (MS+0.2 mg/L NAA) for root initiation and
multiplication. Three new plants were multiplied from
each transgenic plant at least. After robust roots devel-
oped, the plants were transplanted in pots and allowed
to grow with proper shade and moisture.
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1.7 Cytological observation
Chromosome number of the hybrids were deter-
mined using young buds. Pistils of flower buds were
peeled out, treated with 0.002 mol/L 8-hydroxyquino-
line for 4 h, then fixed in Carnoy’s solution for 24h,
and stored in 70% ethanol at 4℃ until use. The pistils
were hydrolyzed in 1 N HCl at 60℃ for about 10 min,
squashed in a drop of modified carbol fuchsin and ob-
served under oil[17].
1.8 Herbicide resistance test and molecular detection
of bar gene
13.5% Basta® at 1:200 dilution was sprayed on the
leaves of synthetic B. napus. Chemical injury of leaves
was observed and recorded. Chemical injury of plants
and number of dead plants were observed in synthetic B.
napus lines grown in culture medium amended with 15
mg/L PPT after 13 d of culture. In both of the above
tests, non-transgenic plants were used as control.
PCR method is the same as that used for detection of
transgenic B. oleracea.
Southern blotting analysis was performed as follows:
20 μg of total genomic DNA was fully digested with
EcoR I, and electrophoretically separated with 0.8%
agarose gel. Prehybridization, hybridization and film
wash procedures follow those reported by Sharpe et
al.[18]. Digoxin labeling kit was used for probe labelling
and detection (Roche Diagnostics, Swiss). A 0.5-kb
fragment of bar gene generated by PCR was used as
template for probe labelling.
1.9 Pollen viability determination
Newly opened flowers were sampled at 9―10 a.m.,
anthers were squashed and pollen grains were stained
with 1% aceto-carmine and observed under microscopy.
Pollen viability rate was calculated as the number of
stained pollen grains/total pollen grains ×100. Three
views per flower were observed with nearly 500―1000
pollen grains. Zhongshuang 6, a common B. napus va-
riety, was used as control.
2 Result and analysis
2.1 Genetic transformation of B. oleracea and analy-
sis of transgenic plants
(i) Agrobacterium mediated genetic transformation
of B. oleracea. Transformation frequency with coty-
ledons and hypocotyls of B. oleracea was calculated
using data collected from three repeats. The result
Chinese Science Bulletin Vol. 51 No. 13 July 2006
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showed that the average transformation frequency was three plants and the inserted bar gene can be stably in-
8.04% with cotyledons as explants, which was higher herited. The three transgenic B. oleracea plants were
than that from hypocotyls (6.81%). Finally, 29 inde- selected as parents for the resynthesis of B. napus.
pendent transgenic plants were established, of which 21
Table 2 Genetic analysis of T1 population
were from infected cotyledons and 8 from infected hy- χ2
Transgenic Sample PPT-resistant PPT-susceptible
pocotyls (Table 1). lines numbers plants (R) plants (S) :
(3 1)a)
QR306 55 41 14 0.006
Table 1 Transformation frequency of cotyledons and hypocotyls medi-
QR307 99 76 23 0.165
ated by Agrobacterium tumefaciens
QR308 156 116 40 0.034
No. of No. of posi- Transformation
Repeats Explant a) χ20.05=3.84.
explants tive shoots frequency (%)
cotyledon 165 14 8.48
Exp I 2.2 Resynthesis of B. napus
hypocotyl 187 13 6.95

Exp II
cotyledon 241 20 8.29 (i) Hybridization between B. oleracea and B. rapa.
hypocotyl 279 20 7.17 Three thousand nine hundred and ninety-two ovaries
cotyledon 231 17 7.36
Exp III were cultured from 8 cross combinations of B. rapa×B.
hypocotyl 254 16 6.30
oleracea, yielding 154 seeds and 92 hybrid seedlings.
67 hybrid lines after culture and propagation were ob-
(ii) Bar gene detection of transgenic B. oleracea.
tained with an average hybrid production rate of 2.30%.
The resistance-susceptiblity separation of T1 plants was There was a great difference between hybrid production
found in PPT resistance screening experiment. Leaf rate among cross combinations (Table 3). For example,
lesions appeared in susceptible plants followed by the hybrid production rate of Huangjin Xiaobaicai×B.
wilting and death of whole plant. The resistant plants, oleracea was 14.8%, which was the highest, whereas
however, grew normally without any symptoms (Fig. that of Huangjindi Xiaobaibai×B. oleracea and Jiu-
2(a)). PCR detection for bar gene was carried out using yuexian Hongcaitai×B. oleracea was zero. Most cross
leaves from T1 resistant plants, and a unique band was combinations had a low hybrid production rate below
amplified from all tested plants as well as the positive 4.0%.
control, whereas no band was amplified from the nega-
tive control (Fig. 2(b)). This result indicates that bar Table 3 Hybrid production rate of B. rapa × B. oleracea by ovary
culturea)
gene has been integrated into the nuclear genome of B. No of Production Hybrid
No of seeds Hybrid
oleracea. Crosses ovary
obtained plants
rate of seeds production
cultured per ovary (%) rate (%)
QF01×J 766 9 5 1.17 0.65
QF04×J 493 19 17 3.85 3.45
QF05×J 378 15 3 3.97 0.79
QF06×J 525 11 0 2.10 0
QF08×J 399 78 59 19.55 14.8
QF10×J 462 3 0 0.62 0
Fig. 2. Transgenic B. oleracea with bar gene. (a) Response to PPT QF11×J 483 8 3 1.66 0.62
spray at seedling stage; (b) PCR amplification of bar gene. M, Marker; 1, QF15×J 486 11 5 2.26 1.03
blank; 2, negative control; 3, positive control; 4―10, transgenic plants. Total 3992 154 92 3.86 2.30
a) J represents transgenic plants with bar genes; Production rate of
seeds per ovary = Number of seeds obtained/Number of ovary cul-
(iii) Inheritance of the transgene in B. oleracea. tured×100%; Hybrid production rate = Hybrid plantlets/ Number of
Resistance evaluation by PPT spray and PCR detection ovary cultured×100%.
of T1 populations inbred from some T0 transgenic B.
oleracea plants showed that the segregation of resis- (ii) Chromosome observation. Chromosome
tance and susceptibility occurred in T1 populations de- counting was performed in 31 PPT resistant synthetic
rived from QR306, QR307 and QR308. The segrega- B. napus plants. The result showed that except one with
tion ratio was 3:1, which conforms to Mendelian 34 chromosomes and three with 19 chromosomes, all
model of one pair of genes (Table 2), indicating that a others had 38 chromosomes (Fig. 3(g)), which is the
single copied bar gene was inserted into all of these chromosome number of natural B. napus. The three

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Fig. 3. Resynthesized transgenic B. napus with bar gene. (a) Seed germination (2n=19) from B. rapa×B. oleracea hybrids cultured on MS media; (b)
seedlings of B. rapa×B. oleracea hybrids cultured on MS media supplemented with 0.2 mg/L NAA(before treated with colchicine); (c) screening of
resistant seedlings from B. rapa ×B. oleracea hybridsin MS medium supplemented with 0.2 mg/L NAA + 15mg/L PPT; (d) flowering plants; (e)
siliques of plants; (f) PCR result for bar gene in resynthesized B.napus. M, Marker; 1, blank; 2, negative control; 3, positive control; 4―10, resynthe-
sized B. napus; (g) chromosomes of resynthesized B. napus; (h) a resynthesized B. napus line at flowering stage.

plants with 19 chromosomes may be a result of chro-
mosome doubling failure, since it is exactly the number
of the addition of the two parental species (B. rapa,
n=10; B. oleracea, n=9).
(iii) Agronomic performance. Synthetic B. napus
grew vigorously with characteristics from both parents.
Most leaves are larger with less divided leaves and light
leaf color, resembling the female parent. Based on the
observation data, plants from 95% of the cross combi-
nations were with smooth and hairless leaves; those
from 80% of the combinations were with short petioles,
big and thick leaves. Plants from 82% of the combina-
tions had smaller siliques containing only 4 ― 8
seeds/pods (data not shown). The flowers of the syn-
thetic B. napus were creamy white (or yellowish white)
and set seeds normally when hybridized with natural B.
napus, showing no cross barrier at all. These synthetic
B. napus had more branches and longer blooming pe-

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riod. The angle between branches and the main stem
was smaller in the synthetic B. napus with stronger re-
sistance to biotic and abiotic stresses. They matured
8―15 d later than natural B. napus (Fig. 3(h)).
(iv) Transgene detection. PCR detection of bar
gene was carried out in 67 synthetic B. napus lines. The
result showed that there were 31 positive lines and 36
negative lines (Fig. 3(f)). These lines were transferred
to MS medium containing 15 mg/L PPT and 0.2 mg/L
NAA, or sprayed with 13.5% Basta® at 1:200 dilution
on leaves after transplantation. The result agreed well
with PCR detection. Three synthetic B. napus lines,
which were positive according to PCR and Basta® de-
tection, derived from the same transgenic male parent,
were randomly chosen for genomic Southern blot
analysis together with their male parent. The result
confirmed that exogenous bar gene had been integrated
into the genome of synthetic B. napus (Fig. 4). The
band pattern of hybridization after enzyme restriction
indicated that the chromosome region harboring bar
gene from the male parent B. oleracea line might have
not been exchanged with A-genome in these three syn-
thetic B. napus lines.
(v) Pollen fertility observation. The result from
stainability of pollen grains of 25 plants from 11 syn-
thetic B. napus lines showed that there was big differ-
ence among plants derived from the same parental line
of synthetic B. oleracea on pollen fertility. The CV of
pollen stainability within three of the lines even
reached over 20% (Table 4). The average pollen
stainability of most lines, however, ranged from 60% to
90%, which enabled nearly normal seed set upon polli-
nation (pollen stainability of natural B. napus control
was 98.88%).
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Fig. 4. Southern blot of resynthesized B. napus. A fragment of bar gene,
generated by PCR amplification of pCAMBIA3300, was used as the
probe (0.5 kb). M, Marker; 1, positive control; 2, negative control; 3,
transgenic B. oleracea DNA; 4―10, resynthesized B. napus DNA.
3 Discussion
Compared to other interspecific hybridization, the
hybridization of B. oleracea and B. rapa followed by
chromosome doubling results in new amphidiploid
plants of a novel type of B. napus that belongs to the
same species as cultivated natural B. napus. To easily
identify the hybridity, morphological markers such as
white flower petals were used for selection. Chromo-
some counting, bar gene detection and other agronomic
characters as well as pollen viability were also invoked
to confirm their hybrid nature. Theoretically, pollen
fertility of these hybrids should be quite good. In fact,
however, a decrease in pollen fertility was often ob-
served[19]. Jørgensen and Andersen[20] reported that
pollen fertility of hybrids was 16%―86%. In our ex-
periment, the fertility observed was between 58.61%―
88.95%.
Some previous studies have revealed that A-genome
and C-genome of B. napus are highly homologous in
sequence, but chromosomes of the two genomes during
meiosis do not match well. Moreover, at the structure

Table 4 Pollen stainability of resynthesized B. napusa)

Cross combination Line code Number of plants Number of pollens (mean) Pollen stainability (%)
QF01×J 1 2 671 71.27±0.97
QF01×J 3 6 635 88.95±33.30
QF01×J 4 2 777 75.30±26.80
QF05×J 1 1 597 79.06
QF08×J 5 1 708 87.57
QF08×J 22 2 723 86.72±1.36
QF08×J 34 2 805 76.90±7.44
QF08×J 39 2 993 58.64±20.82
QF08×J 41 4 856 58.61±15.10
QF08×J 51 1 785 73.89
QF11×J 3 2 787 82.13±1.08
a) Number of pollens (mean)=Total number of pollens of each line/Number of plants.

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level, the homolog of A genome and C genome is only
partial, so the possibility of gene exchange between
them was relatively small[14,18,21,22]. Namai et al.[23] ob-
served 13%―72% trivalents at metaphase stage of
meiosis in the first generation hybrids between B. rapa
and B. napus. Based on this observation, Lu et al.[24]
deduced that the genomic recombination frequency of
C-chromosome and A-chromosome should be less than
0.7%―4.0%. The recombination frequency is possibly
lower in the case of genes located near the centromere.
Results from genomic Southern blot indicated that there
might not be any exchange between A-genome and
C-genome in the region of exogenous bar gene in the
selected synthetic B. oleracea lines. Thus we believe
that it is very likely (with the probability above 95%)
that the resythetic B. napus population obtained in this
study contains the transgene on C-genome. Further
studies on the inheritance behavior of the transgene in
these lines using chromosome-specifc (RAPD) markers,
and molecular cytogenetics by genomic in situ hybridi-
zation (GISH) with exogenous bar gene as probe, will
offer direct evidence for gene exchange frequency be-
tween A-genome and C-genome.
In triploid hybrids obtained from crosses of B. napus
(AACC, 2n=38) and B. rapa (AA, 2n=20), C-chrom-
osome is randomly distributed in AAC hybrids[25]. In
subsequent backcross generations, individual C-chrom-
osomes are transmitted into gametes irregularly. They
are likely to be lost during meiosis because no ho-
mologous chromosome for pairing can be found[26].
Lu[26] observed the decrease of vitality of individual
hybrids with C-chromosomes due to aneuploidy.
Among gametes (n=10―19) formed in interspecific
hybrids during meiosis, the relative adaptability of the
gametes with 10 chromosomes is 10―50 times higher
than that of others (n=11―19). In this way, with the
increase of backcross generation, the number of chro-
mosome in backcross generation tends to be close to 20.
A number of studies indicated that the transmission
frequency of a transgene from A-genome to the next
generation was nearly 50%[8,13,14,25,27]. Metz et al.[8]
found that the transmission frequency of a transgene
from C-genome of two transgenic lines into its BC1 was
26% and 46%, respectively, and that to BC2, BC3 and
BC4 was averaged to 5%, 11% and 9%, respectively.
Lu et al.[14] concluded that the transmission frequency
of transgenes from C-genome to BC1, BC2, BC3 and
BC4 was 39.9%, 7.7%, 1.2% and 0.1%, respectively.
1584
According to Zhu et al.[13], the transgenes from
A-genome and C-genome were segregated at a ratio of
1:1 in BC1 generation, while in BC2 generation and
afterwords, the transmission frequency of transgenes
from C-genome to subsequent generations was obvi-
ously lower than that from A-genome. One line was
found to lose the transgene on C-genome in BC4 gen-
eration. Lu et al.[14] deemed that the transgenes on
C-genome will disappear after several generations of
backcross if the transgenic B. napus is not grown re-
peatedly and the transgenes are not manually selected.
Therefore, genes from A-genome of B. napus could be
more easily transferred into B. rapa (A-genome) or B.
juncea (AB-genome)[8] than genes on C-genome. But
there is still controversy among these authors over the
transmission frequency of transgenes on C-genome to
hybrids and backcross generations. We think that the
inconsistency of transmission frequency of transgenes
from various studies resulted from the fact that the po-
sition of transgenes in B. napus genomes was uncertain.
Tomiuk et al.[28] also proved that the gene insertion site,
whether it was in the C-genome or A genome of the two
transgenic B. napus lines in the experiments conducted
by Metz et al.[8], could not be identified. Hence, the
acquisition of C-chromosome transgenic B. napus is of
great importance for explaining the difference in trans-
mission frequency of genes on C-genome and for un-
derstanding the mechanism behind transmission of
transgenes to hybrids and subsequent backcross genera-
tions.
Resynthesis of B. napus is an important approach for
the enrichment of B. napus gene pool and genetic de-
velopment of new varieties at present. Genetic trans-
formation has become the most efficient measure for
genetic modification of crops. The ecological risk aris-
ing from the release of transgenic plants is a main re-
striction to the development and application of trans-
genic B. napus. Based on the theory of safe integration
sites of exogenous genes in B. napus[12] and the evolu-
tionary relation of Brassica species[29], this study ex-
plored a new way to obtain C-chromosome transgenic
B. napus with bar gene through hybridizing transgenic
B. oleracea var. alboglabra (CC) with non-transgenic B.
rapa, resulting in new materials for ecological risk as-
sessment in the field on genetic drift of transgenic B.
napus with exogenous gene on different geonomes.
This makes it possible to further prove the safety of
C-chromosome transgenic B. napus, and reveal the
mechanism behind genetic exchange between A-ge-
Chinese Science Bulletin Vol. 51 No. 13 July 2006

nome and C-genome of B. napus. In addition, this study
contributed greatly to the application of low ecological
risk transgenic B. napus by creating novel lines with a
transgene of low transmission probability.
Acknowledgements We thank Dr. Lu Changming and Dr.
Lu Guangyuan for modification opinions of the paper. This
work was supported by National 863 Project (Grant Nos.
2002AA212011, 2003AA222101 & 2005AA241030) and the
National Natural Science Foundation of China (Grant
No.30270791).
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