ARTICLES

1578 Chinese Science Bulletin Vol. 51 No. 13 July 2006

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,

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-

ARTICLES

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-

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-

www.scichina.com www.springerlink.com 1579

ARTICLES

1580 Chinese Science Bulletin Vol. 51 No. 13 July 2006

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.

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

ARTICLES

showed that the average transformation frequency was 8.04% with cotyledons as explants, which was higher than that from hypocotyls (6.81%). Finally, 29 inde-pendent transgenic plants were established, of which 21 were from infected cotyledons and 8 from infected hy-pocotyls (Table 1).

www.scichina.com www.springerlink.com 1581

Table 1 Transformation frequency of cotyledons and hypocotyls medi-

ated by Agrobacterium tumefaciens

Repeats Explant No. of explants

No. of posi-tive shoots

Transformation frequency (%)

cotyledon 165 14 8.48 Exp I

hypocotyl 187 13 6.95 cotyledon 241 20 8.29

Exp II hypocotyl 279 20 7.17 cotyledon 231 17 7.36

Exp III hypocotyl 254 16 6.30

(ii) Bar gene detection of transgenic B. oleracea.

The resistance-susceptiblity separation of T1 plants was found in PPT resistance screening experiment. Leaf lesions appeared in susceptible plants followed by wilting and death of whole plant. The resistant plants, however, grew normally without any symptoms (Fig. 2(a)). PCR detection for bar gene was carried out using leaves from T1 resistant plants, and a unique band was amplified from all tested plants as well as the positive control, whereas no band was amplified from the nega-tive control (Fig. 2(b)). This result indicates that bar gene has been integrated into the nuclear genome of B. oleracea.

Fig. 2. Transgenic B. oleracea with bar gene. (a) Response to PPT spray at seedling stage; (b) PCR amplification of bar gene. M, Marker; 1, blank; 2, negative control; 3, positive control; 4―10, transgenic plants.

(iii) Inheritance of the transgene in B. oleracea. Resistance evaluation by PPT spray and PCR detection of T1 populations inbred from some T0 transgenic B. oleracea plants showed that the segregation of resis-tance and susceptibility occurred in T1 populations de-rived from QR306, QR307 and QR308. The segrega-tion ratio was 3:1, which conforms to Mendelian model of one pair of genes (Table 2), indicating that a single copied bar gene was inserted into all of these

three plants and the inserted bar gene can be stably in-herited. The three transgenic B. oleracea plants were selected as parents for the resynthesis of B. napus.

Table 2 Genetic analysis of T1 population

Transgenic lines

Sample numbers

PPT-resistant plants (R)

PPT-susceptible plants (S)

χ2

(3:1)a)

QR306 55 41 14 0.006QR307 99 76 23 0.165QR308 156 116 40 0.034a) χ2

0.05=3.84.

2.2 Resynthesis of B. napus

(i) Hybridization between B. oleracea and B. rapa. Three thousand nine hundred and ninety-two ovaries were cultured from 8 cross combinations of B. rapa×B. oleracea, yielding 154 seeds and 92 hybrid seedlings. 67 hybrid lines after culture and propagation were ob-tained with an average hybrid production rate of 2.30%. There was a great difference between hybrid production rate among cross combinations (Table 3). For example, the hybrid production rate of Huangjin Xiaobaicai×B. oleracea was 14.8%, which was the highest, whereas that of Huangjindi Xiaobaibai×B. oleracea and Jiu-yuexian Hongcaitai×B. oleracea was zero. Most cross combinations had a low hybrid production rate below 4.0%.

Table 3 Hybrid production rate of B. rapa × B. oleracea by ovary

culturea)

CrossesNo of ovary

cultured

No of seeds obtained

Hybrid plants

Production rate of seeds

per ovary (%)

Hybrid production

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 QF11×J 483 8 3 1.66 0.62 QF15×J 486 11 5 2.26 1.03

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-tured×100%; Hybrid production rate = Hybrid plantlets/ Number of ovary cultured×100%.

(ii) Chromosome observation. Chromosome

counting was performed in 31 PPT resistant synthetic B. napus plants. The result showed that except one with 34 chromosomes and three with 19 chromosomes, all others had 38 chromosomes (Fig. 3(g)), which is the chromosome number of natural B. napus. The three

ARTICLES

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-

1582 Chinese Science Bulletin Vol. 51 No. 13 July 2006

ARTICLES

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.

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%.

(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%).

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.

www.scichina.com www.springerlink.com 1583

ARTICLES

1584 Chinese Science Bulletin Vol. 51 No. 13 July 2006

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.

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-

ARTICLES

www.scichina.com www.springerlink.com 1585

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).

References

1 Wilkinson M J, Elliott L J, Allainguillaume J, et al. Hybridization between Brassica napus and B. rapa on a national scale in the United Kingdon. Science, 2003, 302: 457―459 [DOI]

2 Mikkelsen T R, Jensen J, Jørgensen R B. Inheritance of oilseed rape (Brassica napus) RAPD markers in a backcross progeny with Brassica Campestris. Theor Appl Genet, 1996, 92: 492―497 [DOI]

3 Snow A A, Andersen B, Jørgensen R B. Costs of transgenic herbi-cide resistance introgressed from Brassica napus into weedy Bras-sica rapa. Mol Ecol, 1998, 8: 605―615 [DOI]

4 Hauser T P, Shaw R G, Østergård H. Fitness of F1 hybrids between weedy Brassica rapa and oilseed rape (B. napus). Heredity, 1998, 81: 429―435 [DOI]

5 Warkwick S I, Simard M J, Légère A, et al. Hybridization between transgenic Brassica napus L. and its wild relatives: B. rapa L., Raphanus raphanistrum L., Sinapis arvensis L., and Erucastrum gallicum (wild.) O. E. Schulz. Theor Appl Genet, 2003, 107: 528―539 [DOI]

6 Metz P L J, Nap J P. A transgene-centered approach to the biosafety of transgenic plants: Overview of selection and reporter genes. Acta Bot Neerl, 1997, 46: 25―50

7 Hauser T P, Jørgensen RB, østergárd H. Fitness of backcross and F2 hybrids between weedy Brassica rapa and oilseed rape (B. napus). Heredity, 1998, 81: 436―443 [DOI]

8 Metz P L J, Jacobsen E, Nap J P, et al. The impact on biosafety of the phosphinothricin-tolerance transgene in inter-specific B. rapa×B. napus hybrids and their successive backcrosses. Theor Appl Genet, 1997, 95: 442―450 [DOI]

9 Jørgensen R B, Andersen B, Hauser T P, et al. Introgression of crop genes from oilseed rape (Brassica napus) to related wild species: An avenue for the escape of engineered genes. Acta Horticulturae, 1998, 459: 211―217

10 Scheffler J A, Dale P J. Opportunities for gene transfer from trans-genic oilseed rape (Brassica napus) to related species. Transgenic Res, 1994, 3: 263―278 [DOI]

11 Eastham K, Sweet J. Genetically modified organisms (GMOs): The significance of gene flow through pollen transfer. Environmental Issue Report No 28, Copenhagen: European Environment Agency, 2002. 75

12 Mikkelsen T R, Andersen B, Jørgensen R B. The risks of crop

transgene spread. Nature, 1996, 380: 31 [DOI] 13 Zhu B, Lawrence J R, Warwick S I, et al. Inheritance of GFP-Bt

transgenes from Brassica napus in backcrosses with three wild B.rapa accessions. Environ Biosafety Res, 2004, 3: 45―54 [DOI]

14 Lu C M, Kato M, Kakihara F. Destiny of a transgene escape from Brassica napus into Brassica rapa. Theor Appl Genet, 2002, 105: 78―84 [DOI]

15 Snowdon R J, Friedrich T, Friedt W, et al. Identifying the chromo-somes of the A- and C-genome diploid Brassica species B. rapa (syn. campestris) and B. oleracea in their amphidiploid B. napus. Theor Appl Genet, 2002, 104: 533―538 [DOI]

16 Doyle J J, Doyle J I. Isolation of plant DNA from fresh tissue. Fo-cus, 1990, 12: 13―15

17 Li Z, Liu H L, Luo P. Production and cytogeneric hybrids betwer-assica napus and Orychophragmus violaceus. Theor Appl Genet, 1995, 91: 131―136

18 Sharpe A G, Parkin I A, Keith D J, et al. Frequent nonreciprocal translocations in the amphidiploid genome of oilseed rape (Bras-sica napus). Genome, 1995, 38: 1112―1121

19 Beversdorf W D, Weiss-Lerman J, Erickson Lr, et al. Transfer of cytoplasmically inherited triazine resistance from bird’s rape to cultivated oilseed rape (Brassica campestris and B. napus). Can J Genet Cytol, 1980, 22: 167―172

20 Jørgensen R B, Andersen B. Spontaneous hybridization between oilseed rape (Brassica napus) and weedy B. campestris (Brassica-ceae): A risk of growing genetically modified oilseed rape. Am J Bot, 1994, 81: 1620―1626 [DOI]

21 Parkin I A P, Sharpe A G, Keith D J, et al. Identification of the A and C genomes of amphidiploid Brassica napus (oilseed rape). Genome, 1995, 38: 1122―1131

22 Parkin I A P, Lydiate D J. Conserved patterns of chromosome pair-ing and recombination in Brassica napus crosses. Genome, 1997, 40: 496―504

23 Namai H, Sarashima M, Hosoda T. Interspecific and inter-generic hybridization. In: Tsunoda S, Hinada K, Gomez-Campo C, eds. Brassica Crops and Wild Allies Biology and Breeding. Tokyo: Japanese Science Society Press, 1980. 191―202

24 Lu C M, Xiao L, Wu Y H. Ecological risk assessment of transgenic rapeseed in China. J Agricul Biotechnol, 2005, 13(3): 267―275

25 Nozaki T, Mishiba K, Mii M, et al. Construction of synteny groups of Brassica alboglabra by RAPD markers and detection of chro-mosome aberrations and distorted transmission under the genetic background of B. campestris. Theor Appl Genet, 2000, 101: 538―546 [DOI]

26 Lu C M. Fertilization fitness and relation to chromosome number in interspecific progeny between Brassica napus and B. rapa: A comparative study using natural and resynthesized B. napus. Breeding Science, 2001, 51: 73―81 [DOI]

27 Snow A A, Andersen B, Jørgensen R B. Costs of transgenic herbi-cide resistance introgressed from Brassica napus into weedy B. rapa. Mol Ecol, 1999, 8: 605―615 [DOI]

28 Tomiuk J, Hauser T P, Jørgensen R B. A- or C-chromosomes, does it matter for the transfer of transgenes from Brassica napus. Theor Appl Genet, 2000, 100: 750―754 [DOI]

29 U N. Genomic analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertiliza-tion. Japan J Bot, 1935, 7: 389―452