Euphytica

, Volume 162, Issue 1, pp 81–89 | Cite as

Improving ovary and embryo culture techniques for efficient resynthesis of Brassica napus from reciprocal crosses between yellow-seeded diploids B. rapa and B. oleracea

  • Jing wen
  • Jin-xing Tu
  • Zai-yun Li
  • Ting-dong Fu
  • Chao-zhi Ma
  • Jin-xiong Shen
Article

Abstract

The primary aim of this study was to optimize in vitro culture protocols to establish an efficient reproducible culture system for different Brassica interspecific crosses, and to synthesize yellow-seeded Brassica napus (AACC) for breeding and genetical studies. Reciprocal crosses were carried out between three B. rapa L. ssp. oleifera varieties (AA) and five accessions of B. oleracea var. acephala (CC). All the parental lines were yellow-seeded except one accession of B. oleracea. Hybrids were obtained through either ovary culture from crosses B. rapa × B. oleracea, or embryo culture from crosses B. oleracea × B. rapa. A higher rate of hybrid production was recorded when ovaries were cultured at 4–7 days after pollination (DAP). Of different culture media, medium E (MS with half strength macronutrients) showed good response for ovaries from all the crosses, the highest rate of hybrid production reaching 45% in B. rapa (1151) × B. oleracea (T2). In embryo culture, the hybrid rate was significantly enhanced at 16–18 DAP, up to 48.1% in B. oleracea (T3) × B. rapa (JB2). The combinations of optimal DAP for excision and media components increased recovery of hybrids for ovary and embryo culture, and constituted an improved technique for B. rapa × B. oleracea crosses. In addition, yellow seeds were obtained from progenies of two crosses, indicating the feasibility of developing yellow-seeded B. napus through the hybridization between yellow-seeded diploids B. rapa and B. oleracea var. acephala.

Keywords

Brassica oleracea var. acephala B. rapa Synthetic Brassica napus Embryo culture Ovary culture Yellow seed color 

Abbreviations

BA

6-Benzylamino purine

DAP

Days after pollination

MS

Murashige and Skoog medium

NAA

α-Naphthaleneacetic acid

Introduction

Rapeseed Brassica napus (AACC, 2n = 38) is widely cultivated as an oilseed crop in China, Canada, Australia and Europe. It evolved as a natural allopolyploid following hybridization between B. rapa (2n = 20, AA) and B. oleracea (2n = 18, CC) (UN 1935). B. oleracea has also been suggested as the cytoplasmic donor in the evolution of B. napus (Erickson et al. 1983; Palmer et al. 1983). As compared to its progenitor species B. oleracea and B. rapa which exhibit extensive morphological and physiological diversity, B. napus has limited variability (Prakash and Hinata 1980). Intensive breeding has also exhausted this variability to a considerable extent. Thus, resynthesis of B. napus by utilizing the wider gene pool of the extant diploids offers new avenues for extending the range of genetic variation available to breeders. The potentials for combining agronomically desirable traits such as self-incompatibility, earliness, resistance to aphids and Verticillium wilt and yellow-seed coat from diploids in synthetic B. napus are well documented, as also the importance of newly synthesized B. napus in genetic analysis, evolutionary studies and heterosis breeding (Rahman 2005; Prakash and Raut 1983; Quazi 1988; Happastadius et al. 2003; Chen et al. 1988; Chen and Heneen 1992; Zhang and Zhou 2006; Lukens et al. 2006; Seyis et al. 2006).

Yellow-seeded cultivars have higher oil and protein but lower fibre contents as compared to brown- and black-seeded ones (Stringam et al. 1974). Development of yellow-seeded genotypes has been one of the major objectives of rapeseed breeders in quality breeding programs. Hybridization between yellow-seeded B. rapa and yellow-seeded B. oleracea is the most direct and simple strategy to generate B. napus with yellow seeds. Though many investigations to breed yellow-seeded B. napus have been carried out in the last several years, only few stable yellow-seeded forms have been developed. Several reasons have been attributed for the failures to obtain truly yellow-seeded B. oleracea genotypes (Chen et al. 1988). Yellow-seeded accessions of B. oleracea var. acephala are cultivated as ornamental plants for their purple or white leaves in southwest of China. They also possess other desirable traits, such as drought and cold tolerance and self-incompatibility. The present investigation is the first attempt to hybridize reciprocally both the yellow-seeded constituent diploid species viz. B. rapa and B. oleracea var. acephala to develop yellow-seeded B. napus, to transfer the useful characters and also to study the reciprocal cytoplasmic effect in synthetic B. napus.

Embryo rescue techniques considerably help in obtaining wide hybrids where hybrid embryos abort in early stages of development. Since its first use in Brassica by Nishi et al. (1959), extensive investigations have been carried out to improve the techniques for obtaining higher seed set (Inomata 1976, 1978, 1979; Zhang et al. 2003, 2004). However, the success rate is very low particularly in crosses where B. oleracea is used as female parent (Stewart 2004). Furthermore, scanty information is available on successful reciprocal synthesis of B. napus (Song et al. 1993). In this study, we aimed at developing an efficient model of ovary and embryo culture for the reciprocal resynthesis of B. napus, and providing preliminary evidence for the feasibility of developing yellow-seeded B. napus through the hybridization between yellow-seeded diploids B. rapa and B. oleracea var. acephala.

Materials and methods

Plant material and crosses

Three varieties of B. rapa L. ssp. oleifera (codes: DB, JB2, 1151) (2n = 20, AA) and five accessions of B. oleracea var. acephala (codes: T1, T2, T3, T4, T6) (2n = 18, CC) were used in the investigation. These are all inbred strains with yellow seed coat color except brown-seeded T1. B. oleracea var. acephala accessions from Southwest University, China, exhibit considerable morphological variability, such as purple (T1) or white (T4 and T6) leaves, lack of petals (T2) and white flowers (T3). B. rapa plants were sown at the end of October in 2004 in Wuhan, forty days later than B. oleracea plants to ensure synchronous flowering. Due to degeneration following selfing, only few plants from each B. oleracea accession survived for hybridizations and incomplete reciprocal crosses were made in March 2005. So five B. rapa × B. oleracea and five B. oleracea × B. rapa crosses were used for ovary and embryo culture.

Ovary culture

Ovaries of the combination B. rapa × B. oleracea were removed after 4, 7, 10 and 14 days respectively after pollination and surface sterilized by soaking in 70% ethanol for 1 min, followed by a 15 min rinse with 0.1% HgCl2 solution and, subsequently, three rinses with sterile water for 5 minutes each time. For ovary culture, parts of the ovary stalks were cut off transversely on autoclaved filter paper in Petri dishes and the ovaries were placed with the basal cut end on the media.

The basal MS medium (Murashige and Skoog 1962) supplemented with 3% (w/v) sucrose, 8% agar at pH = 5.8, was autoclaved for 22 min at 121°C. For obtaining seeds, effects of five kinds of media were studied (Table 1). Media A (Inomata 1979), B (Shi and Meng 1995) and D (Zhang et al. 2004) were reported effective for ovary culture, whereas media C and E were used for ovary culture for the first time. Medium C contained organic components of NLN (Lichter 1982) and the mineral salts and cofactors of MS, and E had MS with half strength macronutrients (1/2 MS). The organic components of medium C (from NLN) were filter-sterilized and added after other components were autoclaved.
Table 1

Composition of various media used for ovary culture, embryo culture and hybrid plant development

Composition

Ovary culturea

Embryo culture

Plant regeneration

Chromosome doubling

Root induction

A

B

Cb

D

Ec

Basal medium

MS

MS

MS + NLN

MS

1/2 MS

MS

MS

MS

MS

BA (mg l−1)

0

0.2

0

3

0

0.2

1.5

1.5

0

NAA (mg l−1)

0

0

0

0.1

0

0

0.25

0.25

0.1

Colchicine (mg l−1)

0

0

0

0

0

0

0

100

0

aMedia used for ovary culture. Medium A, B and D were reported effective for ovary culture in other studies, whereas C and E were used for ovary culture for the first time

bMedium C contained organic components of NLN (Lichter 1982) and the mineral salts and cofactors of MS

cMedium E had MS with half strength macronutrients (1/2 MS)

For each cross, a total of about 200 ovaries were inoculated. They were kept in culture room at 25 ± 2°C with automatically timed white fluorescent lights (34W each) of a 16 h photoperiod. Cultured ovaries were taken out from the media after 35 days of explantation. Seed sets and naked-embryos in the capsules were excised aseptically and cultured on basic MS medium supplemented with 0.2 mg l−1 BA (Table 1). The number and rate of hybrids were calculated.

Embryo culture

Siliques of pollinated B. oleracea plants were removed and examined from 10 to 25 days after pollination (DAP) to assess embryo development and abortion. On the basis of these results, optimal stage for sampling explants was determined for embryo culture, and pods collected were surface sterilized as described above. Ovules were carefully dissected on filter paper in Petri dishes under sterile conditions, and the excised embryos were cultured on MS medium supplemented with 0.2 mg l−1 BA (Table 1).

Cytological and morphological confirmation

After plantlet formation, nodal segments with an intact leaf of each hybrid were transferred to medium for plant regeneration (Table 1). Multiple apical meristem and axilary shoots arose ten days later. The shoot tips were used to determine the chromosome number of the hybrids, after treated with 2 mM 8-hydroxyquinoline for 3–4 h at 22°C and fixed in Carnoy’s solution. Chromosome preparations were made according to Li et al. (1995).

Plant regeneration and chromosome doubling

All the hybrids obtained through ovary and embryo culture after cytological confirmation were multiplied by culturing the shoot tips or nodal segments on the plant regeneration medium (Table 1). Subculturing was done every 20–30 days interval. After several subcultures, averagely 30–40 cloned shoots were obtained from one hybrid. Subsequently, the shoots were transferred to medium with colchicine for chromosome doubling to restore fertility (Table 1). Ten days later, they were shifted back to MS medium with 1.5 mg l−1 BA and 0.25 mg l−1 NAA. The shoots were rooted on MS with NAA (0.1 mg l−1). In the middle of October, they were thoroughly washed to remove the adhering gel and transplanted directly to the field using sun-shading net method as described by Liu et al. (2003).

Preliminary screening for yellow seeds in selfed progenies of synthetic B. napus

Selfed progenies of synthetic B. napus were obtained from each doubled hybrid plants (A1 generation) through bud pollination. For each cross, only brown and yellow seeds were sown and advanced for one (A2) to two (A3) generations until May, 2007. Seed color was assessed visually and classified as yellow, yellow–brown, brown and black, following the criteria described by Rahman (2001).

Results

Ovary culture

Effect of different DAPs

In crosses B. rapa × B. oleracea, highly significant (data not shown, P < 0.01) differences in rates of hybrid production were observed among ovaries excised at four different DAPs. Generally, the ovaries at 7 DAP had a higher tendency for hybrid production (Table 2), and mean rate of hybrid production from ovaries cultured at 7 DAP across combinations was maximal (22.8%) and significantly higher than those at other DAPs. In crosses 1151 × T2, DB × T4 and JB2 × T6, significantly higher rates of seed sets were recorded at 7 DAP, being 42, 30 and 20%, respectively. While the Chi-square test of general contingency table (data not shown, P < 0.01) indicated that not all crosses followed the same pattern across DAPs. For cross DB × T3, the seed set response of ovaries cultured at 4 DAP was significantly better than that at other DAPs.
Table 2

Rate and mean of hybrids per ovary (%) through ovary culture at different days after pollination (DAP) in B. rapa × B. oleracea crosses

Cross

DAP

Mean

4

7

10

14

1151 × T2

14

42

6

2

16

JB× T3

4

18

8

0

7.5

DB × T4

0

30

2

0

8

JB× T6

0

20

6

0

6.5

DB × T3

18

4

0

0

5.5

Mean

7.2

22.8

4.4

0.4

8.7

Effect of different media

Response of five media was evaluated for ovary enlargement and hybrid survival. The rate and mean of hybrid production for different cross combinations and media are given in Table 3. After ten days of culture, the ovaries on medium A, B and E enlarged, whereas those on medium D had a tendency to form callus at the basal cut end, from which shoots regenerated three weeks later. The seed formation was inhibited in those cultures where callus was formed. Thus no seeds were obtained from crosses JB2 × T3, DB × T4 and DB × T3 when the ovaries were cultured on medium D. In contrast, the ovaries of cross JB2 × T6 had highest seed set on medium D. Similarly, it was found that ovaries cultured on medium C had no response in terms of seed formation. All the crosses showed good response on medium E, for mean rate of hybrids (25.1%) was significantly higher than those cultured on other media. There was a significant variation in the response of five crosses, and the cross 1151 × T2 was more responsive for seed formation (Table 3). All the five media were effective for the cross 1151 × T2, and medium E was optimal with the rate of hybrids per ovary reaching 45%.
Table 3

Rate and mean of hybrids per ovary (%) through ovary culture inoculated on different media in B. rapa × B. oleracea crosses

Cross

Media

Mean

A

B

C

D

E

1151 × T2

7.5

17.5

7.5

2.6

45

16

JB× T3

0

2.5

0

0

35

7.5

DB × T4

5

5.2

10

0

20.5

8

JB× T6

5

2.5

0

15

10

6.5

DB × T3

5

5

2.5

0

15

5.5

Mean

4.5

6.5

4

3.5

25.1

8.7

The number of naked-embryos and seeds obtained in each cross is summarized in Table 4, together with the results of hybrid identification. A total of 56 naked-embryos and 31 seeds were obtained from all cross combinations.
Table 4

The number and rate (in parentheses, per ovary × 100%) of hybrids obtained and confirmed, and the number of plants germinated in different cross combinations of B. rapa × B. oleracea through ovary culture

Cross

Ovaries cultured

Hybrids obtained

Plants germinated

Hybrids confirmed

Seeds

Naked-embryos

Total

1151 × T2

199

14y

18

32 (16.1%)

29

26(13.1%)

JB× T3

200

0

15

15 (7.5%)

13

13(6.5%)

DB × T4

198

7y

9

16 (8.1%)

16

10(8.1%)

JB× T6

200

10y

3

13 (6.5%)

12

11(5.5%)

DB × T3

200

0

11

11 (5.5%)

9

9(4.5%)

Total

997

31

56

87

79

69

y: Yellow seed coat color of the seeds obtained

Embryo culture

Studies on embryo development at different stages after pollination helped to decide the optimal time of embryo culture. As early as 16–20 days after pollination, ovules in the crosses with T1 and T4 as female parent started to degenerate. First, the ovules grew pale and a dark spot appeared inside. The integument began to collapse and shriveled completely by turning dark brown to black in color. Eventually, there were only embryos inside without endosperm left. Few intact ovules were found by the 18th day and none was ever found later than 20 DAP. At 16 DAP, about 72% of the ovules were viable inside the pods, and embryos in the same pod were in globular or heart-shaped stage, as well as some invisible to the naked eyes. While in crosses with T3 as female parent, we could obtain viable ovules during 16–22 days after pollination.

The number and rate of embryos isolated and cultured from different crosses, the number and rate of hybrid plants produced are presented in Table 5. No hybrid seeds were harvested under natural conditions in reciprocal crosses. The rate of hybrid plants was low in cross T1 × JB2 (7.9% per ovary), but was higher than 19.3% in other crosses, reaching 48.1% in T3 × JB2.
Table 5

The number and rate of seeds, embryos and true hybrids per ovary (%) obtained through conventional field pollination and embryo culture in B. oleracea × B. rapa crosses

Cross

No. of flowers pollinated

No. of seeds obtained in field

No. of ovaries dissected

No. of embryos obtained

No. of hybrids confirmed

Rate of embryos obtained

Rate of hybrids confirmed

T1 × JB2

523

0

38

3

3

7.9

7.9

T1 × DB

711

0

52

19

17

36.5

32.7

T3 × JB2

808

0

27

14

13

51.9

48.1

T3 × DB

747

0

44

15

11

34.1

25

T4 × DB

672

0

31

7

6

22.6

19.3

Total

3461

0

192

58

50

30.2a

26a

aThe mean frequency of hybrids obtained per ovary over different cross combinations

Cytological and morphological confirmation

Mitotic metaphase revealed that 69 hybrids from ovary culture and 50 from embryo culture were true hybrids containing 19 chromosomes as expected (Fig. 1a). The young hybrid plants were morphologically intermediate between the two parents with many matroclinous characters. They had hairy leaves as in B. rapa and curled leaves and purple or white stems as in B. oleracea. However, the leaf color was close to the female parent. It was darker in crosses involving B. oleracea as the female parent than in the reciprocal crosses.
Fig. 1

(a, b) Mitotic metaphase in F1 hybrid (2n = 19) and synthetic allopolyploid (2n = 38), respectively. The bar indicates 5 μm

Plant regeneration and chromosome doubling

Hybrid plants which were cytological confirmed were cultured on the medium with 100 mg l-1colchicine for chromosome doubling, and the average doubling frequency was 75.3%. Leaf mitotic metaphase from some of the doubled plants with 2n = 38 revealed the success of chromosome doubling (Fig. 1b).

Preliminary screening for yellow seeds in selfed progenies of synthesized B. napus

Self-pollinated seeds of A2 or A3 plants from all the ten crosses were visually examined for color. Progenies from 1151 × T2 were advanced to A3 generation because of an additional growing season (summer–autumn) in Lanzhou, China in 2006, thus the seeds derived from A3 plant were subjected to seed color analysis. In this cross, three A3 families that segregated for black, brown and yellow–brown seeds were obtained, together with one A3 plant producing almost yellow seeds (Fig. 2c, d). In the three reciprocal crosses (Tables 4, 5), A2 families produced black seeds except in crosses involving JB2 and T3 as parents. The A2 plants of JB2 × T3 produced only yellow A3 seeds (Fig. 2g), whereas the A2 plants of its reciprocal cross T3 × JB2 produced brown seeds (Fig. 2h) or segregated for black and brown seeds. A2 plants of the remaining crosses produced black or a mixture of brown and black seeds, and no yellow seeds were observed in this generation.
Fig. 2

Seed color of B. rapa (1151, JB2), B. oleracea (T2, T3) and synthetic B. napus. (a) 1151. (b) T2. (c, d ) Seeds of two A3 plants derived from 1151 × T2. (e) JB2. (f) T3. (g) Seeds of one A2 plant from JB2 × T3. (h) Seeds of one A2 plant from T3 × JB2

Discussion

Due to the occurrence of crossing barriers, the frequency of hybridization success for synthetic B. napus had been reported early to be too low to be of significant use in normal breeding program (Nishi et al. 1959). Application of in vitro culture techniques would appreciably enhance the frequency of hybrid production. Inomata (1978) reported that ovary culture was markedly effective for the production of hybrids in B. rapa × B. oleracea but not in the reciprocal combinations. In contrast, Takeshita et al. (1980) proposed that hybrids were more easily obtained through embryo culture when B. oleracea was the female parent. Based on these reports, we planned to obtain hybrids through ovary culture in crosses B. rapa × B. oleracea var. acephala, and embryo culture in B. oleracea var. acephala × B. rapa.

The results have shown that the ovaries excised at 7 DAP had a higher tendency to form seeds than those at younger or older stages in four of the five crosses. This was consistent with reports in the hybridization of B. juncea × B. hirta and B. juncea × B. campestris (Mohapatra and Bajaj 1987, 1988), but Zhang et al. (2004) suggested that ovaries at 9 DAP were optimal for the culture in crosses B. rapa × B. oleracea. Our results also indicated that not all crosses followed the same pattern across DAPs, the ovaries from cross DB × T3 at 4 DAP were most responsive for seed formation. In wide hybridization of Brassiceae, the production of hybrid seeds through culture of pollinated ovaries at 4 DAP has been well documented (Delourme et al. 1989; Inomata 2003), indicating early degeneration of hybrid embryos in these crosses. It seemed that the optimal development stage for ovary culture varied among different crosses and the earliest ovaries were not always the responsive targets.

Media composition has been proved to influence the efficiency of embryo rescue techniques in many crops. In this study, addition of vitamins and amino acids to the medium (C) did not improve the growth of seeds in cultured ovaries. Our findings demonstrated that MS medium, with half strength of macronutrients (medium E in Table 1), was a better medium for hybrid recovery in ovary culture than MS with hormones or addition of more organic components. Ovaries from all the crosses cultured on medium E showed good response. In this context, several authors have reported the extensive effect of reducing the salt concentration in MS media with or without hormones, such as callus formation (Morard and Henry 1998), root induction (Khalafalla and Hattori 1999), somatic embryo formation (Chen and Chang 2001) and shoot induction (Pedraza-Santos et al. 2005). Our results strongly suggest that reducing the salt concentration in MS media also promotes the development of zygotic embryos in ovary culture, which has not been investigated earlier. Similar results were reported for B. juncea × B. hirta (Mohapatra and Bajaj 1987), where White’s medium supplemented with casein hydrolysate showed better response than MS or MS supplemented with indoleacetic acid, kinetin and/or casein hydrolysate. Inomata (1976) suggested that ovaries from different genotypes or crosses had different requirement for inorganic salts and ovaries from diploid plants required lower nitrate content for seed formation. These results lead to the speculation that lower macronutrients content in medium E or White seemingly, has a salutary effect on hybrid embryo development in ovary culture and medium E may be superior to White, because of more comprehensive nutrition element contained in the former.

In this study, no hybrid was obtained under natural condition in B. oleracea × B. rapa crosses, as in the research by Song et al. (1993). However, the rate of hybrid production was markedly increased when the timing of embryo culture was earlier than those (Takeshita 1980; Happastadius et al. 2003), showing the importance of identifying optimum development stage for culture. Embryos from our crosses should be taken out 16–18 days after pollination, for early embryo abortion or later embryo degeneration in crosses involving B. oleracea var. acephala as the female parent. The lowest rate of hybrids per ovary (7.9%) in this study was still higher than others (Sarashima and Matsuzawa 1986) in B. oleracea × B. rapa, whereas the rates of the remanent crosses were best in the same cross directions compared with the published data available (Takeshita et al. 1980; Lu et al. 2001; Happastadius et al. 2003).

The fact that yellow seeds were obtained from two crosses indicates that hybridization between the two yellow-seeded diploids is an amentable way to resynthesize yellow-seeded B. napus. The molecular mechanisms underlying the absence of yellow-seeded character in the progenies of some crosses remain obscure. It may be explained by functional complementation of intergenomic pigmentation systems as suggested by Chen et al. (1988). They obtained black seeds in crosses between light brown-seeded B. alboglabra and yellow-seeded B. rapa. Parallel attempts to disclose the genetics of seed color have been made in B. rapa and B. oleracea, but different or controversial results have been documented (Stringam 1980; Chen and Heneen 1992; Heneen and Brismar 2001). It was concluded that different pathways for the pigmentation of seed color may take place in/between B. rapa and B. oleracea, parallel with the highly polymorphic variation occurring in the two species (Chen and Heneen 1992), thus accounted for the inheritance complexity of seed color in resynthesized B. napus. Meanwhile, genetic and epigenetic variations in newly resynthesized B. napus (Lukens et al. 2006) may also contribute to the segregations of seed color in synthetic B. napus.

Hybrids were obtained reciprocally in three crosses (Tables 4, 5). New B. napus with the cytoplasm of B. rapa or B. oleracea may be useful for studying the origin of the cytoplasm of natural B. napus (Erickson et al. 1983; Palmer et al. 1983) or the cytoplasmic effect of agronomic traits, considering the long history of controversy regarding the maternal control of some agronomically desirable traits and the fact that no information on maternal and cytoplasmic effect on resynthesized Brassica amphidiploid is available. Oil composition of cultivated rapeseed (Brassica napus L.) was reported to be determined mainly by the embryo, but maternal effects have also been found (Kondra and Stefansson 1970; Thomas and Kondra 1973). Similar situation was reported in the inheritance of seed coat color in Brassica (Chen and Heneen 1992; Heneen and Brismar 2001). In this study, A2 plants of JB2 × T3 produced yellow seeds, whereas in its reciprocal crosses no yellow seeds but brown or black seeds were obtained, indicating cytoplasmic effect on seed color. Further work aiming at studying the inheritance of seed color in resynthesized B. napus is under way. The synthetic B. napus will also provide a better knowledge of cytoplasmic or maternal effect on genome evolution following allopolyploidization.

To conclude, embryo rescue technique was significantly improved either with ovaries excised between 4 and 7 DAP in B. rapa × B. oleracea or with embryos dissected during 16–18 DAP in B. oleracea × B. rapa and at rates sufficiently high to be practical for breeding purposes. Specially, MS basal medium with half strength of macronutrient salts was very effective for the production of B. rapa × B. oleracea hybrids. These results will be helpful in developing synthetic B. napus of reciprocal origins by in vitro culture to expand the germplasm pool for breeding, genetic and evolutionary analysis.

Notes

Acknowledgments

The authors thank Prof. Shyam Prakash from Indian Agricultural Research Institute, New Delhi for critical reading and suggestions for improving the manuscript. This research was financed by funds from the High-tech program “863” (20060110z1093), the High-tech program “863” (2006AA10A), the Program for Changjiang Scholar and Innovative Research Team in university (IRT0442), and the Program for “948” (2003-Q04).

References

  1. Chen BY, Heneen WK (1992) Inheritance of seed colour in Brassica rapa L. and breeding for yellow-seeded B. napus L. Euphytica 59:157–163CrossRefGoogle Scholar
  2. Chen BY, Heneen WK, Jonsson R (1988) Resynthesis of Brassica napus L. through interspecific hybridization between B. alboglabra Bailey and B. rapa L. with special emphasis on seed colour. Plant Breed 101:52–59CrossRefGoogle Scholar
  3. Chen JT, Chang WC (2001) Effects of auxins and cytokinins on direct somatic embryogenesis on leaf explants of Oncidium ‘Gower Ramsey’. J Plant Growth Regul 34:229–232CrossRefGoogle Scholar
  4. Delourme R, Eber F, Chevre AM (1989) Intergeneric hybridization of Diplotaxis erucoides with Brassica napus. I. Cytogenetic analysis of and BC1 progeny. Euphytica 41:123–128CrossRefGoogle Scholar
  5. Erickson LR, Straus NA, Beversdorf WD (1983) Restriction patterns reveal origins of chloroplast genomes in Brassica amphiploids. Theor Appl Genet 65:201–206CrossRefGoogle Scholar
  6. Happastadius I, Ljungberg A, Kristiansson B, Dixelius C (2003) Identification of Brassica oleracea germplasm with improved resistance to Verticillium wilt. Plant Breed 122:30–34CrossRefGoogle Scholar
  7. Heneen WK, Brismar K (2001) Maternal and embryonal control of seed colour by different Brassica alboglabra chromosomes. Plant Breed 120:325–329CrossRefGoogle Scholar
  8. Inomata N (1976) Culture in vitro of excised ovaries in Brassica campestris L. I. Development of excised ovaries in culture media, temperature and light. Japan J Breed 26:229–236Google Scholar
  9. Inomata N (1978) Production of interspecific hybrids between Brassica campestris and Brassica oleracea by culture in vitro of excised ovaries II. Effect of coconut milk and casein hydrolysate on the development of excised ovaries. Japan J Genet 53:1–11CrossRefGoogle Scholar
  10. Inomata N (1979) Production of interspecific hybrids in Brassica campestris × B. oleracea by culture in vitro of excised ovaries II. Development of excised ovaries on various culture media. Japan J Breed 29:115–120Google Scholar
  11. Inomata N (2003) Production of intergeneric hybrids between Brassica juncea and Diplotaxis virgata through ovary culture and the cytology and crossability of their progenies. Euphytica 133:57–64CrossRefGoogle Scholar
  12. Khalafalla MM, Hattori K (1999) A combination of thidiazuron and benzyladenine promotes multiple shoot production from cotyledonary node explants of faba bean (Vicia faba L.). J Plant Growth Regul 27:145–148CrossRefGoogle Scholar
  13. Kondra ZP, Stefansson BR (1970) A maternal effect on the fatty acid composition of rapeseed oil (Brassica napus). Can J Plant Sci 50:345–346Google Scholar
  14. Li Z, Liu HL, Luo P (1995) Production and cytogenetics of intergeneric hybrids between Brassica napus and Orychophragmus violaceus. Theor Appl Genet 91:131–136Google Scholar
  15. Lichter R (1982) Induction of haploid plants from isolated pollen of Brassica napus. Z Pflanzenphysiol 105:427–434Google Scholar
  16. Liu XP, Liu ZW, Tu JX, Chen BY, Fu TD (2003) Improvement of microspores culture techniques in Brassica napus L. Hereditas (Beijing) 25:433–436 (in Chinese with English summary)Google Scholar
  17. Lu CM, Zhang B, Kakihara F, Kato M (2001) Introgression of genes into cultivated Brassica napus through resynthesis of B. napus via ovule culture and the accompanying change in fatty acid composition. Plant Breed 120:405–410CrossRefGoogle Scholar
  18. Lukens LN, Pires JC, Leon E, Vogelzang R, Oslach L, Osborn T (2006) Patterns of sequence loss and cytosine methylation within a population of newly resynthesized Brassica napus allopolyploids. Plant Physiol 140:336–348 PubMedCrossRefGoogle Scholar
  19. Mohapatra D, Bajaj YPS (1987) Interspecific hybridization in Brassica juncea × Brassica hirta using embryo rescue. Euphytica 36:321–326CrossRefGoogle Scholar
  20. Mohapatra D, Bajaj YPS (1988) Interspecific hybridization in Brassica juncea × Brassica campestris through ovary culture. Euphytica 37:83–88CrossRefGoogle Scholar
  21. Morard P, Henry M (1998) Optimization of the mineral composition of in vitro culture media. J Plant Nutr 21:1565–1576CrossRefGoogle Scholar
  22. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 15:473–497CrossRefGoogle Scholar
  23. Nishi S, Kawata J, Toda M (1959) In the breeding of interspecific hybrids between two genomes “c” and “a” of Brassica through the application of embryo culture techniques. Japan J Breed 5:215–222Google Scholar
  24. Palmer JD, Shields CR, Cohen DB, Orton TJ (1983) Chloroplast DNA evolution and the origin of amphidiploid Brassica. Theor Appl Genet 65:181–189CrossRefGoogle Scholar
  25. Pedraza-Santos ME, López-Peralta M, González-Hernández VA, Engleman-Clark E, Sánchez-García P (2005) In vitro regeneration of Alstroemeria cv. ‘Yellow King’ by direct organogenesis. Plant Cell Tissue Organ Cult 84:169–178Google Scholar
  26. Prakash S, Hinata K (1980) Taxonomy, cytogenetics and origin of crop Brassicas, a review. Opera Bot 55:1–57Google Scholar
  27. Prakash S, Raut RN (1983) Artificial synthesis of Brassica napus and its prospects as an oilseed crop in India. Indian J Genet 43:282–290Google Scholar
  28. Quazi MH (1988) Interspecific hybrids between Brassica napus L. and B. oleracea L. developed by embryo culture. Theor Appl Genet 75:309–318CrossRefGoogle Scholar
  29. Rahman MH (2001) Production of yellow-seeded Brassica napus through interspecific crosses. Plant Breed 120:463–472CrossRefGoogle Scholar
  30. Rahman MH (2005) Resynthesis of Brassica napus L. for self-incompatibility action, inheritance and breeding potential. Plant Breed 124:13–19CrossRefGoogle Scholar
  31. Sarashima M, Matsuzawa Y (1986) Interspecific hybridization between Chinese cabbage and cabbage. Bull Coll Agr, Utsunomiya Univ 13(1):1–19Google Scholar
  32. Seyis F, Friedt W, Lühs W (2006) Yield of Brassica napus L. hybrids developed using resynthesized rapeseed material sown at different locations. Field Crops Res 96:176–180CrossRefGoogle Scholar
  33. Shi SW, Meng JL (1995) Studies on in vitro culture of pollinated ovary in interspecific crosses between Brassica species. J Wuhan Botany Res 13:8–14 (In Chinese)Google Scholar
  34. Song K, Tang K, Osborn TC (1993) Development of synthetic Brassica amphidiploids by reciprocal hybridization and comparison to natural amphidiploids. Theor Appl Genet 86:811–821CrossRefGoogle Scholar
  35. Stewart A (2004) A review of crossing relationship between cultivated Brassica species. Cruciferae Newsl 25:25–26Google Scholar
  36. Stringam GR (1980) Inheritance of seed color in turnip rape. Can J Plant Sci 60:331–335CrossRefGoogle Scholar
  37. Stringam GR, Mcgregor DI, Pawlowski SH (1974) Chemical and morphological characteristics associated with seed coat colour in rapeseed. In: Proceedings 4th International Rapeseed Conference, Giessen, West Germany, 99–108Google Scholar
  38. Takeshita M, Kato M, Tokumasu S (1980) Application of ovule culture to the production of intergeneric and interspecific hybrids in Brassica and Raphanus. Japan J Genet 55:373–387CrossRefGoogle Scholar
  39. Thomas PM, Kondra ZP (1973) Maternal effects on the oleic, linoleic, and linolenic acid content of rapeseed oil. Can J Plant Sci 53:221–225Google Scholar
  40. UN (1935) Genome-analysis on Brassica with special reference to the experimental formation and peculiar mode of fertilization. Jpn J Bot 7:389–452Google Scholar
  41. Zhang GQ, Zhou WJ (2006) Genetic analyses of agronomic and seed quality traits of synthetic oilseed Brassica napus produced from interspecific hybridization of B. campestris and B. oleracea. J Genet 85:45–51PubMedCrossRefGoogle Scholar
  42. Zhang GQ, Tang GX, Song WJ, Zhou WJ (2004) Resynthesizing Brassica napus from interspecific hybridization between Brassica rapa and B. oleracea through ovary culture. Euphytica 140:181–187CrossRefGoogle Scholar
  43. Zhang GQ, Zhou WJ, Gu HH, Song WJ, Momoh EJJ (2003) Plant regeneration from the hybridization of Brassica juncea and B. napus through embryo culture. J Agron Crop Sci 189:347–350CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Jing wen
    • 1
  • Jin-xing Tu
    • 1
  • Zai-yun Li
    • 1
  • Ting-dong Fu
    • 1
  • Chao-zhi Ma
    • 1
  • Jin-xiong Shen
    • 1
  1. 1.National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed ImprovementHuazhong Agricultural UniversityWuhanP.R. China

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