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Studies on Microspore Culture of Hybrid Parents in Brassica napus

H.Z. Wang, X.F. Wang, G.H. Liu, Y.B. Zheng and Q. Yang

Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan 430062, P R China

Abstract

In this study, totally 42 accessions of Brassica napus, most of them were maintainers and restorers of pol-CMS, were used to investigate the influence of genotypes on embryogenesis of microspore culture. The influence of genotypes, agar concentration in solid B5 medium and culturing temperature on direct plantlet formation from embryoids was also studied. The results showed that there were significant genotypic differences for embryogenesis among these genotypes. But the restoring or maintaining genes of pol-CMS did not affect embryogenesis of microspore culture. The efficiency of direct plantlet formation from embryoids was related to genotype, agar concentration in medium and culturing temperature. The results indicated that embryoids transferred to solid B5 medium with 1.5% agar and cultured initially in 4℃ for 10d could develop easily into normal haploid plantlets.

Key words

Brassica napus L;Maintainer; Restorer;Microspore culture; Direct plantlet formation;

Introduction

Since Lichter (1982) firstly reported the story of obtaining haploid plants from microspore culture in Brassica napus, extensive researches have been carried out to investigate embryogenesis of microspore culture, and some primary isolated microspore culture and chromosome doubling technology have been developed (Charne and Beversdorf, 1988; Yu and Liu, 1995; Wang et al, 1999; Shi et al, 2002; Wang et al, 2002)。However, the technology for direct plantlet formation from embryoids is still being improved since the efficiency for direct plantlet formation from embryoids is quite low. The utilization of heterosis is a successful strategy to increase rapeseed yield. For F1 hybrids to express strong heterosis, it is very important that the parents be kept homozygous. Microspore culture and chromosome doubling are unique tools to establish completely homozygous genotypes rapidly. Therefore, the exploration of using microspore culture technology to improve hybrid parents will be helpful in hybrid breeding. The objective of this study was to detect genotypic differences on embryogenesis of microspore culture and to analyze effects of some factors on the frequencies of plantlets produced directly from embryoids, and then to establish a highly effective system of microspore culture in Brassica napus hybrid breeding.

Methods

Totally 42 genotypes of Brassica napus were used in this study. These materials included 10 maintainers and 13 restorers of pol-CMS, 9 offspring lines of interspecific hybridization, 4 lines with yellow seed coat, 6 F4 lines of 9327583170 (93275 was the restorer of pol-CMS, and 83170 was a line with large seed and long pod). All these lines were planted in the Experimental Farm of Oil Crops Research Institute, CAAS.

Microspore culture protocol was modified from the methods described by Yu et al (1995), Wang et al (1999) and Shi et al (2002). After the cotyledonary embryoids were formed, they were transferred to solid B5 medium contained different agar concentrations and fortified with 0.1 mg/L GA3 and 2% sucrose, then cultured at 4 ℃ from 0 d to 14 d. Finally, these embryoids were cultured with 16 h/8 h light cycle at 25℃ for one month. Plantlets with more than 2 cm height and normal leaves developed from cotyledonary embryoids were counted as cases of direct plantlet formation.

SPSS software was employed to test differences among genotypes.

Results

Genotypic differences on embryogenesis of microspore culture

Table 1 showed that genotypes had significant effects on the embryogenic frequencies. The average embryogenic frequency for each genotype ranged from 0 to 11.67 per bud. Of the 42 lines, 22082, the maintainer of pol-CMS, had the highest embryogenesis (11.67 per bud), while F4 lines of the cross 9327583170 had the lowest embryogenesis (0-0.14 per bud)

Table 1 Differences of embryogenic frequency among different genotypes1)

Year

Type

Genotype

Number of embryoids/bud

Year

Type

Genotype

Number of embryoids/bud

2002

Pol-CMS

P54-2

0.380.021a

2003

F4 lines of 9327583170

D105

00a

P25-7

0.340.042a

D103

0.140.026b

12597

0.100.015b

D108

0.0620.0067c

12127

0.460.021a

D110

0.0680.0047c

12167

1.220.13c

D115

0.0120.0021a

Pol-CMS

P14-2

3.190.11a

D125

0.0620.010c

P15-1

00b

pol-CMS

22028

2.500.29a

P18-1

3.480.33a

22039

1.290.16a

P27-1

0.680.061c

22046

00a

W17-1

3.010.25a

22082

11. 671.91b

W18-1

2.020.081d

22084

0.440.091a

P10-2

2.410.28d

pol-CMS

24221

5.120.51a

Offsprings of interspecific hybridization

15461

0.970.14a

24276

2.260.30b

15463

0.140.025b

24110

00c

15447

0.630.047c

24220

0.540.064bc

2003

Offsprings of interspecific hybridization

25007-1

1.560.15a

24272

0.470.045bc

25029-1

0.490.040b

24691

8.081.24d

25056-2

1.450.13a

Lines with yellow seed coat

27173

00a

25063-1

5.440.32c

27250

0.680.021b

25025-1

0.830.040b

27254

0.0430.0036c

25060-1

1.390.12a

27253

0a

1) Number of embryoids/bud in the table is mean SE;

a, b and c represent significance at 0.05 probability level

Difference on embryogenesis between restores and maintainers of pol-CMS

The mean of embryoid yield of 13 restorers of pol-CMS was 2.40/bud, while that of 10 maintainers was 1.84/bud. Statistical analysis showed that there was no significant difference of embryoid yield between the restorers and maintainers of pol-CMS. This might mean that genes of restoring or maintaining of pol-CMS did not affect embryoid regeneration of microspore culture.

Effect of low temperature on direct plantlet formation from embryoids

Data from culturing cotyledonary embryoids in B5 medium contained 1.2 % agar at 4 ℃ for 0-14 d showed that low temperature was advantageous for plantlet development. From Table 2, it is easy to see that haploid plantlets at 4℃ were 2-2.6 times of those at 25℃ (i.e.: for 0 d at 4 ℃). The treatment of embryoids cultured for 10 d at 4 ℃ provided the highest ratio of plantlets developed from embryoids, which reached to 81.2%.

Table 2 Effect of low temperature on the development of plantlet from embryoids for line 22028

Days at 4℃

Number of embryoids

Number of plantlets

Ration of plantlet developed from embryoids (%)

0(CK)

42

13

31.0

7

45

32

71.1

10

64

52

81.2

14

53

33

62.3

Influence of agar concentration in B5 medium on direct plantlet formation from embryoids

Embryoids with the same size of line 25007, one offspring of interspecific hybridization, were transferred to solid B5 medium contained agar of 0.6 %, 0.8 %, 1.2 % and 1.5 % (w/v). Results showed that agar concentration had strongly influenced on haploid plantlet production (Table 3). The suitable agar concentration in B5 medium was 1.2%-1.5%.

Interaction between agar concentration and low temperature

Embryoids of 22082, the maintainer of pol-CMS, were cultured for 0 d or 10 d at 4 ℃ in B5 medium with different agar concentration from 0.6 % to 1.5 % (Table 4). The results showed that all cotyledonary embryoids deposited at 4 ℃ for 10 d with 1.5 % of agar were developed into plantlets, and the frequency.

Table 3 Effect of agar concentration on direct plantlet formation of line 25007 of plantlets formed from embryoids in B5 medium contained 1.5% agar was much higher than those in the medium contained 0.6%, 0.8% and 1.2% agar whether at 4 ℃ or not.. This meant that the embryoids, once formed from microspores, cultured initially in B5 medium with 1.2-1.5% agar at 4℃ for 10d were easy to develop into haploid plantlets.

Agar concentration (%)

Number of embryoids

Number of plantlets

Ration of plantlets/embryoids

0.6

55

0

0

0.8

60

8

13.3

1.2

76

43

56.5

1.5

66

41

62.1

Effect of donor plant genotypes on the direct formation of plantlet

Frequency of plantlets produced directly from cotyledonary embryoids was ranged from 0 to 100% among 7 genotypes (Table 5) when they were culture at 4 ℃ for 10 d with 1.5 % of agar. This meant that genotypes had strong effect on direct plantlet production.

Table 4 Effect of low temperature and agar concentration on direct plantlet formation of line 220821)

Agar concentration

Low temperature treatment

Number of embryoids

Number of plantlets

Ratio of plantlet/embryoids

0.6

NT

60

8

14.3

LT

58

21

36.2

0.8

NT

37

15

40.5

LT

58

30

57.7

1.2

NT

42

23

54.8

LT

68

55

80.9

1.5

NT

39

36

92.3

LT

57

57

100.0

1) NT: cultured at 25℃; LT: cultured for 10d at 4℃

Table 5 Effect of genotypes on direct plantlet formation

Genotype

Number of embryoids

Number of haploid plantlets

Ratio of Plantlets/embryoids

22028

48

42

87.5

22082

50

50

100.0

24221

31

3

10.0

24272

32

4

12.5

27250

35

0

0

25056

46

16

34.8

25025

32

4

19.0

Discussion

Results from this study showed that both pol-CMS maintainers and restorers could generate haploid embryos through microspore culture under the same inoculation conditions, though significant genotypic differences on embryogenesis existed. This indicated that both maintaining or restoring genes of pol-CMS might not be directly related to embryogenesis. Therefore, it was possible to use microspore culture technology to purify the hybrid parents of pol-CMS rapidly. This no doubt will be very useful in hybrid breeding. Up to now, several improved hybrid parents of pol-CMS system have been developed in our lab through microspore culture technology, and three of their hybrids have been released.

The results also showed that low temperature (4 ℃) and agar added to B5 medium were helpful for embryoids to develop directly and speedily into plantlets with rapid and healthy growth. These techniques were effective to avoid regenerating weak seedlings from embryoids, and to escape from a long time of subculturing and from somatic cell mutation. But it is still unknown how low temperature and agar could improve the frequency of plantlets generating from embryoids directly.

References

Charne D G, Beversdorf W D. (1988) Improving microspore culture as a rapeseed breeding tool: the use of auxins and cytokinins in an induction medium. Canadian Journal of Botany, 66 (8): 1671-1675

Lichter R. Induction of haploid plants from isolated pollen of Brassica napus L. Pflanzenphysiol,1982,105:427-734

Shi S W, Wu J S, Zhou Y M, Liu H L. (2002) Diploidization techniques of haploids from in vitro culture microspore of rapeseed (Brassica napus L.). Chinese Journal of Oil Crop Sciences, 24(1): 1~5

Wang H Z, Zheng Y B, Yang Q, Liu Z, Li J. (1999) Application of microspore culture technology in the breeding of rapeseed hybrids. Proceeding of 10th International Rapeseed Congress, (Canberra, Australia), 1999, 264~269

Wang Y F, Lu R J, Sun Y F, Zhou R M, Liu C H, Huang J H. (2002) The high frequency embryos induction and plant regeneration derived from microspores for rape grow in field. Chinese Agricultural Science Bulletin, 18(1): 20~23

Yu F Q, Liu H L. (1995) Effects of donor materials and media on microspore embryoid yield of Brassica napus. Journal of Huazhong Agricultural University, 14(4): 327~332

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