Development of self-incompatible double low winter oilseed rape lines by means of doubled haploid system

Vratislav Kučera, Miroslava Vyvadilová, Dagmar Tomášková

Research Institute of Crop Production, Department of Applied Genetics, Drnovská 507, 161 06 Praha 6 – Ruzyně, Czech Republic 

    E-mail:Kucerav@hb.vurv.cz

 

 

Abstract:

The F1 hybrids of self-incompatible (SI) lines with self-compatible (SC) donors of double low quality were used for development of doubled haploid (DH) lines with the required combination of these characters. The pollen embryogenesis method in microspore cultures was used for obtaining DH regenerants. SI degree and stability of individual DH plants were tested by repeated self-pollination in flowers and buds during the whole period of flowering. The average number of seeds per developed pod, after self-pollination in flowers (SF) and buds (SB), were determined as a measure of SI. The number of SI plants ranged from 10 to 27 %, partially SI from 0 to 8 % and completely SC from 70 to 90 %. Although the expected ratio of SI to SC regenerants is approximately 1:1, considerable shift to self-compatibility has been observed, probably due to gametic selection in microspore cultures against SI genotypes. Highly SI plants showing SF data from 0.0 to 3.2 proved erucic acid content from 0.44 to 0.82 % and glucosinolate content from 3.5 to 31 mmol per g of seeds. Obtained results indicate that there is the possibility to achieve DH lines with the combination of the desired characters from F1 hybrids of parental lines with recessive determined traits in case of deriving larger amount of regenerants.

 

 

Keyword

 

rapeseed; doubled haploid lines; self-incompatibility; seed quality

 

Introduction

 

 One of the possibilities to increase the rapeseed yield for 15 to 20 % is the hybrid breeding. Hybrid cultivars production is based on the utilization of cytoplasmic male sterility (CMS) or sporophytic self-incompatibility. Although use of CMS appears to be more successful in the present times (Renard et al., 1997) some rapeseed hybrids have been released recently on the basis of SI (Kott, 1995).

 

 Natural occurrence of SI plants in winter oilseed rape is very low, about 0.1 % (Kučera et al.,1995), modern cultivars are entirely self-pollinating lines. Active S alleles conditioning the high and stable degree of SI could be transferred to the Brassica napus genome by means of resynthesis from the original species Brassica oleracea and B. rapa. Some transformation experiments have been done for S alleles introduction (Hinata et al., 1993).

 

Much easier way is detecting SI plants in cultivars or breeding materials of winter oilseed rape. SI plants selected by this way and their inbred progenies manifest usually various levels of pseudo-compatibility due to the recessive disposition of SI.  Our SI lines produced until now had a high glucosinolate content. The aim of our work was to verify the possibility of obtaining homozygous  lines with a high degree of SI and 00 quality by means of doubled haploid system. This procedure could be complicated by the occurrence of gametic selection in microspore cultures that may  cause lower frequency of desired genotypes.

 

 

 Material and methods

 

The experimental material used were self-incompatible lines obtained by inbreeding of SI plants selected from winter oilseed rape cultivars and subsequently derived SI DH lines with higher glucosinolate content (Kučera et al.,1995). The F1 hybrids of these SI lines with  self-compatible  donors of double low quality were utilised to achieve DH lines with the combination of recessively determined SI and 00 quality. SI degree and stability of individual plants in DH regenerants were tested by repeated self-pollination in flowers and buds during all the period of flowering. The average number of seeds per developed pod after self-pollination in flowers (SF) and buds (SB) were determined. The relative number of seeds Rs=SF/SB x 100 or only SF number were chosen as a measure of SI degree. As SI were considered plants with Rs ranging from 0,0 to 20,0 or SF from 0,0 to 3,0, as partially SI with Rs from 20,1 to 80.0 or SF from 3,1 to 5,0 and the completely self-compatible (SC) with Rs above 80 or SF above 5.

 

The donor plants for microspore cultures were grown in 19x19 cm plastic containers in a  glasshouse from January to April with day/night temperature 20-25/10-15 0C, day length 16 hours under the artificial lighting approximately 250 mE .m2/sec. For obtaining DH regenerants the methods of pollen embryogenesis in microspore cultures and diploidization of haploids by the colchicine treatment in vitro were used (Vyvadilová et al., 1992). The regenerants from in vitro cultures were transferred to a peat compost in 8 cm pots and after 7 - 8 weeks of vernalisation and transplanting to 19x19 cm plastic containers were grown in an insect protected glasshouse.  Haploids and aneuploids detected using karyological analyses of young plants were excluded. The determination of erucic acid and glucosinolate content was made by the standard methods modified for a low number of seeds.

 

One way analysis of variance using confidence level 95% was used for statistical evaluation of SI to SC degree differences.

 

 

 Results and discussion

 

Self-incompatibility degree and seed quality characters segregation in DH regenerants

 

Comprehensive results of SI level testing in DH regenerants originated from four F1 combinations of SI lines and SC donors of 00 quality are given in Table I. The number of SI plants in individual groups of regenerants ranged from 10 to 27 %, partially SI plants from 0 to 8 %, and completely SC from 70 to 90 %. Fig.1 shows a broad range from completely and partially SI to highly SC plants that was observed in all groups of regenerants. Considering that there are no significant differences between partially SI and SC individuals in all cases it is recommended to select only plants with a high SI level verified in R2 generation again for DH lines deriving.

 

Although the expected ratio of SI to SC regenerants is approximately 1:1, considerable shift to self-compatibility has been observed. It could be caused by gametic selection in microspore cultures against SI genotypes. The data of SI degree level and 00 quality characters for selected DH R1 regenerants are demonstrated in Table II. Highly SI plants derived from three F1 hybrids showing SF data from 0.0 to 3.2 proved erucic acid content from 0.44 to 0.82 % and glucosinolate content from 3.5 to 31 mmol per g of seeds. Obtained results were verified in the R2 generation again. Repeated tests during the whole period of vegetation proved a partial weakening of SI at the end of flowering. Quality analyses of larger seed samples showed a little higher glucosinolate content in comparison with R1 generation. It could be due to different growing conditions of plants in R1 and R2 generation and a genetic instability of parental inbred lines.

 

 Further about 250 DH regenerants were derived from four F1 hybrids of self-incompatible DH lines with a high glucosinolate content (Table III.) and self-compatible DH donors of 00 quality (glucosinolate content from 9 to 10 mmol per g of seeds) to verify gametic selection against SI and segregation in quality traits. The number of highly SI regenerants ranged from 10 to 20 %, which confirmed the previously detected occurrence of gametic selection against SI in microspore cultures. Glucosinolate content in individual plants varied from 20 to 87 mmol per g of seeds. The results indicate that there is a possibility to obtain DH lines with the combination of desired characters from F1 hybrids of parental lines with recessively determined traits in case of deriving a large amount of regenerants. 

 

Self-pollination of four experimental hybrids of different SI DH lines by bees in isolation cages unexpectedly proved their entire self-compatibility. This indicates that the DH lines homozygous for recessive S alleles could be used only for the production of single-cross hybrids.

   

 
References

Hinata, K., Watanabe, M., Toriyama, Isogai, A. (1993). A review of recent studies on homomorphic self-incompatibility. International Review of Cytology, 143, 257-296.

Kott, L. S. (1995). Hybrid production systems based on self-incompatibility in oilseed Brassica. Proceedings of  9th International Rapeseed Congress, Cambridge, UK, 4-7 July, 73-78.

Kučera, V., Vyvadilová, M., Tomášková, D. (1995). Development of self incompatible lines of winter rape by means of doubled haploid systém Proceedings. of  9th International Rapeseed Congress, Cambridge, UK, 4-7 July,  89-91.

Renard, M., Delourme, R., Vallée, P., Pierre, J. (1997). Hybrid rapeseed breeding and production. Proceedings of the International Symposium on Brassicas, Rennes, France, 23-27 September, 291-298.

Vyvadilová, M., Zelenková, S. (1992). Responsiveness in microspore cultures of some cultivars and Czech breeding materials of rapeseed (Brassica napus L.). Genetika a  Šlechtění, 28, (4), 243-252.

 

 

I. Evaluation of self-incompatibility degree in DH regenerants

    of four F1 hybrids of SI lines and 00 quality donors

F1 combination

No of evaluated regenerants

Plants counted as

 

 

SI

Partially SI

SC

 

 

No

%

No

%

No

%

OP 600

56

15

26.8

2

3.6

39

69.6

OP 584

25

4

16.0

2

8.0

19

76.0

OP 645

18

2

11.1

1

5.6

15

83.3

14 x 309

10

1

10,0

0

0,0

 9

90.0

Abbreviations:

SI - self-incompatible

SC - self-compatible

 

II. Evaluation of SI degree and seed quality in selected DH regenerants

 derived from F1 hybrid generation of AI and 00 quality donors

Regenerant no.

 

SF

SB

Rs

 

erucic acid

(%)

glucosinolates

(mmol.g-1 seed)

OP 600-36

0,0

5,6

0,00

0,61

3,5

OP 600-41

0,0

1,7

0,00

0,82

26

OP 600-48

0,1

3,4

2,94

0,59

10

OP 600-54

0,3

4,1

7,32

0,64

14

OP 600-56

0,7

8,3

8,43

0,44

20

OP 645-4

2,3

12,2

18,85

0,46

31

OP 584-34a

1,9

10,5

18,09

0,65

17

OP 584-34b

3,2

13,5

23,70

0,73

23

Abbreviations:

SF - mean number of seeds after self-pollination in opened flowers

SB - mean number of seeds after self-pollination in buds

Rs -relative number of seeds (SF/SB x 100)

 

III. Self-incompatibility degree and seed quality parameters of SI DH lines

Regenerant no.

SF

SB

Rs

erucic acid

(%)

glucosinolates

(mmol.g-1 seed)

START 5/27

0,1

12,0

0,83

0,60

32

WRG 12/2/13

0,2

12,2

1,63

0,68

46

WRG 12/3/15

0,1

  8,6

1,16

0,70

49

WRG 12/5/5

0,1

11,7

0,85

0,40

90

1.      Range from self-incompatibility to self-compatibility in DH regenerants

derived from individual F1 hybrid combinations