Abstract

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 93275×83170 (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 93275×83170 had the lowest embryogenesis (0-0.14 per bud)

Table 1 Differences of embryogenic frequency among different genotypes¹⁾

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

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.

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⁾

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

Table 5 Effect of genotypes on direct plantlet formation

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

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