¹Dept. of Agricultural Sciences, KVL 40 Thorvaldsensvej DK-1871 Denmark

²Dept. of Mathematics and Physics, KVL 40 Thorvaldsensvej DK-1871 Denmark

³Institute of Oil Crops, Chinese Academy of Agricultural Sciences 430062 Wuhan China

E-mail: ocgil@public.wh.hb.cn

ABSTRACT

Protoplast fusions between B. napus and two related species, Orychophragmus violaceus and Xinjiang wild rape were made for transfer of genes for desirable fatty acid composition into B. napus. Many somatic hybrids were recovered and their hybridity was confirmed on morphological, cytological and molecular levels. Fertility of the hybrids between B. napus and Xinjiang wild rape varied depending on the B. napus variety used. Erucic acid content of seeds derived from hybrids after self-pollination and backcross to B. napus showed biased distribution towards high content parent. Fertile hybrids between B. napus and O. violaceus were recovered from asymmetric fusions with X-ray irradiated protoplasts of the donor parent O. violaceus. These hybrids released viable pollen grains and yielded seeds. The potential of somatic hybridization to enrich the accessible gene pool for rapeseed quality improvement is discussed in this paper.

INTRODUCTION

Oil quality improvement of oilseed rape has led to a marked increase of edible rapeseed oil consumption. However, further improvement of high quality rapeseed varieties has been hampered by the narrow genetic background of high oil quality Brassica napus materials. All these high quality varieties were derived from the original low erucic acid content resources, “Liho” or its derivatives (Qian, 1985; Kneen, 1992; Guo, et al 1993). The enlargement of B. napus diversity by exploiting related species with desired characters has attracted considerable interest.

Orychophragmus violaceus and Xinjiang wild rape are two of the promising species for this purpose. O. violaceus, a member of the Cruciferae family, has superior oil quality with low content of erucic acid (0.94%) (Luo, et al. 1994). Xinjiang wild rape, which was found in northwestern China (Wang, et al 1989) and has a similar morphology of Sinapis arvensis L., possesses lower content of erucic (15.81%) acid (average of 203 accessions) than other Chinese Brassiceae landraces and wild relatives (unpublished date). By self-pollination and single seed analysis, Chen et al.(1995) reported the occurrence of zero-erucic seeds (less than 1%) in the progeny of a plant containing 11% erucic acid.

In order to transfer the useful genes in these species for oilseed rape breeding, sexual hybridization has been performed between them and Brassica species with the aid of in vitro techniques. Hybrids between Xinjiang wild rape and Brassica species were either sterile or yielded only shrunken seeds with low germination capacity. Partially fertile hybrids between Brassica spp. and O. violaceus have been produced. However, genome separation has resulted in instability of subsequent progenies and prevented exchange of genetic material of the two genomes (Li, et al., 1995).

Somatic hybridization provides an alternative way for transfer of traits between distantly related species. Interspecific or intergeneric even intertribal somatic hybrids with targeted traits within Brassicaceae have recently been produced. In many cases, introgression of nuclear located genes from the donor parent to somatic hybrids was accomplished (Sjödin et al., 1989; Gerdemann-Knörck et al., 1995; Hansen and Earle 1994b, 1995, 1997; Sigareva and Earle, 1999; Heath and Earle, 1997; Fahleson et al., 1994; Kirti et al., 1997; Sakai, et al., 1996).

The concept of transfer of the valuable traits from these two species, especially their good oil quality to Brassica napus by somatic hybridization was applied in our work. The production and characterization of the somatic hybrids are reported in this paper.

MATERIALS AND METHODS

Plant material, protoplast fusion and culture, analysis of hybrids

Plant materials include three B. napus varieties (Zhong S, Zhong 4, Zhong 821), two accessions of Xingjiang wild rape (W4265, W4296), one of O. violaceus (O4834) provided by Prof. Qian, X.Z. from the mid-term oilseed germplasm gene bank in Institute of Oil Crops, CAAS in Wuhan, China, and one synthesized rapid cycling B. napus (designated as RCBN).

Protoplasts were isolated enzymatically from newly expanded leaves of three weeks old in vitro plants (Hansen & Earle, 1994a). In symmetric fusions, protoplasts of the regenerable parent of each fusion combination were treated with iodoacetate prior to fusion to prevent division and regeneration of unfused protoplasts. In asymmetric fusions, protoplasts of the donor parent was irradiated by X-ray for eliminating part of its genome while the recipient was treated with iodoacetate. Polyethylene glycol (PEG) was used for inducing protoplast fusion as described by Hansen and Earle (1994b). The protoplasts were cultured on a series of media (B, C, E, F) (Pelletier et al., 1983) using a feeder cell system according to Walters and Earle (1994) with modifications as described in Hansen (1998). .

Morphology of the regenerants were observed in the glasshouse. RAPD analysis using ten base oligonucleotide primers and nuclear DNA content determination by flow cytometry were according to Hu and Quiros (1991) and Hansen (1995), respectively. Flower buds were used for chromosome observation in somatic cells of style (Li, et al., 1995). Fatty acid composition was analysed after half seed extraction with gas chromatography modified from a method described by Thies (1971).

RESULTS AND DISCUSSION

Hybridization between B. napus and Xinjiang wild rape

Seven fusions of five parental combinations were made between B. napus and Xinjiang wild rape with three and two varieties in each species, respectively. The shoot regeneration efficiency ranged from 0.5-3.5%. After root induction and acclimatization, 60 regenerants were established in the glasshouse.

Except one plant which was identified as an escape regenerated from unfused protoplast of B. napus var. Zhong S, all the other plants were morphologically intermediate to their parents. Their leaves were divided with two pairs of small side leaflets and a big head-shaped top leaflet, which is characteristic to the B. napus parent. But the trichomes and waxlessness on the surface of leaf resembled their Xinjiang wild rape parent. The flower, pod and adult plant habit were close to B. napus.

Nuclear DNA content was determined in 57 of the plants. The majority had the same nuclear DNA content as the sum of their parents, accounting for 87.7%. Few had more or less than the sum of parental DNA. All plants had nuclear DNA content higher than either of the parents, which infers the integration of genetic material from parents to hybrids.

Hybrid identity of the plants obtained from fusions between Zhong S and W4296 was also confirmed by RAPD analysis. All banding patterns of 22 hybrids amplified with primer GEN-37 comprised the three bands from Zhong S and 2-4 bands from W4296.

Fertility of the plants varied among parental combinations and also among plants from the same fusions estimated by pollen release and seed set. The B. napus genotype had significant impact on the fertility of hybrids. The self-compatibility indices (the percentage of number of seeds in relation to number of flowers pollinated) of the hybrids derived from fusion between Zhong 821 and W4296 was much higher than those from fusion between Zhong S and W4296. Seven hundred thirty and 749 seeds were obtained from hybrids after selfing (S1) and backcross to B. napus parent (BS1), respectively. Many of the seeds were abnormal being either shrunken or very small or they germinated in the pod before maturity.

Fatty acid composition of seeds harvested from somatic hybrids of two fusion combinations, one with low erucic acid content B. napus parent (Zhong S), and the other with high erucic acid content B. napus parent (Zhong 821) were compared (Table 1). The result showed biased distribution of the main fatty acids towards the high erucic acid content parent in S1 seeds. This is consistent with the result obtained from fusions between B. oleracea and B. rapa in which both of the parents had high level of erucic acid (Heath and Earle, 1995). Seeds with very low erucic acid content were found in backcross progeny of the hybrids between Zhong S and W4296. This suggests that low erucic acid B. napus varieties should be preferred for initial introduction of new genes for low erucic acid content into B. napus.

Table 1 Fatty acid composition of seeds harvested from somatic hybrids after self (S1) or cross (BS1) pollination and their parents

Sample ID	#Sample	Palmitic16:0	Stearic18.0	Oleic 18.1	Linoleic 18:2	Linolenic 18:3	Eicosenoic 20:1	Erucic22:1
W4296	4	4.4	1.6	24.7	25.8	14.4	10.6	11.1
Zhong S	7	5.1	1.7	55.2	25.1	11.2	0.6	0.3
S1	38	4.0	1.0	29.2	20.9	14.6	16.1	10.3
BS1	50	3.6	0.8	36.6	18.4	16.4	15.8	5.3
W4296	4	4.4	1.6	24.7	25.8	14.4	10.6	11.1
Zhong 821	3	2.0	1.0	16.8	10.9	9.6	10.9	46.7
S1	35	2.3	1.0	13.3	13.0	10.9	9.6	43.7
BS1	26	2.1	0.9	11.8	11.0	12.3	9.6	46.3

Hybridization between B. napus and O. violaceus

Three B. napus varieties (Zhong 8, Zhong 4 and RCBN) were used in symmetric fusions with O. violaceus (O4834). Shoot regeneration efficiency of three fusions varied from 3.3-35.8%. One hundred and eleven plants regenerated from 92 calluses were established in the glasshouse. Only one of these plants was identified as a regenerant from an unfused protoplast of B. napus. All others had a morphology intermediate to their parents. Morphological variation could be found both among and within fusion combination. While O4834 has purple flower petals and B. napus has yellow petals, their somatic hybrids had different combination of these colours. The colour varied according to the B. napus parent used. Nearly all hybrids from fusions with Zhong 821 as B. napus parent had yellow flowers with purple colour on the edge of petals. The flowers of hybrids from RCBN were light yellow without any trace of purple colour while flowers of hybrids from Zhong 4 were yellow-white with light purple petal edges. All these symmetric hybrids were sterile. Their flowers had unelongated stamens and wedge-shaped yellow-brown anthers. Pollen release was not observed from their flowers. Stainable microspores were observed in flower buds prior to flowering. Backcross to B. napus parent did not yield seeds.

Nuclear DNA content determination, chromosome counting and RAPD analysis confirmed their hybrid nature. A majority of the hybrids had the same level of nuclear DNA content as the sum of the parents. Mixaploid and plants having nuclear DNA content higher or less than the sum of parental DNA were also identified. Hybrids from fusion with RCBN had wider variation on ploidy level probably due to the interaction among the three newly brought-together genomes involved. RAPD analysis with primer OPR-11 showed that all but one hybrid between Zhong 821 and O4834 tested generated bands from both fusion parents, providing evidence of introgression of DNA fragments from parents to hybrids. About 60 chromosomes were observed in somatic cells of styles from one hybrid. As B. napus has 38 chromosomes and O. violaceus has 24, this hybrid possesses chromosomes from both of the parents.

Two asymmetric fusions between Zhong 821 and O4834, one applied 10 Krad and the other, 20 Krad X-ray irradiation to protoplasts of O4834, were made and fertile hybrids were recovered from both fusions. Morphologically, the asymmetric hybrids were closer to B. napus than symmetric ones. Their growth habit was similar to B. napus but with more secondary branches. Among nearly 200 regenerants, which were characterized as somatic hybrids by morphology and ploidy level, two plants from callus #13, one from callus #35, 18 out of 22 from callus #31 of the first fusion and 3 from callus #8 of the second fusion were identified with stainable pollen. Selfing and backcross seeds were obtained from some of the plants and analysis of these seeds is underway.

CONCLUSION

Intergeneric hybridization between B. napus and Xinjiang wild rape, and between B. napus and O. violaceus can be achieved by protoplast fusion and fertile hybrids of both parental combinations can be recovered. The parental genotype has significant effect on the efficiency of somatic hybrid production as well as the fertility of the hybrids. Asymmetric fusion has proved to be efficient for increasing fertility of hybrids compared to symmetric fusions. Introgression of the genes for low erucic acid by somatic hybridization has great potential for the enrichment of B. napus diversity. Identification of the desired modification of fatty acid composition presently awaits analysis of further progenies from the hybrids.

Acknowledgements

We are grateful to Dr. A.Gertz for his advise and help on fatty acid composition analysis. We also thank B.K. Hansen for taking care of plants. This study was supported by the Chinese National Fund for Natural Sciences (39570442), Danish Government Scholarship, Daloon Foundation and KVL-stipend.

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