CREATION OF NEW GERMPLASM IN B.NAPUS USING LANDRACES OF B.CHINENSIS AND B.OLERACEA VAR. ALBOGLABRA FROM SW CHINA

Niu Yingze  Wang Liangzhong  Liu Yuzhen

Sichuan Agricultural University, Yaan, Sichuan, P.R. China  625014

ABSTRACT

Brassica napus L. was originated in Europe and first introduced to China in 1930's. Since 1950's it has been rapidly popularized and developed into a major oil crop in China. However, almost all cultivars and resources of B.napus in China are exotic. Creation of locally originated B.napus, which is more adaptable to the various native natural and cultivation conditions, is of great importance.

In the present study a group of landraces (including wild or semi-wild races) of B.chinensis in SW China were used to hybridize with a local vegetable cabbage "Bai-Hua-Jie-Lan (B.oleracea var.alboglabra), followed by ovary and embryo culture and chromosome doubling. A big number of new B.napus lines were developed. These lines possess a unique SW China genetic background, and showed different valuable genetic characteristics, including various maturity types, plant types, long and big siliques, large seeds and high disease resistance (especially tolerance to Sclerotinia), or vigorous growth.  A new source of cms lines has also been obtained via crosses between the resynthesized B.napus lines and exotic varieties. The new germplasm is of great value for rapeseed breeding.

KEY WORDS: Rapeseed; B.napus L.; B.chinensis; B.oleracea var.alboglabra; resynthesized lines

    Brassica napus L. was originated in Europe and first introduced into China in 1930's(Liu Houli, 1985). It was first demonstrated in production in Sichuan, China, in 1953. Since then, this new oilseed crop has been quickly extended to large area cultivation and become a major oilseed crop along the Yangtze river valleys of China. This region has become one of the three largest B. napus growing regions in the world.

However, almost all cultivars and resources of B.napus are exotic in China. The climatic and soil conditions are widely various. Most foreign varieties and resources are late maturing in China. They do not adapt well to most of the multiple cropping system regions and the cooler and poorer regions. A big number of earlier varieties have been developed from the exotic resources since 1960's.,but they are still limited in part of the regions.

In 1980's, rapeseed researchers in China started resynthesis of B.napus to creat new breeding materials(Li Gen-yi,1988; Chen Bao-yuan,1989). This opened a new way for rapeseed breeding. There is an abundant resource of B. campestris L., B.chinesis (Syn. B.campestris ssp.chinensis (L.) Makino) and B.oleracea L. in China. Therefore, Creation of locally originated B.napus which is more adaptable to the various native natural and cultivation conditions, is of great significance.

Sichuan Agricultural University is located in the Southwest of China. The landorms and climate conditions in this region are specially complex and the transportation is very poor. There are still large number of landraces, wild or semi-wild Brassica forms naturally conserved there. The most important species is B.chinensis (Syn. B.campestris ssp.chinensis (L.) Makino). Since 1986, we have collected a number of landraces and wild or semi-wild forms of B.chinensis(including some B.campestris races from East Tibet ) for resynthesis of new B.napus types. Through the work of resent years, we have developed a group of resynthesized lines and many of them are of good values for breeding. The main results are reported in this paper .

    1. Materials and Methods

    1.1 Materials

A group of landraces of B.chinensis (including oil-seed type and vegetable type) and B. campestris have been collected in SW China since 1986. Seven of them were used to cross with a cabbage vegetable "Bai-Hua-Jie-Lan" (B.oleracea var.alboglabra Bailey). The parents used are listed in table 1

Tab. 1           Parent materials used in the resynthesis of Brassica napus

Material Name	Species	Chromosome Number	Location of collection	Remarks
Ya Huang	B. chinensis	10	Yaan,Sichuan	Cultivated, yellow seed, high oil content, big silique
Le Huang	B. chinensis	10	Leshan,Sichuan	Cultivated, yellow seed, high oil content
Jiu Ye	B. chinensis	10	Jiulong, Sichuan	Wild, hairy leaves and stem
Bao Ye	B. chinensis	10	Baoxing,Sichuan	Wild, small plant, hairy leaves
Xi Zang	B.campestris	10	Naidong,Tibet	Cultivated
Piao Bai	Not defined	10	Yaan,Sichuan	Vegetable, broad and thick leavese
San Bai	B. chinensis	10	Baoxing,Sichuan	Vegetable, without leafy head
Bai-Hua-Jie-Lan	B.oleracea var.alboglabra	9	Kunming, Yunnan	Vegetable, leaves similar to kale, without leafy head, white or yellow flower

   1.2 Hybridization

   The maternal parents were emasculated in general method. Before pollination, the stigmas were treated with 50-60 ppm gibberellin (GA3). Pollination was conducted 2-3 min later. In 1990, we treated some stigmas with distilled water as a control to see the effect of GA3. Reciprocal crosses were made for most of the combinations.

   1.3 In vitro culture of ovaries and young embryos and doubling of chromosomes

   The ovary culture was done 4-8 d after the pollination according to the air temperatures. They were first surface sterilized and then inoculated in the solid White medium containing 10% of sucrose added with 500mg/L hydrolyzed cacein(CH). The temperature of ovary culture was 25℃. After 20-25 d, some ovaries showed signs of seeds. They were opened and the young seeds and embryos were taken out and then cultured on a modified MS medium containing 3% of sucrose, added with 6-BA 0.2mg/L or NAA 0.5mg/L ,KT 2.5mg/L. The day light was used for 1 week and then transferred to the light of 40 W fluorescent lamp until plantlets were formed. Clonal multiplication of the plantlets was made during the off season to enlarge the hybrid population.

Doubling of chromosomes: Before transplanted into the field, the plantlets were transferred onto MS medium containing 3% sucrose, added with 6-BA 1mg/L, NAA0.1mg/L and 30 or 40 ppm colchicine for 7-10 days; then transferred to 1/2MS medium containing 2% sucrose with NAA 0.03-0.1mg/L. Later the plantlets were transferred for 1-2 times in every 7 days until roots were formed. When the young plants were 3-4 cm tall, they were hardened and then transplanted in the field.

    1.4  Selection

Single plant pedigrees were established in F1. Selection in F1 was done when doubled plants were obtained. There were undoubled plants in each cross. Auxiliary pollinations were made only for the normally doubled plants (or branches) to enhance seed setting. In each generation, the superior plants were selected and bagged for selfing and for auxiliary pollinations. The selection  was based on the important characteristics including plant height, plant type, shape of leaves, number of branches, Silique size, disease and stress resistance etc. At harvest the bagged plants with good seed set were collected and the final selection was made based on an indoor seed examination.

2. Results

2.1 Cross compatibility

From the pollinated ovaries left on plants in the field, It was observed that GA3 treatment stimulated the elongation and swelling of the ovaries and resulted in indication of seeds in the siliques. However, no mature and viable seeds were obtained at harvest. Evidently the development of  embryos was hampered and died before maturity. In contrast, most of the ovaries treated with distilled water became dead and dropped, or just remained unswollen on the plant. This indicated that GA3 treatment enhanced the cross compatibility for the interspecific hybridizations.

From the results of ovary culture ( table 2 ), large difference in rate of hybrid seedling formation was observed among the different crosses, especially between the reciprocal crosses. In  the present study, the rate of seedling formation was always higher when B. chinensis was used as the maternal parent. The cultivar “Ya Huang” (B.chinensis) showed the highest  compatibility to “Bai-Hua-Jie-Lan”

Tab 2                  Results of Ovary and Young Embryo Culture

Crosses	Ovaries	No. of seedlings from young embryo	No. of seedlings. from bare embryo	No. of  seedlings. from germ seed	Total no. of seedlings	Rate of green seedling (%)
JL×Ya huang	668	12	3	8	23	3.44
Ya huang × JL	140	0	42	5	47	33.57
JL × Le huang	836	19	3	0	22	2.63
Le Huang × JL	68	1	1	0	2	2.94
JL × Baoxing	156	5	7	0	12	7.69
Baoxing × JL	218	5	5	13	23	10.55
JL × Jiu Ye	156	1	0	1	2	1.28
Jiu Ye × JL	136	0	13	16	29	21.32
JL × Xizang	78	1	0	0	1	1.28
Xizang × JL	239	3	0	0	3	1.26
JL × PiaoBai	150	1	4	2	7	4.61
JL × San Bai	112	1	0	1	2

JL=Bai-Hua-Jie-Lan

   2.2  Performance of the offspring and their classification status

   In F1, the doubled plants showed a normal appearance, a complete floral structure and plump anthers. The undoubled plants showed a giant appearance, being lack of stamens or stamens less than 6 pieces in the flowers, with anthers shrunken, and free of pollens. The total doubled plant rate among the different combinations ranged from 8.5-32.6%, in which only 1.7-6.1% was wholly doubled.

   All interspecific combinations showed a strong heterosis in F1. The F₁ proginy of different combinations were conspicuously different in growth vigor and plant characters. In F₂, large segregation appeared and a diversity of genetic variation occurred in plant height, growth vigor, plant type, duration, leaf shape, leaf color, flower color, branch number, silique shape and size, seed size etc. New variants also occurred in F₃ and later generation. Some lines got basically stable and uniform at F4-F5.

   All of the resynthesized lines possess three kinds of leaves, i.e. long-stalk leaves, short-stalk leaves and non-stalk leaves. In 1991, the number of chromosomes of lines “88-3”(Ya Huang × JL), 89-1-2, 89-1-23 (Le Huang × JL)were microscopically investigated at miosis of the pollen mother cells. It was found that most of the miotic cells investigated showed n=19 at MI stage. Part of the cells showed n=10 and also other abnormal numbers. During the process of selection, some crosses were made between existing B. napus lines and the resynthesized  B. napus lines. No generative isolation was observed between the two types. These results suggested that the resynthesized B. napus and the existing B.napus belong to the some species.

2.3 Important materials with breeding values obtained

By continuous selfing and selection, a total of over 90 resynthesized lines have been obtained. Near half of them have been stable and apparently uniform. The lines are, respectively, derived from the crosses between “Bai-Hua-Jie-Lan” and Ya Huang, Le Huang, Jiu Ye, PiaoBai, Xizang. The following important types of lines deserve further selection and can be used in breeding and genetic research.

(1) Long silique, large seed and many seed lines(H225, H233, H042, H039, H1931, H311, etc.): These lines have siliques longer than 10 cm, the longest being 16.5 cm. The average number of seeds per silique was normally more than 20, with some lines being more 23. The 1000-seed weights were normally above 4.5g.

(2) Many-branch lines(H301, H313, etc): These lines normally possess more than 15 first-order branches, 40-50% more than the normal lines. The highest line H313 showed plants with 28 first-order branches.

(3) Specially early lines(H045,H201,H236,H251,etc.): These lines showed a maturity 7-10d earlier than the early varieties released in production. They are as early as, or even earlier than the local B. Chinensis cultivars and they possess a higher yield potential.

(4) Lines with high resistance (or tolerance) to sclerotinium(H042, H135, H210, H322,etc.): Sclerotinium is the No.1 diseasein rapeseed  in China, especially in the Yangze River Velleys. Highly resistant varieties have been rare so far. These resynthesized lines showed  very good resistance to sclerotinium during the years of selection and also in a resent leaf test experiment.

(5) White Flower Lines(H129, H151, H239, etc): These lines have pure white flowers. A few crosses have been made between the white flower lines and yellow flower lines. The F₁ offspring are wholly white flowered. In F2 the majority of plants are white flowered. This character can be used as a marker trait.

(6) Vegetable and fodder type lines(H129,H238,etc.): These lines grow very fast and produce a very high biomass. They have large leaves and thick stems, with very tender stem tops. The tender stem and branch tops can be used for vegetable and the remaining part can be used for fodder or manure.

(7) Other types: dwarf lines, dense silique lines, yellow-seed lines and lodging resistant lines, etc.

In addition, a new source of cms line has been obtained from a cross between a resynthesized line “He 3-17” and an F₃ progeny of cross “Brongor × Westar”. A group of new cms lines have been developed from the new source of sterile cytoplasm.

3.  Discusssion

Since the artificial B. napus was first synthesized in 1930's by U,Nagahara, many rapeseed reesearchers have made similar studies thereafter and have successfully resynthesized B.napus (Liu Hou-li,1985). These studies provided with very important experimental evidences for the understanding of the natural process of evolution of B napus L. However, few researchers exploited this technique for breeding purpose until 1970's. In 1980's, the techniques for ovary and embryo culture were established and the resynthesis of amphidiploid Brassica was made easier and more successful(Wang Liangzhong, 1991). Rapeseed researchers begin to exploit the technique to create new germplasm for rapeseed breeding. Chen Bao-yuan (1989) successfully resynthesized new B. napus and obtained yellow seed materials in the resynthesized rapeseed. He also obtained new lines of high linoleic acid content and new source of cms lines from the crosses between existing B.napus lines and resynthesized B.napus lines. Luhs W. and Friedt W. (1995) obtained high-erucic acid rapeseed by means of resynthesis. Becker W. and Engqvist G. M. discussed the potential of resynthesized rapeseed for hybrid breeding. These results, among others, showed a good perspective of resynthesized rapeseed in breeding.

In the present study, a big number of useful B. napus lines have been developed from the resynthesized rapeseed using the landraces and wild or semi-wild forms of Brassica resources from SW China. These lines possess a unique genetic background from the local Brassica resources and have showed a very good adaptation to the poorer productive conditions. These lines are of important values for rapeseed breeding.

Furthermore, the basic species and the compound species in Brassica are evolving in a parallel way. They both are distributed in different regions and eco-environments. A big diversity of eco-types with various favorable genes are available, and new genes will also occur in both of the species. Therefore, it is of great potential to continuously use the various basic species to resynthesize new types of compound species for either breeding or theoretical researches.

Reference:

Becker, H. C. and Engqvist G. M. (1995), Potential of resynthesized rapeseed for hybrid breeding, Proceedings of 9th International Rapeseed Congress, A40:113-115.

Chen Bao-yuan (1989), Resynthesized Brassica napus L.:A potential in breeding and research, Abstract (Chinese), translated by Liu Hou-li, Annual report of rapeseed, 1989, Huazhong Agricultural University, 54-55.

Li Gen-yi (1988), Studies on the production of artificial synthetic rape (Brassica napus L.) and its value in rape breeding (abstract of Ph.D. thesis, Chinese), Annual report of rapeseed, 1988, Huazhong Agricultural University, 28-30.

Liu Hou-li (1985), Rapeseed Genetics and Breeding (Chinese), Shanghai Science and Technology Publishing House, 9-43.

Luhs, W. and Friedt, W. (1995), Breeding high-erucic acid rapeseed by means of Brassica napus resynthesis, Proceedings of 9th International Rapeseed Congress, D18:449-451.

Wang Liangzhong and Liu Yuzhen (1992), Synthesis of Brassica napus L. (I), (Chinese, English abstract), Southwest China Journal of Agricultural Sciences, 5(3): 12-17.