【作者】 张国庆；

【导师】 周伟军；

【作者基本信息】 浙江大学 ， 作物学， 2005， 博士

【摘要】 1.利用41个微卫星引物对11个中国甘蓝型油菜和12个欧洲甘蓝型油菜进行PCR分析,共产生了50个多态性位点。对50个位点分析,等位基因在1到14个,平均为2.7个。SSR标记分析表明,一个标记可以区分14个不同的DNA片断。对图谱的聚类分析清楚表明,可将所有油菜基因型分为3组,其中一组全为瑞典油菜,另外两组是中国油菜。且中国油菜较瑞典油菜具有广泛的遗传多样性。瑞典油菜品种间的遗传相似性在69.5-95.6%之间,且遗传距离较小;而中国油菜的相似性则在57.1-81.6%,且遗传距离较大。试验结果表明,可以通过选择合适的微卫星引物对甘蓝型油菜杂交亲本进行选择,并对杂交优势进行预测。 2.将甘蓝型油菜与芥菜型油菜进行正反交,通过对杂交子房培养获得其再生植株。试验结果表明,芥菜型油菜与甘蓝型油菜(Ames 24521×沪油15,Vittasso×浙双72)杂交子房培养效果好于其反交。取授粉后15d的杂交子房进行培养,其植株再生率高于授粉10d后的杂交子房培养,而产生的愈伤组织则少于授粉10d的杂交子房培养。同时,对培养基筛选结果表明,以MS为基本培养基,附加0.3mg/L NAA和1.5 mg/L BAP,与0.3 mg/L NAA和2.0 mg/L BAP,子房培养效果较好,其愈伤形成率和植株再生率分别达到55.43%和26.65%与66.98%和24.61%。 3.以白菜型油菜Hauarad(708)、茅山-3(714)及油白(715)和甘蓝京丰一号(6012)为供体材料,对白菜型油菜与甘蓝杂交种进行胚胎挽救研究。结果表明,取授粉后不同天数的子房进行培养,获得的种子数不同。不同杂交组合所获得的种子数存在明显差异。结果还表明,结籽率与培养基类型和组合方式有关。对所获得的种子在分化(萌发)培养基上培养表明,来自于杂交715×6012的种子萌发效果最好,种子萌发率达66.7%;并在生根培养基上得到了再生植株,对获得的再生植株进行初步细胞学鉴定,结果表明其单倍体的染色体数均为19,为真杂种。 4.以白菜型油菜和甘蓝杂交子房培养获得的再生苗为供体材料,研究影响再生苗生根和成活率的因素。再生根数和长度与培养基类型和培养时间有关,且当培养时间超过14 d,根数与根长则不再发生明显变化。试验还表明,不同基因型和培养时间的再生苗的叶片数和最大叶宽差异不显著。将再生苗转移到炼苗室进行锻炼,炼苗8d,再生苗的成活率可以达到80%以上,好于炼苗4d的再生苗的成活率。试验利用秋水仙碱直接对再生植株浸根进行加倍处理,结果表明,随着秋水仙碱浓度提高和处理时间的延长,其加倍率也随之提高。 5.试验以42份不同油菜材料为研究对象,进行小孢子培养,结果表明,不同材料其小孢子培养反应能力存在差异,胚产量不同。直接从大田取材研究了温度对小孢子胚产量的影响,试验结果表明,材料ZU5138、ZU5218、ZU5313和ZU5325在8-10℃取其花蕾进行小孢子培养,效果最好,而材料ZU5207则在11-15℃取材进行培养效果最好,其胚产量达到了219.3胚/花蕾。试验还表明,花蕾大小也是影响小孢子培养效果的一个重要原因,对于材料ZU5207和ZU5325,取处于花瓣与雄蕊比例为1/2的花蕾进行小孢子培养效果最好,其胚产量分别达到了314.0和48.6胚/花蕾。另外,热击组合试验表明,材料ZU5138,ZU5218和ZU5313的小孢子分离后,在32℃下热击培养3 d的效果好于30℃下热击培养7 d。更多还原

【Abstract】 1. Eleven Chinese and twelve European/Swedish rapeseed (B. napus) genotypes were analysed by PCR with 41 microsatellite primers, generating a total of 50 polymorphic loci. For the 50 loci, the number of alleles ranged from 1 to 14, and the average number of alleles per loci was 2.7. As an example of SSR scoring in Metaphor agarose gel, a single marker could distinguish 14 different DNA profiles. The dendrogram clearly distinguished 3 clusters, a cluster with exclusively Swedish genotypes, and two clusters with Chinese genotypes. The genetic diversity within the Chinese genotypes was broad compared to the genetic diversity within the Swedish material. The genetic similarity within the Swedish breeding lines ranged from 69.5-95.6 %, while that of Chinese genotypes ranged from 57.1-81.6 %. In a combined Swedish/Chinese breeding programme the similarity range was 51.5-81 % and 15 % of the genotypes would have less than 60 % DNA in common. The results in this report will permit to establish a set of microsatellite primers that can be used for selecting appropriate parents for Brassica napus hybrids and for monitoring hybridity level.2. Crosses were made to produce interspecific hybrids between Brassica napus × B. juncea and their reciprocal with the aid of embryo culture techniques. A better response of. hybrid embryo culture was obtained from two cross combinations between B. juncea × B. napus (Ames 24521 × Huyou 15, and Vittasso × Zheshuang 72) than from their reciprocals. Embryo culture was more effective in terms of plant regeneration when embryos were cultured in vitro at 15 days after pollination (DAP), while more calli were initiated when embryos were excised and cultured at 10 DAP. A better response was observed on the MS medium with 0.3 mg/L NAA + 1.5 mg/L BAP, and with 0.3 mg/L NAA + 2.0 mg/L BAP. Callus formation and plant regeneration on these two media reached 55.43 % and 26.65 %, and 66.98 % and 24.61 %, respectively.3. Using three varieties of B. campestris, Hauarad (708), Maoshan-3 (714) and Youbai (715), as the maternal plants and one variety of B, oleracea Jingfeng-1 (6012) as paternal plants, crosses were made to produce interspecific hybrids through ovary culture techniques. The ovaries from the cross between B. campestris × B. oleracea (708x6012 and 714x6012) were cultured and ovary culture was more effective in terms of seeds obtained when ovaries were in vitro culture at 9 days after pollination (DAP). While for the cross of 715x6012 it was better when ovaries in vitro culture at 12 DAP. Among three cross combinations, the cross of 714x6012 showed best response and 43 seeds per ovary were obtained. Of the media studied, the ovaries from the cross of 708 × 6012 were cultured on MS media supplemented with 3.0 mg/L BA+0.1 mg/L NAA showed better response, and its rate of seeds per ovary reached 44.0%. While the ovaries from the other two crosses (714x6012 and 715x6012) showed best response with cultured on B5 media supplemented with 3.0 mg/L BA+0.2 mg/L NAA, and the rates of seeds per ovary reached 72.0% and 60.0%, respectively. All seeds obtained from the three cross combinations were cultured on the MS media supplemented with 1.0 mg/L BA+0.05 mg/L NAA, and the seeds from the cross of 715x6012 showed best germination response and the percentage ofgerminations reached 66.7 %. The regenerated plantlets were obtained from these seedlings after cultured on the MS media supplemented with 0.05 mg/L NAA. Cytological study showed that these regenerated plants were all true hybrids of B. campestris × B. oleracea.4. Using the regenerated plants obtained from interspecific crosses between B. campestris and B. oleracea through ovary culture techniques, the experiment was conducted to examine the factors affecting root initiation and survival rate of these regenerated plants. Of the media studied, the best response was observed from the MS media supplemented with 1.0 mg/L BA + 0.1 mg/L NAA. Among three cross combinations, the regenerated plants from the cross between B. campestris x B. oleracea(715x6Q12) showed the best result, with the number of roots and length of roots reaching 9.51 and 4.7 cm, respectively. Of the culture durations studied, better response was obtained from the regenerated plants cultured for 14 days than for 7 days. However, there was no obvious difference of the number of roots and length of roots, when the regenerated plants cultured for over 14 days. The result also showed no significant difference of the leaf number and the width of maximum leaf between various genotypes and culture durations. The regenerated plants from three cross combinations were hardened for 8 days and their survival rate reached over 80 %, which was obviously better than those hardened for 4 days. In addition, the roots of regenerated plants were treated with colchine directly, within a colchicine concentration, the doubling efficiency increased with increasing treatment time.5. Conditions for reliable induction of embryogenesis from isolated microspores were studied in 42 genotypes of oilseed rape grown in field conditions. The results showed that there was a significant variation in the response of various genotypes to microspore culture. The genotypes ZU815 and ZU816 were the highest responsive accession studied and their normal embryo yield reached 264.50 and 320.04 embryos/bud, respectively. Four genotypes (ZU5138, ZU5218, ZU5313 and ZU5325) showed better responses in terms of embryo production when the day temperature was in the range of 8 - 10 °C, and their embryo yields reached 147.3, 90.8, 209.0 and 42.8 embryos/bud respectively. The genotype ZU5207 showed better response at 11 - 15 °C with the embryo yield of 219.3 embryos/bud. No embryo was induced in all the genotypes when the day temperature was over 20 °C. In genotypes ZU5207 and ZU5325, the flower buds with a petal/anther length ratio of 1/2 were the most effective for the embryogenesis and their embryo yields reached 314.0 and 48.6 embryos/bud respectively. In genotype ZU5313 high embryo yields (208.2 - 212.0 embryos/bud) were observed from the flower buds with the petal/anther ratio of both 1/2 and 3/4. The genotypes ZU5138, ZU5218 and ZU5313 produced more embryos when incubated at 32 °C for 3 d than that when incubated at 30 °C for 7 d.6. The present study evaluated the effects of chilling, partial desiccation, cotyledon excision and successive subculture of microspore-derived embryos on plant development in oilseed rape. The results showed that out of the five media, all the genotypes showed the best response when the embryos were cultured on the half-strength Murashige and Skoog medium with 2.0 mg/L benzyiaminopurine. A cold treatment for 3 or 5 days further increased frequencies of embryo germination (90.0 %) and plantlet development (58.46 %). Desiccation for one day also increased the embryo germination and plantletdevelopment in all genotypes tested. Cutting the cotyledons of the embryos at late cotyledonary stage significantly increased the frequency of plantlet development. The highest rate of plantlet development was obtained from cultures of embryos sampled with size of less than 4.0 mm. The microspores from the rapeseed genotypes ZU805, ZU816 and ZU817 were treated directly with colchicine for doubling chromosomes. It was showed that embryo yields were different with the various colchicine concentrations and there was the best results when the microspores were trerated directly with 0.8 mg/L colchicine. The doubling efficiencies of all the genotypes were increased with the treatment of colchicines compared to the control. In addition, the roots of regenerated plants derived from isolated microspores of genotypes ZU805, ZU816 and ZU817 were treated with colchine directly. The results showed that within the suitable concentrations of colchicines, the doubling efficiencies were increased with the increase of treatment durations and concentrations.7. Using 4 oilseed rape Fi hybrids (7039, 7040, 282 and 5102) as donor plants for microspore culture, the experiment were conducted to select glyphosate and haloxyfop resistant embryos through glyphosate- and haloxyfop-contained cultural media with microspore-derived embryos in vitro respectively. The genotypes 7039 and 7040 were used to select regenerated plants which showed glyphosate resistance and the other two genotypes were used -for haloxyfop resistance selection. The embryos at cotyledonary stage were taken to grow on glyphosate- and haloxyfop-contained MS/2 medium for two weeks. The embryo which was not resistant to glyphosate and haloxyfop collapsed in short time, while those which was resistant to glyphosate and haloxyfop turned green and survived within two weeks. The green embryos were then transferred into the normal MS-2 medium for further plant regeneration. All regenerated plants showed tolerance to glyphosate after tested by spraying 0.25% glyphosate herbicide, indicating that the present in vitro selection method of glyphosate resistance was effective and reliable. However, when the regenerated plants selected from 0.02 % haloxyfop were sprayed with 0.05 % haloxyfop, most of these regenerated plants grew well, while the survival rate of the regenerated plants from 0.01 % haloxyfop contained medium were lower. The present experiment indicated that the method of using 0.02 % haloxyfop to select was better. The chromosome doubling efficiency of regenerated plants reached 34 % and 52 % after directly treated with 170 mg/L colchicine for 20 and 30 h, respectively.8. Characters from the Fl plants of doubled haploid (DH) populations in B. napus were analyzed and it was showed that the means of each agronomic trait were between their parents, but they were nearer to the paternal in 6 agronomic traits (plant height, branch position, number of pods in the main raceme, length of pod, number of pods/plant and number of seeds/pod). The number of genes controlling each agronomic trait was analyzed based on the DH populations. The results showed that the number of genes controlling number of pods in the main raceme was the most (15.6), and the least number of genes was involved for the stem width (only 7.8). According to estimated coefficients of skewness and kurtosis of the traits tested, gene interaction was found to be absent for stem width, plant height, length of main raceme, number of primary and secondary branches, pod density in the main更多还原