Interspecific Hybridization, Plant Regeneration And Its Genetic Evaluation Within Some Brassica Species

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...