Studies On Microspore Culture Technology Of Brassica Napus And Its Comparison Of Physiological Characterization With Allohexaploid Brassica

Brassica napus is an important oil crop in China, with the improvement of people’s living level, there is an urgent need for high-yield and high-quality varieties to meet the demand for healthy rapeseed oil. Two conventional tetraploid varieties of B. napus and one synthetic allohexaploid Brassica were used as materials in this research. We analyzed the factors affecting microspore embryogenesis and plant regeneration of B. napus, observed the process of microspore embryogenesis, doubled the ploidy level of microspore-derived plants and tested their ploidy and compared the physiological parameters in different periods between tetraploid plants and allohexaploid plants. Based on the above research, we hope to explore new germplasm resources for rapeseed breeding. The main results are as follows:1. We examined the effects of different factors on microspore embryogenesis and embryo regeneration of two B. napus varieties (Zheshuang758and Zheshuang72). The results indicated that when the donor bud was between3.5mm and4.5mm, the isolated microspores were cultured for2days at32℃(heat shock) in dark, and then transferred to25℃, it would have a higher embryo production. The low-temperature (4℃) pretreatment inhibited microspore embryogenesis. Adding1mg/L6-BA and100mg/L PEG to NLN medium could promote microspore embryogenesis, while the effect of0.1mg/L2,4-D was not so obvious. In addition, embryo age played an important role in embryogenesis. The results showed that the embryos in cotyledon stage (4weeks) were the best for embryogenesis, the plant regeneration rate reached84.2%and81.5%in Zheshuang758and Zheshuang72respectively. When the concentration of agar in solid differentiation medium was15g/L, two B. napus cultivars had highest plant germination rate,81.0%and79.2%, respectively.2. In the process of microspore embryogenesis of B. napus, we observed that the survival rate of isolated microspores decreased rapidly with prolonged incubation time in1-5days. Part of microspores began to expand in the medium. Most of the inflated microspores could not be divided or stop growing after been isolated, or the growth status was abnormal. A very small part of inflated microspores were divided into2-cells,4-cells, multicellular proembryo, globular embryo, heart-shaped embryo, then developed into torpedo-shaped embryo and eventually formed a visible cotyledonous embryo.3. Flow-cytometric (FCM) was used to analyze the ploidy level of two B. napus varieties, allohexaploid species, and micro spore-derived plants. Ploidy could be well determined from fresh, newly born leaf blades at the seedling stage. An unequivocal identification of haploids, doubled haploids, tetraploids, hexaploids as well as some chimeras, was possible to be detected from their discrete peaks of histograms. The results showed that most of the regenerated plants were haploids and spontaneous diploids.4. By comparing the physiological parameters of different periods (Seedling, bolting and budding stages) between tetraploid plants and allohexaploid plants, the results showed that in different periods, the content of chlorophyll, the net photosynthetic rate, the content of soluble sugar and soluble protein in the leaves of allohexaploid plants were higher than those conventional tetraploid plants. Thus we can take advantage of these good characteristics of allohexaploid plants to improve the relevant traits, increase production and improve quality of Brassica species.