Study On Technical System Of Wheat Microspores Culture

Wheat microspores culture is a highly efficient regeneration system and can get a large number of haploids and doubled haploids (DH group). Wheat microspores culture playes an important part in the haploid breeding, and it provides rich and stable genetic materials for the basic research of genetics and cell biology. Many factors such as culture technology and environment were required in the wheat microspores culture.14wheat varieties at home and abroad such as Jimai20, Shannong664and pavon were adopted in this research. Meanwhile many factors such as the supply of materials, materials pretreatment, separation and purification of microspores, culture medium, density and variety were researched, in order to provide a theoretical basis for techniques of wheat microspores culture. The results were as follows:1. A material supply system for microspores culture was eatablished. The wheat could tassel when planted before February by sowing in different times. The artificial vernalization should be adopted when planted after March, and the vernalization days of weak winterness, winterness and strong winterness varieties should be20days,20-40days and more than40days. Under the condition of field, the supply date of wheat could be prolonged from April to July, so the research has solved the problems of concentrative supply and short time of materials during the microspore culture of wheat.2. The two methods of spikes pretreatment and microspores pretreatment were adopted. When using the microspores pretreatment, the number of active microspores and the embryoid size were significantly higher than the results of spikes pretreatment.3. The preparative process of microspores was optimized. The spikes of wheat should firstly be disinfected with sodium hypochlorite (2.0%) for20min, and then rinsed with water, finally treated with ethanol (75%) for1h. The pollution can be effectively controlled by this method. With the treatment of mashing tissue for30s in the speed of15OOOrpm, the percentage of integrated amount is higher, and the quantity of microspores separated is more. The centrifugal effect was better with the maltose concentration of30%.4. The number of active microspores of different varieties was little, and the diameter of embryoid of different varieties was small, when the culture density of microspores was5×103/ml. When the culture density of microspores was1×104/ml, the number of active microspores and the embryoid size of different varieties increased respectively, and there was significant difference compared with the density of5×103/ml. When the culture density of microspores was2×104/ml, there was no significant difference on the number of active microspores and the embryoid size of different varieties compared with that of the1×104/ml, but there was obvious difference compared with that of the5×103/ml. The results showed that it was more feasible for the microspore culture when the culture density of microspores was from1×104/ml to2×104/ml.5. There were significant differences in the number of active microspores and the embryoid size, when the different varieties were cultivated under the same conditions. The order was Yannong10<Shannong664<Jimai20<pavon, and there was significant difference among varieties. According to the evaluate of winterness, the varieties of Yannong19, Shannong664, Jimai20and pavon belonged to strong winterness, winterness, weak winterness and springness, respectively. It was obvious that the varieties of stronger springness would be in better form in the microspore culture.6. The method for isolating microspore is the key factor for the success of microspore culture. The method of triturating tissues could produce numerous microspores, but it could also produce tissue fragments and death microspores, so it is not propitious to microspore culture. Meanwhile, microspores separated by triturator will be directly exposed to the medium, so there are strict requirements for the concentration of the medium. These hold-up the advanced culture of microspore from abroad which is absorbed and applied.The results showed that the scattering method to obtain microspores was very simple, and which could avoid the negative factors which were produced in the separating of microspore. It could obtain a higher survival rate and the rate of forming plantlets.