| Anther culture is a technique that anthers in which microspore cells have developed to a certain stage were collected and cultured in artificial medium,so that it can change the pollen development path of gametophytes to the path that microspore cells can divide and develop into plant like the totipotent cell.Anther culture is an important means of artificial haploid breeding,and is widely used in plant breeding research because of its preferably operation and high induction rate.The main means of apple haploid breeding also is anther culture.First,the embryoid were obtained by anther culture,and then induced embryoid grow into seedlings,seedlings grow into haploid plants.And the acquisition of embryoid as a complete step in the technical chain is also the most critical step.The main purpose of this study is to study the cytological development of embryoid induced by apple anther culture from the cellular level.The anthers of Danxia and Gala were cultured and these anthers of different culture days were collected.Making the anthers paraffin sections for observation.The number of cells in different developmental stages was collected and analyzed.Finally,the induction rate of embryoid body and callus was calculated statistically.The results are as follows:1.Apple anther optimization of the production technique: The dehydration,time and dyeing time were improved on the basis of the traditional production of paraffin sections,making it suitable for cytological observation of apple anthers.Through the experiment,a complete process of paraffin section preparation for apple anthers was obtained,that is,the dehydration of paraffin sections was changed to 50%,60%,70%,85%,95%,100% gradient of ethanol to ensure dehydration of the anthers will not shrink,and maintain a full state.And dyeing with Ehrlich's hematoxylin dye 15 min to ensure good staining of the slice.2.Apple microspore cell development process observation:Understand the characteristics of apple anther culture embryoid development,that is,there is no sequence between the basal development and apical cell develop in mentpollen embryogenic cell development by through the cytology observation of paraffin sections,and the comparison with the zygotic embryo development process.3.Through the microscopic examination,found the apple pollen cells from a single cell,to the multi-cell group,and then to the spherical embryo,heart-shaped embryonic cell development process.4.By comparing the status of cells in different embryos,and observing the pollen cell development status of Gala and Danxia,we found that the proportion of dividing cells in the process of Gala anther culture was less,and the callus cells were many,and the percentage of dividing cells in the process of Danxia anther culture was more,and the callus cells were less.We also found that the distribution of callus cells in gala was similar to that of Danxia's dividing cells,all of which were active between 15 and 17 days in the 5-60 days process of anther culture.5.By comparing the cell states of the different culture days,all cell states in the process of embryoid formation,ie,normal cells,dividing cells,callus cells,and dead cells,were counted and analyzed for data.Normal cells in the whole embryogenesis in the proportion of the average,no significant changes.The 5-17 days period was the main period of cell division,and 15-17 days was the most vigorous period of cell division.The major developmental stages of the callus cells of the Danxia were after cultured for 30 days,but the major developmental stages of the callus cells of the Gala were cultivated 5 to 10 days.Dead cells account for the smallest percentage among 15 and 17 days,this time is just the strong period of cell division.6.The results of the induction rate were obtained by counting the number of embryos and calli from different cultivars.The induction rate of Danxia embryoid body was 5.2%,and the callus induction rate was3.69%.The induction rate of Gala embryoid body was 1.37%,and thecallus induction rate was 7.94%. |
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