Type Identification And Cytological Study Of Sterile Mechanism In A Male Sterile Line-TaiYu D2 On Maize

The male sterile line TaiYu-D2(D2s),maintain lines of the same nuclear genetic background(D2m),the corresponding restorer lines(D2r), D2r×D2s generation of hybrid and 478-C was experimental materials. Using paraffin sections, from the plant, spikelet, anther morphology area we studied the development of D2s sterility in the form of structural changes in the external in the development of microspores, tapetum and other areas. Using PCR technology, using C, S, T, three pairs of specific primers, we studied the types of infertility and sterile mechanism of D2s through the DNA of D2s for PCR amplification and electrophoretic analysis. Major findings are as follows:1 External morphological changes of D2s'Plant, spikelet and anther There is no difference between plant morphology of D2s and D2m basically, tassel of D2s does not exposed anther and do not loose powder at the period of loosing powder. Tassel branch was evergreen, dry until the last. The spikelet of D2s was thin, shriveled, white wing retaining transparency. Internal anther was clear compared with D2m's through observed contrast to plant material, spikelet and anther Of D2s, D2m, D2r and the restorer line D2r to restore three of the F1. We found D2s was controlled by a pair of gene from statistics of F1 and F2 generation of the separation.2 The pollen changes of D2s, D2m and hybrid We observed only some debris under the microscope, observing the pollen grains of D2s stained through I2-IK, Microscope field of vision is purple. Pollen grains of D2m was observed circular under the microscope, there were both purple and yellow pollen grains after I2-IK staining, purple pollen grains accounting for 86.06%. Pollen grains of D2r was observed circular , pollen grains of D2r was all purple after I2-IK staining;pollen of F1 are all circle, and found 99.93 percent rate of purple pollen grains after staining using I2-IK. We concluded that the pollen of D2s are all sterile, D2r can complete restoring the fertility of D2s.3 Microspore development process of D2s and D2m anther Sampling from the beginning of differentiation of the pistil and stamen primordia to tassel completely out of, through the method of paraffin section, microspore mother cells of the D2s stained less during the dyad stage, microspore was smaller at the stage of initial microspore developmental than D2m's, shows irregular polygon and growth was retarded, nutrient supply on spore was further restricted during this period. Microspore showed some shrinkage of the state at the mononuclear stage and some fragments into irregular, there are many large and small peripheral vacuole, cytoplasm decomposition, nuclear deformation, complete disintegration of the late stage of monocyte. Degradation of pollen microspore mother cell has done at the Phase II of nuclear pollen, only some debris, the space of anther become small as extrusion of tapetal layer. Microspore mother cell completely deteriorated at the stage of mature pollen.4 Development process of D2 s and D2m'tapetal layerThrough observation paraffin sections, tapetal layer begins longitudinally elongating, radial swelling at the stage of dyad, tapetal layer gradually become vacuole, stains become shallow. Cells of tapetal layer become irregular and short and stout at the stage of mononuclear. Pollen cell of tapetal layer has been partly vacuolization during Phase II nuclear, tapetal layer was significantly thickened and radial expansion, accounted for most of the space of anther as radial elongation, the flat surface of anther was circular. Cells of tapetal layer has been thoroughly vacuole at the stage of mature pollen, a loop cells of tapetal layer stick to the center of homopolymerization, occupy all the space of anther as the radial elongation. The four-chamber of anther became into two-chamber of D2s, but the original two-chamber layers of tapetal layer and the anther wall stick together into a large chamber, there appeared as if a layer down the internal tapetal layer. At the same time vascular of anther connecting the two major anther room disappeared, two large rooms linking together like two dumbbells back-to-back .5 PCR identification.We done polymerase chain reaction amplification of the total DNA and mitochondrial DNA of D2s using three sets of specific primer sequences of cytoplasmic male sterility C,T, S. We found a special band in CMS-C specific primers using electrophoresis in accordance with the amplified fragment size of 398bp, while the T and S primers did not amplify any bands.