Cytology And Preliminary Molecular Mapping For Cytoplasmic Male Sterility (CMS) In Radish (Raphanus Sativus)

The heterosis in radish is much significant, especially in increasing yield and fiber quality. Comparing with hand-emasculation and pollination, and genetic male sterile lines, utilization of CMS lines is much more effective and economical in producing commercially hybrid seeds. In this study, light-and electro-microscopy, DD-RT-PCR was performed to detect the mechanism controlling the male cytoplasmic sterility in Chinese radish.The cytology of the microsporogenesis and development of anther wall of cytoplasmic male-sterile (CMS) A2, A4 and its maintainer lines B2, B4 was studied. The results revealed that the tapetal cells of the male sterile A2 developed abnormally at tetrad stage, and they became vacuolated and numerous small vacuoles are present. Exine of microspore was light stained, and cell wall was destructed. Finallywith degeneration of the tapetal cells, the pollen aborted. The tapetal cells of CMS line A4 enlarged abnormally at meiotic phase, pressing microsporocyte seriously. Lately, their cell walls had not been observed, and the cell contents fused to form big lump invading the loculi, and finally leading to pollen grain abortion.The cytological development of male cells and the tapetum of male fertile and combined cytoplasmic-genetic male sterile line A)4 of radish was studied using light, and transmission electron microscopy. The cytoplasmic-genetic male sterile line began to abort when microspores had just released from tetrads by light microscopy investigation, while abnormal behavior of pollen mother cells were observed during meiotic stage on its ultra-structural study, including degeneration of organelles, irregularity of nuclear membrane etc. At the same time, development of tapetal cells was similar to that of the normal anther. With the development of the anther, the tapetal cells of CMS line showed an abnormal increase in size and other appearance, microspores were always distinguishable from the maintainer line. It is inferred that abortion of microspores contributed to mutation of genes controlling male sterility, which further leading to hypertrophy of tapetum and abnormal changes of ultra structure. The total DNA was isolated from young radish leaf. Two DNA fragments about 780bp and 280bp were obtained with PCR. After recovering the fragment about 780bp, the PCR product was sequenced and 705bp was obtained. Blast results shows that the full -length or f138 coding sequence is 417bp and the sequence showed 100% homology with the reported orf1!38 nucleotide sequence. Of the 96 arbitrary primers used in the experiment, the primers which can produce the polymorphism were obtained was used to test the fertility of genetic stocks successfully, which shows the RAPD technique determine the fertility of land races rapidly and precisely to back up seed production strongly.Using leaf and young anther as materials, a highly efficient method system for radish, including total RNA extraction and purification, Reverse Transcription Differential Display-PCR (DD-RT-PCR), PAGE and fast-sensitive silver staining was founded. In order to extract high quality RNA from radish, four protocols were applied. The results indicated that the modified SDS-acidic phenolic method was more suitable for total RNA extraction than CTAB-acidic phenolic method and SDS-alkaline phenolic method. High quality RNA was also obtained with the modified hot borate (HB) procedure that needs more time and higher cost. Total RNA with modified SDS-acidic phenolic method was subjected to purification with DNase 1 and the OD260/OD280 ranged from 2.0 to 2.2. The cDNA bands with higher discrimination were between 100 and 380 bp with DD-PCR.Transcription Differential Display-PCR (DD-RT-PCR) was conducted in fertile and male sterile radishes. The results indicated that cDNA bands of fertile and male sterile materials didn't differ with quantity. Special expressed patterns of flowers were detected in fertile materials, so did leaves. In addition, thereis some extents of dose effect, which are due to some of them, were expressed...