Preliminary Studies On The Mechanism Of Cytoplasmic Male-sterility Onion(Allum Cepa. L)

Allium cepa L., is a biennial cross-pollination plant in onion genera of lily family which heterosis is obvious. The selection and use of sterile lines to make hybrids have been valued by many researchers. Flower organ is too small, flowering is not concentrated and seed-getting rate of one flower is too low in crossbreed seeds production of onion. Using the male sterility line to produce the hybrid seed is an efficient pathway. In onion, cytoplasmic mechanism study for the male sterility production and application of F1 hybrids has an important role. Two sets of onion cytoplasmic male sterile line material were selected for the study. Pollen abortion procedure was observed and analyzed by paraffin section and electron microscopy. The changes of the activity of peroxidase(POD), catalase(CAT) and superoxide dismutase(SOD) were determined at different development stage. Primers were designed according to five mt DNA genes sequence and PCR products were sequenced. Studies on morphology, cytology, physiology and molecular biology were made of the mechanism of male male sterile line.(1)From seedling to flowering stages, there was no obvious difference in plant shape and growth potential between the male sterile plants and fertile plants of onion. But it was easy to distinguish from phenotype after flowering: the flowers of the male sterile DA compared to fertile DB were smaller with very short filaments; anthers were thin and shriveled which were pale yellow or dark green at early stage, but late converted into brown. Filaments of fertile plants were growing normally and anthers were plump and yellow. The inflorescence of DA was sterility smaller than other three materials, and the performance of DA sterile characters was more obvious than JA.(2)The microstructural observations of the anther development process of the male steriles JA、DA and fertiles JB、DB showed that:At the pollen-mother cell stage the pollen-mother cells could develop normally both in fertile anthers and sterile anthers. However in the sterile anther pollen sacs were formed irregularly,anther wall was uneven and pollen mother cells were surrounded tightly by the tapetum; At the tetrad stage, the cell of the middle layer arranged in order and the nuclei were observed clearly.The tapetum developed completely possessing dense cytoplasm in the fertile anther. But at the same time the tapetum was degenerated completely and tapetal cells become vacuolated followed by condensation of the tapetal cytoplasm in the sterile anthers. The tapetum of DA was degenerated faster than the tapetum of JA. At the microspore stage, fertile anthers microsporedevelopment was normal, the tapetum with darker muclei began to degrade in the fertile anthers.However, the microspore cytoplasm appeared concentration morphologically and the tapetum were degraded completely in the sterile anthers. At the pollen grain stage, the mature pollen grains with plump shape were stained deeply and adjacent pollen sac were joined with each other in the fertile anthers. However, the empty pollen grains shrinkaged to a thick line lead to endothecium was stuck together and the pollen sac cannot be joined in sterile anthers.(3)The ultrastructural observations of the anther development process of the male steriles JA、DA and fertiles JB、DB showed that:At the tetrad stage, vacuole like structure appeared with tapetum and mitochondria swelling.Meanwhile the microspore was uneven with concentrated cytoplasm and vacuolations in the sterile anthers. At the microspore stage, the tapetum degraded seriously and the microspore crowded together lead to the cells not be identified and appeared a large number of vesicular structures so the microspore could not developed normally. In the pollen grain stage, in the fertile anther the tapetum degraded to residual and the mature pollen grain had strong density and deep dye. In the sterile anther, the tapetum could not be distinguished and the pollen wall was observed but the microspores were empty, deformed and aborted completely.(4)Anther enzyme activity detection in different microspore developmental stages:The results indicated that along with the development of the anther the enzyme activities of POD、CAT、SOD were lowered than fertile lines.(5)Contrast and detection of mitochondrion-associated genes:Sequencing results showed that there was one poly-morphic locus(C/A) in different onion lines. This polymorphic locus randomly appeared in sterile and main-tainer lines which was not the characteristic difference between two lines. When sequences were compared, some 4 or 5bp deletion were characterized at cob1 in the sterile line DA that suggested a close relationship of onion CMS with the variation in DNA sequence. The sequences of PCR products from at pA, nad4 and cob2 of experimental material were same with onion mt DNA that had been reported that the experimental material chosen were so closely related.