| The fertility of alfalfa (Medicago sativa L.) is closely related to seed yield. To identify the female sterile mutants is important for revealing the mechanisms of floral ontogeny and development of pistil in alfalfa. The cytogenetic analysis of these mutants is useful for loci the genes related to female sterility furthermore. All these above will provide the basis for fertility improvement and seed production in alfalfa breeding programme. In this paper, using a multi-pistil mutant dedigned as mip we have identified in our previous work, its phenotypic character, bud differentiation, the development of embryo sac and female gametophyte, ultrastructure of stigma and pollen tube guidance were investigated by field observation, paraffin serial sections, fluorescence staining and SEM observations. The results are as below:1) From 2009-2011, the rate of multi-pistil of mip under 3-year continuous observations in field showed that there were 45.8%-70.3% florets exhibited 2-3 pistils. With the increasing of the numbers of pistil, the length of pistil decreased, ranged from 5.92 cm to 4.65cm. The numbers of ovule per floret showed the tendency to ascend from 11.4 to 21.8. Accompanied with the multi-pistil mutation, we also found that there was a secondary floret differentitated at the same palce with the main floret in mip, and this extra secondary floret never pouduced pods. This secondary floret was always exhibited multi-pistil characteristics while the main floret may be single pistil or multi-pistil. If the main floret was normal with single pistil, it could produce pods. However, the seed-setting of the main floret in mip was significantly lower than that of wild type plant, the former was only 18.38% and the later was 84.62%. These results indicated that the multi-pistil mutantion negatively affected the female fertility of mip. The seed set of reciprocal crosses between mip and wild type plant was also low, which indicated that pollens fertility of mip was also affected.2) The floral ontogeny was investigated in mip and wild type plant by using scanning electron microscope (SEM). We found that the flower bud differentiation in normal alfalfa plant could be classified into four main stages:the stage of floral primordium differentiation, the stage of calyx primordium differentiation, the stage of petal-stamen-pistil primordium differentiation, and the stage of formation of stamen and pistal organs. Such differentiation and development of floral organs of alfalfa conflicted with the "ABC" model hypothesis proposed in plant species of Arabidopsis and Antirrhinum, in which the initiation of the primordia of the floral organ is a centripetal and sequential process. Here it is unidirectional in all whorls starting from the abxial positon of the flower with a high degree of overlap. In mip, our observations showed that there were no differences between the ontogeny of wild type and mip flowers during the early stages. However, in the late stage, extra carpel in place of the vexillary stamen was visible.3) We found that the MMC in normal alfalfa plant undergoes meiosis to generate four spores, and three of which undergo programmed cell death, leaving only the proximal (chalazal) megaspore as the functional megaspore in each ovule. The megaspore then undergoes three sequential mitotic nuclear divisions to generate the eight nuclei of the mature embryo sac. In mip, MMC could produce normal functional megaspore, but abnormality happened at four-nuclear stage during the nuclear division phase of megagametogenesis. The development of female gametophyte in mip mutant showed the abnormal nuclear numbers and positions, poly-eggs, and degeneration of embryo sac.4) The pollen tube guidance of mip and wild type plant were observed under fluorescence microscopy, the results showed that 8 h after pollinated, significant divergences were detected between the mip and wild type. There were many pollen grains attached to the stigmas and began to germinate in wild type, whereas few pollen germinated on the stigma in mip mutant. After few hours, a large fraction of pollen tubes (PTs) appeared in the style of wild type plant, but fewer PTs went through the style of mutant. There is no callose deposition on ovule of wild type plant during the periods of pre-fertilization and post-fertilization, however, in the case of mip, callose depositon were observed on the ovule. After PTs had reached the ovary, they were guided and move to micropyle in a way of "N" shape in the wild type plant. On the contrast, PTs in mip mutant were failed to be attracted by the micropyle with a kind of sprawled growth. The embryo of wild type showed normal development 72h after pollination, whereas the embryo of mip failed to develop furthermore. The callose deposition observed on the ovules at this stage in mip maybe confirm the unsuccessful fertilization.5) According to the SEM observations of stigma, no honeycomb like stuctures had been found on the surface of stigma in mip compared to that in wild-type plant. Although alfalfa belongs to the typical wet stigma type, almost no exudate was observed on the plication of the stigma in mip mutant. The neutral red staining showed that the stigma of wild type plant was red after the staining, which meant that the cuticle was broken and lots of secretion released. There were only 50% stigmas were stained in mip, which indicated that half of the cuticle was still intact after pollinated.All in all, we concluded that this new spontaneous mutant(mip) identified in alfalfa was a female partial-sterile mutant with multi-pistil mutantion. The abnormal development of embryo sac and female gametophyte in mip happened at the four-nuclear stage, which maybe lead to the failure of pollen tubes guidance after pollination. The female sterility and low seed set of mip also tightly linked to the defected function of the stigma. This is the first report on natural multi-pistil mutant with partial-female sterile and it is a new source of mutation to understand female reproduction development in Medicago sativa L. in the future. |
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