| Objective: The mechanism of sexual reproduction was investigated in Senecio scandens Buch.-Ham.ex D.Don,associated with duration phases of double fertilization and relative transcription expression levels of self-incompatibility genes(class-I SLG and class-II SLG)in leaf,petal and stigma.Methods: 1.The entire structural materials with pistil and stamens were haversted from the diverse duration phases of artificial pollination(time 1,2,4,6,8,10,12,18,24,36,48,72 and 96 h)in Senecio scandens Buch.-Ham.ex D.Don.Having been dyed with aniline blue and the treatment of paraffin sections after 24 h FAA fixation solution,the samples were thus used to investigate the basic structure of flowers and to observe the duration phases of double fertilization.2.The real-time quantitative q PCR was applied to detect the relative expression levels of class-I SLG gene and class-II SLG gene in different tissue such as leaf,petal and stigma,which had been pollinated within 24 h,in Senecio scandens Buch.-Ham.ex D.Don,based on observation of double fertilization.Results: 1.Senecio scandens Buch.-Ham.ex D.Don stigma is two crack,closed style,lower sub-room,a room,synantherous stamens.When the flower of Senecio scandens Buch.-Ham.ex D.Don bloomed,embryo sac was not yet mature.After pollination within 8 h,it was observed that it has an abnormal embryo sac.From 12 hours,it was observed that mature embryo sac contains three antipodal cells,one central cell with 2 polar nucleus(vertical or transverse distribution),one egg cell and the degenerated synergid cell at this time.2.Pollen germination and pollen tube growth: pollen grains are germinated and the pollen tubes are normalized after 2 hours;The pollen tubes develope into stylar canal and go on germinating in the mastoid cells from 4 to 6 hours;At 8 h after pollination,pollen tube is across the receptacle into the ovary;At 10 h after pollination,pollen tube enter the embryo sac and release the contents of pollen grains.3.Male and female gamete fusion: From 12 hours,Two nuclei are close to the egg and secondary nuclei.Phenomenon of fine nucleus near egg cell,fusion of fine nucleus and secondary nucleus appears in 18 hours,including small male nucleolus appearing in secondary nucleus.Zygote is attributable to the combination of sperm and egg cell in 24 h.Two-cell embryos from zygote are observed in 36 h.4.Embryonic development: The pear-shaped embryo was formed after two-cell embryos.With the embryo differentiation,a deep groove appears on the surface of pear-shaped embryo.Finally,the mature embryo was formed.5.Development of endosperm: As early as the egg cell fertilization,the primary endosperm nuclei began to split.From the period of 2,4 free endosperm to the period of free endosperm,more and more of the free endosperm,free endosperm nuclear gradually celluar,embryo sac filled with endosperm cells,ultimately,the endosperm nucleus and cell wall disintegrate,and endosperm is absorbed by the embryo.6.As shown in the results of real-time quantitative q PCR,the relative transcription expression level of self-incompatibility gene,class-I SLG,was higher in petal than in leaf and stigma,and there was no significant difference between leaf and stigma.There was no significant difference in the expression level of class-II SLG gene between leaf and stigma,while the expression level in petal was significantly different from that in leaf and stigma.(p< 0.05).Conclusion: 1.The double fertilization process of Senecio scandens Buch.-Ham.ex D.Don: From 1 h to 10 h,pollen germinate for the pollen tube and is across the stylar canal,through the receptacle and ovary,finally,reach the embryo sac and release the pollen grains;From 12 h to 24 h,the combination of sperm and egg form zygote,another sperm cells and polar nucleus fuse to form the primary endosperm nucleus to complete the dualfertilization process;From 24 h to 36 h,zygotic split of two-cells embryos,and further development of embryos,the primary endosperm nucleus are further divided,cultured and filled with embryo sac to form endosperm,which is gradually absorbed and dissociated during the development of the embryo.2.When the flower of Senecio scandens Buch.-Ham.ex D.Don bloomed,the embryo sac is not yet mature.From 12 hours,mature embryo sac contains three antipodal cells,one central cell with 2 polar nucleus(vertical or transverse distribution),one egg cell.,and the degenerated synergid cell at this time.It can be proved that pollination triggered embryo sac of Senecio scandens Buch.-Ham.ex D.Don gradually mature.3.The class-I SLG gene and class-II SLG gene are present in Senecio scandens Buch.-Ham.ex D.Don and show differences in tissue expression level after pollination,indicating that they may be involved in the process of sexual reproduction. |
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