Study On The Irradiation Effects And Mechanism After Nitrogen Ion Implanting Into Cotton (Gossypium HirsutumL.) Pollen

Ion beam bioengineering technology has been considered as a new method for mutation breeding.With low-energy ions impanted organisms, the biological effects and the function mechanisms of the technology were investigated by some researchers,and the technology has been used into genentic breeding and gene engineering. The researchers have observed some biological effects and revealed some basic mechanisms for the past decade,and the great development and application have been obtained .In this paper ,the biological effects of the cotton pollon implanted by nitrogen ions were studied and discussed.The biological effect of Sumian 12 pollen implanted by N ion beams were studied in present thesis . The viability of Sumian 12 pollen were implanted by nitrogen ion beam (the energy was 20KeV, the doses were 100 units( 0.26×10¹⁶ N⁺/cm²), 150 uints(0.39×10¹⁶ N⁺/cm²), 200 units(.52×10¹⁶ N⁺/cm²),300 units(0.78×10¹⁶ N⁺/cm²) respectively,no-treated was control.) . The effects on viability of cotton pollen , the exterior and interior structure of pollen, pollen germination rate, the growth rate of the pollen tube, the situation of insemination, the mutation aroused by ions implantation in ovule using SSR and diversity gene of the offspring after ions implantation using SSHwre studied . The result showed that:The viability of Sumian 12 pollen before and after nitrogen ion implantation were measured by six methods:In situ pollen germination, Benzidine-α-Naphthol method, pollen tube quick germination, FDA fluorescence method, TTC method and pollen in vitro germination. Both the results of pollen viability before and after N⁺ injecting were zero, so TTC method was not suitable. We found that five methods, except TTC method, be suitable for measuring pollen viability before N⁺ implantation, their results were all about 98%. Pollen tube quick germination couldn't measure the viability of pollen in vacuum, but we know that vacuum not influence pollen viability. Benzidine-α-Naphthol method, pollen tube quick germination and pollen in vitro germination were also incompetent for measuring the viability after N⁺ injecting. The results of FDA fluorescence method were repeatable and consonant with the results of in situ pollen germination. In conclusion, FDA fluorescence method were competent for measuring the viability of cotton pollen before and after N⁺ injecting. Our results showed a significant negative correlation between ion implantation and pollen viability.The pollen germination on the stigma and pollen tube grow in the style by decolorized analine blue methed with fluorescence microscope were observed. A simple methed for truly research the pollen germination on the stigma and pollen tube growth and the suitable temperature of pollen germination were found .The result showed that: The beginning time of in situ pollen germination were consistent with the time of pollen in vitro germination. At 1 hour after pollination of in situ pollen germination, only few pollen grain germinated in stigma. At 4 hour after pollination of in situ pollen germination, all of the pollen grains on stigma were germination. At 12 hour after pollination of in situ pollen germination, large number of pollen tubes have grown to the end of the style. At 2 hour after pollination of in vitro pollen germination, few pollen grains begin germination. At 13 hour after pollination of in vitro pollen germination, a lot of pollen tubes have grown to the end of the style. Otherwise, the style waas growing while the pollen tubes growing. The suitable temperature for incubating pollen germination was 25～35℃. The most suitable temperature was 30℃.In the study , all of the changes after N⁺ injecting cotton pollen were observed by scanning electron microscope(SEM), transmission electron microscope(TEM), decolorized analine blue methed and paraffin section method. The radiation mechanism and its biologic effect after ion implantation were research . The result showed that: The number of break pollen grain were increasing with the increase of the N ion implantation dose, ion beam could distinctly cut pollen wall and produce different holes. The inclusion in pollen was decreasing with the increase of ion implanting dose, the higher the dose was , the more the interspace had, the larger of the starch granules volume were. Ion beam could distinctly decrease pollen germination rate. The number of pollen tubes in style declined after N ion implantation, after pollination, the number of pollen tubes of control pollen were 320±42, after implanting with dosages 100 units,150 units,200 units,300 units, the number of pollen tubes were 145±19.8,114±12.7,94±11.4,51.3±11 respectively. But the pollen tubes growth rate were accordant after implanting different dose. At 13 hours after pollination, all pollen tubes have grown to the end of the style. The fecundation time and insemination time were coinciden with different ion dose. Ovary weight and diameter size were reduce with the increase of implantation doses. SSR (simple sequence repeat) analysis indicated that the molecular genetic polymorphism of ovules, which being pollinated by N ion treated pollens, were changed. This result suggested that ion beam might cause some genetic changes of pollens. The SSH result showed: The BLASTN homology search analysis revealed that 3 nonredundant clones were classified into the following groups : 1 known function genes , 2 cotton ESTps of unknown function . The ion beam implanted could bring on M₁ genetic variation of agronomic character, but there was no orderliness between genetic variation of agronomic character and ion dosage. The result suggested that ion beam could enter into the inner of the cell and changed the structure and the genetic materials, reduced the viability of pollen and the growth rate of ovary, lead M₁ genetic variation of agronomic character and produce obviously biological effects.