| Mutagenesis is the important means to create genetic variation in cotton breeding. A large number of energy rays are frequently applied in mutant breeding, such as X-,β-, and y-rays, neutrons, and protons. In most cases, seeds were used as mutation materials, but it is easy to obtain chimera in the M1 progeny. Pollen grains are haploid, which has a nutritional nucleus and two sperms. Pollen grain is sensitive to all kinds of mutagens. Pollination with irradiated pollen grains could directly transfer the mutation of sperms to the offspring of the pollinated plant. Furthermore, pollen irradiation is the effective means for creating dysploid. Gamma rays belong to the most efficient tools to create mutants in plants, with the advantages of convenient operation, short cycle, and high mutation quantity. Ion beams have been applied as a nuclear technique since the late 1950s, especially in the field of surface modification of materials. In the 1990s, this technology was first applied to improve crop cultivars by Chinese scientists, achieving great success in plant and microorganism. But the short path of ion-implantation was in doubt on whether the low-energy ions could enter plant cells. And the interaction between the organism and these ions is complicated. At present, the biological effect of ion-implantation is mostly emphasized on statistical analysis of phenotypic and genetic effects. Detailed information of interaction between ion beam implantation and the structure, vigor and skeleton system of cell is still insufficient. Alpha-particles, which are directly ionizing particles with a high linear energy transfer (LET), have a strong interaction with the target substance. Alpha-particles have been widely used as a mutagenic source in mammalian cells. However, low doses of ionizing particle radiation have beneficial effects on mammalian cells. Because plant cells have tough cell walls and because alpha-particles transfer energy to very limited regions, it is not known whether alpha-particles could be used as an effective mutagen on plant breeding. In this study, we used upland cotton as irradiation material to compare the effects induced by irradiation of y-rays, nitrogen ions and alpha-particles in the following:the ultrastructure of the pollen exine and interior walls, the germination rate of the pollen grains, the number of pollen tubes in the styles after pollination, and the F-actin distribution in the pollen tubes. The effects of the law of genetic variation of their M1, M2 and M3 progeny induced by 60Co-γray were also studied. The results will be valuable not only for enriching the germplasm resources but also to the foundation of mutation material foreground for the location and clone of correlative genes. The main results were as follows:1. The examination methods of development of upland cotton male gametophyteWe used DAPI labeling and fluorescence microscope to visualize the meiosis of the male gametophyte. The results showed that the size of bud is closely related with the development of pollen mother cell. The buds were harvested in the 7 am-9 am, whose glossy and elongated bell-shaped at the size of (0.4-0.5)×(0.3-0.4) cm were in meiosis. The difference in the stages of meiosis existed not only in different individual plant, different bud of the same in dividual inflorescence, but also in different cell of the same bud. The procedure of detaching the exine of Gossypium hirsutum L. pollen grain was established in this experiment. The pollen grain was fixated with 4% paraformaldehyde solution, oxidated with 10% sodium hypochlorite solution, heat shocked at 55℃for 30 minutes and pressed. After these treatments, the exine could be detached along the full de-exined pollen grains. The pollen grain and de-exined pollen grain were stained with DAPI. The nucleus of cotton pollen grain was not observed, but the nucleus of de-exined pollen grain was showed clearly. The inner wall had not fluorescence. The three nuclei of the pollen grain of Gossypium hirsutum L. were determinated firstly. We used TRITC-phalloidin labeling and laser scanning confocal microscope to visualize the F-actin cytoskeleton of the pollen tubes after germination in liquid medium. The results showed that F-actin bundles oriented along the longitudinal axis in the long pollen tube and extend only 10 to 20μm away from the tip of the pollen tube. Growing pollen tubes were loaded with the Ca2+ sensitive ratiometric dye Fluo-3 am, the result showed that the [Ca2+]i at the tip increased during the peaks of the Ca2+ oscillations, and that the Ca2+ gradient extended further down the shaft of the tube.2. The study on mutation effects of N+、αandγrays on Gossypium hirsutum L. pollen grains and identification of mutation progeny 2.1 To study the radiobiological effects of low-energy nitrogen ions on plant cells, nitrogen ions of varied energies (10 keV,20 keV and 30 keV) were implanted in upland cotton pollen grains. Irradiation effects on pollen grains were assessed in terms of the ultrastructural changes in the exine and interior walls of pollen grains, the germination rate, the number of pollen tubes in styles after pollination, the extent of the tip-focused Ca2+ gradient and the ultrastructural changes in the F-actin of pollen tubes that developed from the treated pollen grains upon germination in liquid medium. The results showed that nitrogen ion passed through the pollen grains by etching and penetrating the exine and interior walls and destroying cell structures. More pollen grains were destroyed as the energy of the nitrogen ions increased. The pollen grain germination rate as well as the number and length of the pollen tubes after nitrogen ion implantation were decreased with increased ion implantation energy. Damaging effects were also observed for the tip-focused Ca2+ gradient and the F-actin of pollen tubes. The function and structure of pollen tubes in pollination and fertilization may be affected by the changes in the ultrastructure of pollen grains and the gene expression profile of pollen tubes.2.2 The SSR-based molecular markers technique was used to determine the polymorphisms of ovule DNA. The ovule was developed after the pistils were pollinated by the pollen grains which had been implanted with nitrogen ions. The result showed that the ovule DNA polymorphisms was changed, which indicated that the DNA sequences of sperm cell were altered. The suppressive subtraction hybridization (SSH) technique was used to isolate the cDNA fragments that showed differential expression between the M1 progeny developed after the pistils were pollinated by the nature and pollen grains implanted with the nitrogen ions. Two differential expression cDNA libraries were constructed using the cDNAs of the treated-M1 progeny as driver and the cDNAs of untreated-M1 progeny as tester, vice versa. The results demonstrated that the insertion fragments of SSH library were ranging from 450 to 700 bp.50 clones were randomly picked for PCR and sequencing. After matching the result in GenBank dbEST and the BLASTN,50 sequences were obtained. Of them,38 had 56%-100% similarity with known genes, which was 76% of the total EST; 5 sequences had their homology with the sequences in GenBank with unknown function, which was 10% of the total EST; 9 were hypothetical protein, which was 18% of the total EST; 4 had no similarity with the sequences in GenBank, which might be new genes; or the gene could not search the similarity with the sequences of other species, because the sequences located in the easy-varied 3’-end, which was 8% of the total EST.2.3 Effect of parameters of ion implantation machine, including ion energy, total dose, dose rate, impulse energy and implantation interval on the pollen grains of upland cotton implanted with nitrogen ions were studied. The best parameters were screened out. The results also showed that the vacuum condition before the nitrogen ion implantation does not affect the pollen viability. From the perspective of effects, the 5 parameters run as energy>dose rate >total dose> impulse dose> interval. Whereby the comprehensive considerations shall be given for the ion implantation based on the test objects. For instance, the high energy-low dose-long interval might be an effective option to improve the offspring vitality.2.4 20 Gy 60Co-y ray irradiation effects on upland cotton pollen grains were tested in terms of the ultrastructural changes in the exine and interior of pollen grains, their germination rate, the single primer amplification reaction polymorphism of ovule developed after the pistils were pollinated with the pollen grains which had been irradiated with 60Co-y ray, and the law of genetic variation of their M1, M2 and M3 progeny. The results showed that 60Co-y ray had no effects on the exine wall of the pollen grains. The interior structure of pollen grain was destroyed significiantly. The interior wall became thin and irregular, and part of it concavitied to the inner. The endoplasmic reticulum depolymerized. The amount and the density of pollen grain inclusions increased. The number of pollen tubes in style decreased by 38%, compared with the control group. The single primer amplification reaction polymorphism of ovule increased. The germination rate of M1 progeny was decreased by 41.03%. In M1 progeny, the length of taproot, longest lateral root, average lateral root, the number of lateral root, and the height of seedling decreased by 22.24%,18.93%,11.80%,28.02%,23.05%, respectively., compared with the control group. The percentage of sterility plants was 56.7%. The coefficients of variations of boll number, lint percentage, perimeter of stem, seed index, fruit branch number, longissimus fruit branch and plant height increased by 103.206%,74.588%、75.96%,69.83%、33.25% and 29.624%,11.843%, respectively., compared with the control group. In M2 progeny, the percentage of sterility plants was 56.7%. And the coefficients of variations of boll number, cotton yield, fruit branch number, plant height, boll weight, and lint percentage increased by 21.944%,16.261%、3.827%.3.986%.7.25% and 0.497%, respectively. Compared with the control group, the coefficients of variations and change range of agronomic traits in M2 progeny were less than them in M1 progeny. In M3 progeny, the means of plant height, fruit branch number, boll number, cotton yield and boll weight were all less than that of the control group, respectively. And the lint percentage was more than that of the control group. The coefficients of variations of the agronomic traits in M3 progeny was least, compared with that in the progeny of M1 and M2. The mutants were inherited stably. Therefore, in the progeny selection, the M1 seeds are mixed harvested, and single-plant harvested in M1 seeds.2.5 We compared the effects of irradiation by y-rays and alpha-particles on the structure and function of upland cotton pollen grains during pollination. We used mature pollen grains of upland cotton as irradiation material to observe changes in the following: the ultrastructure of the pollen exine and interior walls, the germination rate of the pollen grains in culture medium, the number of pollen tubes in the styles after pollination, and the F-actin distribution in the pollen tubes in medium. We found that alpha-particle and y-ray irradiation have different biological effects on the structure and function of cotton pollen grains.2.6 Because the quantity limitation of irradiated pollen grains every time, alpha-particles do not apply to mutation breeding (Field pollination need a mass of pollen grains.). Nitrogen ions have obviously damage effect on the pollen grains, but the mutation progeny have no evident variation.20 Gy 60Co-γray is suitable for cotton mutation breeding. The pollen grains irradiated by 20 Gy 60Co-y ray were pollinated in the field, a large number of mutants was obtained in the mutation progeny. |
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