| Rice is the one of the staple food crops in the world.Hybrid rice contributes a lot to the increase of the global food production,and the two-line hybrid rice based on the PTGMS line has been widely used in agricultural development.However,diploid hybrid rice varieties as the host,hybrid indica japonica subspecies is still emerging,the productive situation are plagued by rice scientists more than 10 years.How to break through the diploid rice of chromosome is very small and fine genes are mostly exist in wild rice,so breeding high resistance and high yield rice has a lot of difficulties.Facing the severer food crisis,a requirement for rice breeding must be proposed.To breed super rice by making a mixed advantage of polyploid and distant hybridization is a new rice breeding strategy.From the evolutionary perspective,polyploid rice has more possibility of favorable genetic combination,so it has potential high resistance and production.Meanwhile,with the development of molecular biology,the molecular mechanism of PTGMS has always been a popular area of agricultural study.In 2011,Zhang Qifa' s team discovered a PGMS gene pms3 in Nongken58S of the PGMS line,which is a long non-coding RNA related to the PGMS,called LDMAR(long-day-Specific male-fertility-associated RNA).This gene is in chromosome 12 with a whole length of 1236bp.The RNA in Nongken58S produced a C?G SNP after spontaneous mutation,which increased the methylation in the promoter region of this sequence and decreased the transcriptional level in long day condition thus programmed cell death in the pollen happened untimely and caused photoperiod sensitive male sterility.After polyploidization,however,whether the pms3 can show the fertility regulation?So,the typical Photoperiod-and thermo-sensitive genic male sterility rices are created by autopolyploid to recognize the features and functions of genic sterile gene and creating indica japonica subspecies of the hybrids has great significance.The thesis uses the representative Nongken58S of PGMS,Peiai64S of deuterogenic TGMS and HD9802S of TGMS as experimental materials to conduct the experiment:1)Produce polyploid.Use the mature embryo of the three materials to induce the callus by tissue culture and inject the double liquid with colchicin to conduct suspension culture.And then cultivate to a shoot by dividing and rooting thus doubling a diploid rice to a tetraploid rice.The result of this process is that autopolyploid is gained successfully in both Nongken58S and Peiai64S while failed in HD9802S.The result suggest that different genotype of lines have various in polyploidization.2)After obtaining polyploid,conduct ploidy detection in the polyploid plants by morphological observation and cytological experiments to ensure the production of the polyploid materials.It was found that the chromosome was 2n=24 when detecting the root tip chromosomes of Nongken58S-4X and Peiai64S-4X,which showed that the polyploid doubled the original number.Considering the density and size of the stomata,the tetraploid has bigger stomata and smaller density.There are obvious differences between autotetraploid and diploid about explant morphology,anthers,tassels and seeds.Compared with diploid,Nongken58S-4X has less tillers,obvious dwarf and its seeds have clear arista while the size of the seeds is similar to the diploid;however,Peiai64S-4X has similar plant height with the diploid but its seeds are bigger with arista and have less tillers.3)After planting Nongken58S-4X and Peiai64S-4X in Wuhan oil filed in June,2015,the statistics of agronomic characters(height of the plants,length and width of the leaves,the number,length of tassels,the length and width of the seeds,number of total seeds,actual seeds and setting rate in each tassel)and pollen fertility observation were conducted respectively to the contemporaneous diploid and the tetraploid.In November,the rice was planted in Nan Fan planting base in Lingshui county in Hainan province and agronomic characters and pollen fertility observation were conducted.The differences between the growing of diploid and tetraploid in Wuhan(high temperature and long day sunshine)and Lingshui(low temperature and short day sunshine)were found.The result of planting in Wuhan showed that the fertility of Nongken58S-2X and Peiai64S-2X are consistent with the original research roles.Male sterility is still in July-in early September.In mid-late September,non-fertility became partly-fertility and in early October became all-fertility.In Lingshui,the fertility in March became partly-fertility in early April,and complete sterility in mid-late April.However,two autotetraploids had alwaays been non-fertility in Wuhan and its fertility had not been influenced by the temperature and sunshine.In Lingshui,it had always been partly-fertility,and it changes sterility in mid-late April which was similar to the diploid plants.However,the seed set rate is always low on autotetraploids.Probably,it is lacked PMes gene in polyploidy.4)Firstly,it showed that the sequence of pms3 remained quite well and it wasn't influenced by the tissue culture,cell mutation and the colchicin after detecting pms3 in the four sample plants by normal PCR.Especially in the two autotetraploid materials,there were no chromosome missing or cell mutation.Then,the detection of the expression amount difference of the pms3 intetraploid was conducted using real-time PCR and it showed that there was difference in the expression amount in the leaves and young pancicle RNA.The result shows the expressions is various in autotetraploid rice.The research suggest that it is available to creat autotetraploids derive from parent male-sterility diploid.The diploid and autotetraploid is different in all aspects,but the photoperiod-and thermo-sensitivity is still presence in autotetraploid.Furthermore,different autotetraploids also show various.This phenomenon shows scientists could choose better autotetraploid in accordance with parent diploids in breeding seed.Finally,I find PA64s-4x is more suitable for used as intermediate materials in distant hybridization. |
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