苗期快速分选水稻人工无融合生殖克隆种子

【目的】人工无融合生殖系统的建立为水稻杂种优势固定提供了途径。当前人工无融合生殖系统在产生克隆种子的同时会伴随大量的四倍体种子，有必要开发简易高效的分选克隆种子方法。【方法】通过不同温度、盐浓度处理春优84和MiMe（同时突变PAIR1、REC8和OSD1获得的可将减数分裂转化成有丝分裂的材料）种子萌发的幼苗，观察它们形态学上的差异，确定最佳筛选条件。在最佳条件下，处理Fix材料（通过基因编辑技术同时突变PAIR1、REC8、OSD1和MTL基因获得的可固定杂种优势的材料）种子萌发的幼苗，根据形态差异分选克隆种子。利用流式细胞术确定分选的效率。【结果】无融合生殖材料Fix产生4.4%的克隆种子和95.6%的四倍体材料；而MiMe材料的后代全是四倍体。利用滤网在23℃、28℃和32℃水温条件下萌发春优84和MiMe种子48h,Mi Me材料的初生根直径在3种温度处理下均显著大于春优84，以28℃处理最优。在28℃水温条件下，利用0%、0.05%、0.1%、0.2%和0.5%NaCl溶液培养春优84和MiMe种子7 d,Mi Me材料的根长、苗长与春优84无显著性差异，但春优84材料第1片...

Rapid Identification of Rice Clonal Seeds Generated by Synthetic Apomixis at Seedling Stage

【Objective】 The establishment of synthetic apomixis provides a way to fix heterosis in hybrid rice. However, synthetic apomixis only produces few clonal seeds with many tetraploid seeds. Thus, it is necessary to develop a simple and efficient method to select cloned seeds through the synthetic apomixis.【Method】 The seedlings of Chunyou 84 and MiMe (the PAIR1, REC8 and OSD1 were simultaneously mutated, which can turn meiosis into mitosis in rice) were exposed to different temperature and salt concentrations, and their morphological differences were identified to determine the best treatment conditions. Then, seedlings germinated from Fix plants(The PAIR1, REC8, OSD1 and MTL were simultaneously mutated in hybrid rice Chunyou 84 by genome editing technology) were treated in the optimal treatment conditions, and cloned seeds were sorted according to morphological differences. Finally, flow cytometry was used to determine the efficiency of the distinguishing cloned seeds method.【Results】 Fix plants produce 4.4% clonal seeds and 95.6% tetraploid plants, while the descendants of MiMe are all tetraploid materials. The seeds of Chunyou 84 and MiMe were germinated on a net floating on deionized water at 23 ℃, 28 ℃and 32 ℃for 48 h, and the primary root diameter of MiMe was found significantly larger than that of Chunyou 84 under all the three treatments at the most favorable temperature of 28 ℃. Then, the seedlings of Chunyou 84 and MiMe were cultured with 0%, 0.05%, 0.1%, 0.2% and 0.5% NaCl solutions for 7 days. Though the root and shoot length did not show significant difference between Chunyou 84 and MiMe plants, the first complete leaf from Chunyou 84 exhibited severe atrophic and necrosis phenotype in 0.1% NaCl solution, while the MiMe plants were slightly affected by salt damage. According to the above results, firstly, we germinated the Fix seeds on a net floating on deionized water at 28 ℃for 48 h, and screened the putative cloned Fix plants by observing the primary root. Then, the putative cloned Fix plants were cultured with 0.1% salt solution for 7 days, and the putative tetraploid plants were discarded based on the salt damage of the first complete leaf. Finally, the selected individual plants were verified by flow cytometry with screening efficiency of 61.1%. The cloned plants were grown in field and showed significant difference with tetraploid plants at maturity stage.【Conclusion】 After temperature and salt treatment of apomixis material Fix seeds at seedling stage, the clonal Fix plants can be roughly screened by morphological differences of root and leaf phenotype.