苗期快速分选水稻人工无融合生殖克隆种子

doi:10.16819/j.1001-7216.2022.220509

摘要/Abstract

摘要：

【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48 h，MiMe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，MiMe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片完全叶在0.1%盐浓度下严重萎缩坏死，而MiMe材料受盐害影响较小。以28 ℃水温条件下先处理Fix种子48 h，先通过初生根根直径差异进行初次筛选，再利用0.1% NaCl溶液培养7 d，观察第1片完全叶形态差异再次分选，最后挑选出来的单株利用流式细胞术验证，分选效率达61.1%。将获得的克隆植株和四倍体对照种植田间，至成熟期表现明显的植株形态差异。【结论】在水稻苗期，对无融合生殖材料Fix进行温度及盐处理后，通过初生根、叶片等形态上差异可达到初步分选克隆植株的目标。

关键词: 人工无融合生殖, 克隆种子分选, 温度处理, 盐处理, 形态差异

Abstract:

【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.

Key words: synthetic apomixes, clonal Fix screening, temperature treatment, salt treatment, morphological difference

曹跃炫, 严绘景, 王克剑, 刘朝雷. 苗期快速分选水稻人工无融合生殖克隆种子[J]. 中国水稻科学, 2022, 36(6): 656-662.

CAO Yuexuan, YAN Huijing, WANG Kejian, LIU Chaolei. Rapid Identification of Rice Clonal Seeds Generated by Synthetic Apomixis at Seedling Stage[J]. Chinese Journal OF Rice Science, 2022, 36(6): 656-662.

图/表 4

图1 创制MiMe和Fix材料 A—转基因植株突变统计；B，C—MiMe和Fix材料靶位点突变情况。蓝色字母、横杠表示突变碱基；红色字母表示PAM序列。D―缺失；I―插入。

Fig. 1. Generation of MiMe and Fix materials. A, Mutation statistics of transgenic plants; B and C, Mutation of the target-sites in the selected MiMe and Fix materials; Blue lowercase letters and stripes indicate mutational bases; Red letters represent PAM sequences; D, Deletion; I, Insertion.

图2 不同温度处理下二倍体与四倍体幼苗形态表型 A~F—23、28和32℃下春优84和MiMe幼苗表型，标尺=2 cm；G~I—初生根宽度统计(n=10)，**表示采用t检验差异达0.01极显著水平。

Fig. 2. Morphological phenotype of diploid and tetraploid seedlings under different temperature treatments. A-F, Morphological phenotype of Chunyou 84 and MiMe seedlings under temperature treatments of 23, 28 and 32℃. Bar=2 cm. G-I, Statistics of primary root width(n=10). Ten biological replicates, ** indicates significant difference at 1% level according to the t test.

图3 不同盐浓度处理下二倍体与四倍体幼苗形态 A—0%、0.05%、0.1%、0.2%和0.5% NaCl溶液处理春优84和MiMe幼苗表型，标尺 = 5 cm；B—芽长和根长统计，10次重复，*表示采用t检验差异达到0.05显著水平；C—0.1%盐处理下春优84和MiMe幼苗第一片叶表型。CY84—春优84。

Fig. 3. Morphological phenotype of diploid and tetraploid seedlings under different salt treatments. A, Morphological phenotype of Chunyou 84 and MiMe seedlings under 0%, 0.05%, 0.1%, 0.2% and 0.5% NaCl solution treatments. Bar = 5 cm. B, Statistics of shoot and root length. Ten biological replicates, * indicates significant difference at 5% level according to t test. C, The first leaf phenotype of Chunyou 84 and MiMe seedlings under 0.1% salt treatment. CY84, Chunyou 84.

图4 分选无融合生殖克隆种子 A—500粒Fix种子在28℃下萌发处理后，根据初生根粗细筛选78个假定的克隆植株；再通过0.1%盐处理7 d，根据第1片叶枯死情况筛选36个假定的克隆植株；最后采用流式细胞术鉴定出22株克隆植株；B—克隆Fix和四倍体植株流式细胞术结果；C—田间克隆Fix和四倍体成熟期植株，标尺=20 cm；D—田间克隆Fix和四倍体成熟期植株分蘖数、株高、穗长、一次枝梗数和结实率(n=6)，*和**分别表示采用t检验差异达到0.05和0.01显著水平。

Fig. 4. Identification of clonal seeds generated by synthetic apomixis. A, Firstly, a total of 500 Fix seeds were germinated at 28℃, and 78 putative clonal Fix plants were screened according to the root thickness. Then, these 78 plants were treated with 0.1% NaCl solution for seven days, and 36 of them were screened as putative clonal Fix plants based on the first leaf phenotype. Finally, the selected 36 plants were further confirmed using flow cytometry, and 22 of them were clonal Fix plants. B, Represented ploidy results of clonal Fix and tetraploid plants. C, The plants of clonal Fix and tetraploid grown in field at maturity. D, Statistics of tiller number, plant height, panicle length, primary branch number (PBN) and seed setting rate(n=6), * and ** indicate 5% and 1% significant difference at 5% and 1% levels according to the t test, respectively.

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