凤丹牡丹胚培养体系建立及初代畸形苗甲基化变异分析  

Establishment of Paeonia ostii‘Fengdan’Embryo Culture System and Analysis of Methylation Variation in Malformed Embryo Culture Seedlings

作　　者：廉小芳 李昱莹 张婉青 郭丽丽[1,2] 张有福 侯小改[1,2] LIAN Xiaofang;LI Yuying;ZHANG Wanqing;GUO Lili;ZHANG Youfu;HOU Xiaogai(College of Agriculture/College of Tree Peony,Henan University of Science and Technology,Luoyang 471023,China;Henan Province Oil Peony Engineering Technology Research Center,Luoyang 471023,China)

机构地区：[1]河南科技大学农学院/牡丹学院,河南洛阳471023 [2]河南省油用牡丹工程技术研究中心,河南洛阳471023

出　　处：《河南农业科学》2020年第11期110-119,共10页Journal of Henan Agricultural Sciences

基　　金：国家自然科学基金项目(U1804233);河南省高校重点科研项目(19A180002)。

摘　　要：为提高凤丹牡丹组培技术在生产实践中的应用效率,以当年采收油用牡丹凤丹成熟种胚为外植体进行离体培养,设置不同光照及温度培养条件、不同大小成熟种胚、不同植物生长调节剂配比、不同质量浓度外源添加物等处理,完善凤丹牡丹胚培养体系;运用甲基化敏感多态性技术(MSAP)对培养过程中出现的畸形组培苗进行甲基化水平及模式变异的分析。结果表明,试验范围内最优培养条件为接种后进行7 d暗培养随即转入光照培养,适宜培养温度为(25±1)℃;适宜外植体为≥3 mm的成熟种胚;最优植物生长调节剂配比为0.8 mg/L NAA+1.0 mg/L 6-BA,其基础培养基为改良MS(Ca^2+加倍)培养基;与不添加外源物质相比,凤丹牡丹胚乳的添加对成苗率具有极显著抑制作用,添加75 mL/L椰汁处理组培养效果较好,成苗率为85.66%,添加1.0 g/L活性炭处理组成苗率最高,为90.46%。相较于正常组培苗,畸形苗总扩增位点数增多21.20%,总甲基化位点数减少12.97%;正常组培苗总甲基化率为85.38%,畸形苗总甲基化率为74.30%;畸形苗甲基化多态型变异占主导(82.37%),其中去甲基化变异类型占比最多,为46.45%。综上,建立凤丹牡丹胚培养体系适宜培养方案如下:改良MS(Ca^2+加倍)+0.8 mg/L NAA+1.0 mg/L 6-BA+1.0 g/L活性炭(或75 mL/L椰汁),培养条件为(25±1)℃下7 d暗培养后光照培养。相较于正常组培苗,凤丹牡丹胚培养畸形苗总甲基化率较低,以多态型去甲基化变异为主。In order to improve the application efficiency of oil peony Paeonia ostii‘Fengdan’tissue culture technology in production practice,the experiment was carried out with tree peony seed embryo as material.Different light and temperature culture conditions,different sizes of P.ostii‘Fengdan’mature seed embryos,different plant growth regulator ratios and different concentrations of additives were set to perfect the P.ostii‘Fengdan’mature seed embryo culture system.Methylation-sensitive polymorphism technology(MSAP)was used to analyze the methylation level and pattern variation of malformed embryo culture seedlings that appeared during the cultivation process.The results showed that the optimal culture conditions in the test range were dark culture for 7 d after inoculation and then switched to light culture.The suitable culture temperature was(25±1)℃.Suitable explants were mature embryos≥3 mm.The plant growth regulator ratio was 0.8 mg/L NAA combined with 1.0 mg/L 6-BA,and the basic medium was modified MS(double Ca^2+).Compared with no exogenous additives,the addition of P.ostii‘Fengdan’endosperm had a significant inhibitory effect on the seedling rate;75 mL/L coconut milk treatment had better effect with seedling rate of 85.66%;1.0 g/L activated carbon treatment significantly had the highest seedling rate of 90.46%.Compared with normal primary cultured seedlings,the total amplification sites number of malformed seedlings increased by 21.20%,and the total methylation sites number decreased by 12.97%.The total methylation rate of normal tissue cultured seedlings was 85.38%,and the total methylation rate of malformed seedlings was 74.30%.The variation of malformed seedlings methylation dominated by polymorphisms(82.37%),among which demethylation variation accounted for the largest proportion of 46.45%.The results of comprehensive analysis showed that the optimal culture protocol for aseptic culture of P.ostii‘Fengdan’seed embryos were modified MS(double Ca^2+)+0.8 mg/L NAA+1.0 mg/L 6-BA+1.0 g/L

关 键 词：凤丹牡丹 成熟种胚 外源添加物 胚培养体系 胚培养畸形苗 甲基化 

分 类 号：S685.11[农业科学—观赏园艺]