常英英,张启香,宋晓波,査巍巍,裴东.早实核桃体细胞胚发生和植株再生的影响因素研究[J].林业科学研究,2019,32(3):34-39
早实核桃体细胞胚发生和植株再生的影响因素研究
Analysis on Key Factors Affecting Somatic Embryogenesis and Plant Regeneration in Precocious Walnuts
投稿时间:2018-03-05  修订日期:2019-03-21
DOI:10.13275/j.cnki.lykxyj.2019.03.005
中文关键词:  早实核桃  体细胞胚  植株再生
英文关键词:precocious walnut  somatic embryogenesis  plant regeneration
基金项目:国家自然科学基金"RNAi介导的山核桃嫁接砧穗基因沉默信号的表达机理研究"(项目编号31470682);国家自然科学基金"核桃CRISPR/Cas9-GA20ox基因定点突变信号的砧穗应答机制"(项目编号31670682)和国家自然科学基金项目"核桃复幼提高不定根发生能力的多基因作用机制解析"(项目编号31672126)
作者单位E-mail
常英英 林木遗传育种国家重点实验室, 国家林业和草原局林木培育重点实验室, 中国林业科学研究院林业研究所, 北京 100091  
张启香 浙江农林大学林业与生物技术学院, 浙江农林大学亚热带森林培育国家重点实验室培育基地, 浙江 临安 311300  
宋晓波 林木遗传育种国家重点实验室, 国家林业和草原局林木培育重点实验室, 中国林业科学研究院林业研究所, 北京 100091  
査巍巍 林木遗传育种国家重点实验室, 国家林业和草原局林木培育重点实验室, 中国林业科学研究院林业研究所, 北京 100091  
裴东 林木遗传育种国家重点实验室, 国家林业和草原局林木培育重点实验室, 中国林业科学研究院林业研究所, 北京 100091 peigu@caf.ac.cn 
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中文摘要:
      [目的]建立2种早实核桃高效和稳定的体胚发生体系,为离体快繁技术及其基础研究提供技术和理论支撑,也为核桃遗传转化体系的构建奠定基础。[方法]分别以实生核桃‘林早香’和嫁接核桃‘中林6号’花后42~77 d的幼胚为试材进行离体培养,通过统计体胚诱导率,筛选体胚的最佳诱导时期;统计多次继代的体胚畸变率,并筛选最适ABA调节浓度;选取发育成熟的体胚进行饱和NH4NO3溶液脱水处理,分别处理0、24、48、72、96和120 h后统计失水量和转株率,探讨脱水程度对体胚植株再生的影响。[结果]外植体的发育时期对体胚发生至关重要,以‘林早香’和‘中林6号’幼胚为外植体进行体胚诱导,最佳诱导时期分别为花后约49 d和56 d,体胚诱导率分别为88.32%和86.67%。体胚经多次继代培养后产生较高畸变率,适量ABA处理不仅降低体胚畸变率,且提高体胚发生率,最佳ABA调节浓度为1.0 mg·L-1。随着体胚脱水时间的延长,体胚失水量逐渐增加,植株再生率先升高后降低,脱水72 h时,‘林早香’和‘中林6号’的体胚萌发率达到最高,分别为56.67%和53.33%,此时失水量分别为38.73%和40.56%。[结论]早实核桃幼果发育过程中,合子胚的体胚发生率随着发育时期变化,适宜发育时期是提高体胚诱导发生的关键;适量ABA在体胚多次继代培养过程中对畸形胚起重要调节作用;适度的脱水处理对核桃体胚再生具有良好的促进作用。
英文摘要:
      [Objective] To establish an efficient and stable somatic embryogenesis system, to provide technical and theoretical support for in vitro rapid propagation and its further research, and to lay a foundation for the construction of gene transformation of walnut.[Method] Two early-bearing walnut young fruits were sampled during 42-77 days after blooming. Somatic embryogenesis percentages were counted to screen the optimum development stage of the fruit. After subcultured for several times, the somatic embryos distortion percentage was calculated and then the optimum ABA concentration was tested. The somatic embryos were desiccated respectively for 0, 24, 48, 72, 96 and 120 h with saturated NH4NO3, and the germination rate of somatic embryo can be obtained.[Result] The highest somatic embryogenesis percentage of ‘Zao Lin Xiang’ and ‘Zhong Lin 6’ reached 88.32% and 86.7%, respectively, when the young fruits were sampled at 49 d and 56 d after flowering, respectively. Moderate concentration of ABA supplement not only reduced the rate of somatic embryos aberration, resulted from multiple subcultivation, but also increased the rate of somatic embryogenesis, and the optimum ABA concentration was 1 mg·L-1. As the dehydration time was prolongated, the water loss increased gradually, while the germination rate increased at first and decreased later on. After dehydrated for 72 h, the germination rate of ‘Zao Lin Xiang’ and ‘Zhong Lin 6’ were up to the highest (56.67% and 53.33%), while the water loss was 38.73% and 40.56%, respectively.[Conclusion] In the process of young fruit development of early-bearing walnut, the inducing rate of somatic embryogenesis is changed with the development period. The suitable development period is the key to improve somatic embryo induction. Proper amount of ABA also plays an important role in regulating deformed embryo in the process of somatic embryo multiple subculture. What is more, moderate dehydration treatment has a well promoting effect on the regeneration of somatic embryo of walnut.
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