文献类型：期刊文献

中文题名：白栎茎段离体再生体系与VIGS体系的构建

英文题名：Construction of in Vitro Regeneration and VlGS Systems for Quercus fabri from Stem Segments

作者：蔡羽晴[1,2] 吴立文[1] 熊仕发[1] 施翔[1] 石洋鑫[1] 汪阳东[1]

第一作者：蔡羽晴

机构：[1]中国林业科学研究院亚热带林业研究所,浙江杭州311400;[2]南京林业大学,江苏南京210037

年份：2025

卷号：38

期号：6

起止页码：21-32

中文期刊名：林业科学研究

外文期刊名：Forest Research

收录：;北大核心:【北大核心2023】；

基金：浙江省“十四五”育种专项林木协作组课题(2021C02070-9);国家重点研发计划课题“林木高效体胚发生关键技术研究”(2023YFD2200602)。

语种：中文

中文关键词：白栎;组织培养;再生体系;VIGS体系

外文关键词：Quercus fabri;tissue culture;regeneration system;VIGS system

分类号：S792.184

摘要：[目的]探究在白栎组织培养中不同因素对外植体选择、腋芽萌发、不定芽增殖和不定根诱导的影响,建立白栎组织离体再生技术体系;利用再生体系培育的白栎幼苗,初步建立烟草脆裂病毒(TRV)介导的VIGS基因沉默技术体系。将白栎的组培再生和瞬时转化技术体系结合,实现对白栎基因功能的精准解析,为推动白栎遗传改良与分子育种奠定基础。[方法]以白栎茎段为外植体,通过对外植体、消毒时间和培养基进行选择,建立白栎组织离体再生技术体系;以白栎PDS基因(QfPDS)为报告基因,构建以TRV介导的VIGS技术体系。[结果](1)外植体宜选择5月采集的幼嫩茎段为材料,0.1%(质量分数)升汞消毒4.5 min或6月采集的半木质化茎段,0.1%升汞消毒6.0 min;(2)最佳的腋芽启动培养基配方为:WPM+0.5 mg·L^(-1)6-BA+80 mg·L^(-1)抗坏血酸,腋芽启动率为80%,启动时间为8.83 d;(3)最佳的不定芽增殖培养基配方为:WPM+0.03 mg·L^(-1)NAA+1.00 mg·L^(-1)6-BA,出芽率为85%,增殖系数3.13,伸长量2.83 cm;(4)最佳不定根诱导培养基配方为:1/4 MS+0.05 mg·L^(-1)NAA+0.50 mg·L^(-1)IBA,生根率为93.33%,平均根数3.93条,平均根长2.86 cm;(5)利用GV3101农杆菌介导TRV,配置10 mmol·L^(-1)氯化镁+10 mmol·L^(-1)2-吗啉乙磺酸+200 mg·L^(-1)乙酰丁香酮侵染液(pH=5.2),OD600=0.8的侵染液真空侵染白栎叶片,25°C室温培养22 d。[结论]在组织培养中,白栎外植体茎段易发生褐化与污染,可以通过选择外植体样品、消毒时间或添加抗褐化试剂以减缓褐化过程;在白栎再生体系建立过程中,白栎展现出较强的适应性和对NAA较高的敏感性;白栎VIGS体系虽然在基因表达分析中体现了极显著(p<0.01)的沉默效率,但在表型上表现出沉默不完全、不均一的现象。
[Objective]This study aimed to explore the effects of various factors on explant selection,axillary bud sprouting,adventitious bud proliferation,and adventitious root induction in the tissue culture of Quercus fabri,and to establish an in vitro regeneration system for Q.fabri.Additionally,using plantlets of Q.fabri cultivated through regeneration systems,a preliminary TRV(Tobacco rattle virus)-mediated VIGS(Virus-Induced Gene Silencing)system was developed for Q.fabri.By integrating the tissue culture regeneration system with transient transformation techniques,this approach enables precise functional analysis of Q.fabri genes,laying a foundation for genetic improvement and molecular breeding.[Method]Using stem segments of Q.fabri as explants,the regeneration system was established by optimizing explant type,sterilization duration,and medium composition.The Q.fabri PDS gene(QfPDS)was used as a reporter for constructing the TRV-mediated VIGS system.[Results](1)The explants should be selected from tender Semi-lignified stem segments collected in May with optimal sterilization achieved using 0.1%(w/v)mercuric chloride for 4.5 minutes,or from semi-lignified stem segments collected in June,with disinfection using 0.1%mercuric(2)The optimal medium for axillary bud initiation was WPM supplemented with 0.5 mg·L^(?1)6-BA and 80 mg·L^(?1) ascorbic acid,resulting in an 80%bud initiation rate and an average initiation time of 8.83 days.(3)The best proliferation was achieved on WPM supplemented with 0.03 mg·L^(?1) NAA and 1.00 mg·L^(?1)6-BA,with an 85%budding rate,a proliferation coefficient of 3.13,and an elongation length of 2.83 cm.(4)The optimal rooting medium was 1/4 MS supplemented with 0.05 mg·L^(?1) NAA and 0.50 mg·L^(?1) IBA,with a rooting rate of 93.33%,an average of 3.93 roots per explant,and an average root length of 2.86 cm.(5)For TRV-mediated VIGS,Agrobacterium tumefaciens strain GV3101 strain GV3101 was used with an infection solution containing 10 mmol·L^(?1) MgCl2,10 mmol·L^(?1) MES,and 200 mg·L^(?1) acetosyringone(pH=5.2),at an OD600 of 0.8.Leaves were vacuum-infected and cultured at 25°C for 22 days.[Conclusion]During in vitro culture,Q.fabri stem explants are prone to browning and contamination,which can be mitigated by selecting appropriate explant samples,adjusting sterilization time,or adding anti-browning agents.In the establishment of the regeneration system,Q.fabri exhibited strong adaptability and high sensitivity to NAA.Although the VIGS system demonstrated significant(p<0.01)gene silencing efficiency in expression analyses,phenotypic observations revealed incomplete and uneven silencing.