林业科学研究2025，Vol.38Issue(6)：21-32,12.DOI:10.12403/j.1001-1498.20240460
白栎茎段离体再生体系与VIGS体系的构建
Construction of in Vitro Regeneration and VlGS Systems for Quercus fabri from Stem Segments
摘要
Abstract
[Objective]This study aimed to explore the effects of various factors on explant selection,axil-lary 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 regen-eration 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 re-porter 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 acet-osyringone(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 adaptabil-ity and high sensitivity to NAA.Although the VIGS system demonstrated significant(p<0.01)gene silen-cing efficiency in expression analyses,phenotypic observations revealed incomplete and uneven silencing.关键词
白栎/组织培养/再生体系/VIGS体系Key words
Quercus fabri/tissue culture/regeneration system/VIGS system分类
农业科技引用本文复制引用
蔡羽晴,吴立文,熊仕发,施翔,石洋鑫,汪阳东..白栎茎段离体再生体系与VIGS体系的构建[J].林业科学研究,2025,38(6):21-32,12.基金项目
浙江省"十四五"育种专项林木协作组课题(2021C02070-9) （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) （2023YFD2200602）
