农杆菌介导的香榧幼胚遗传转化体系  

Agrobacterium tumefaciens-mediated genetic transformation system of Torreya grandis‘Merrillii’immature embryos

作　　者：周佳君 胡恒康[1,2] 龚丽 干安格 喻卫武[1,2] 吴家胜[1,2] 黄坚钦[1,2] 张启香[1,2] ZHOU Jiajun;HU Hengkang;GONG Li;GAN Ange;YU Weiwu;WU Jiasheng;HUANG Jianqin;ZHANG Qixiang(College of Forestry and Biotechnology,Zhejiang A&F University,Hangzhou 311300,Zhejiang,China;State Key Laboratory of Subtropical Silviculture,Zhejiang A&F University,Hangzhou 311300,Zhejiang,China)

机构地区：[1]浙江农林大学林业与生物技术学院,浙江杭州311300 [2]浙江农林大学省部共建亚热带森林培育国家重点实验室,浙江杭州311300

出　　处：《浙江农林大学学报》2022年第1期13-21,共9页Journal of Zhejiang A&F University

基　　金：浙江省重点研发计划项目(2021C02037);中央财政林业科技推广示范项目(〔2014〕TS01);浙江省科技厅重大专项(2016C02052-12);浙江省科技厅重点研发项目(2018C02004)。

摘　　要：【目的】以香榧Torreya grandis‘Merrillii’幼胚为受体材料,开展农杆菌Agrobacterium tumefaciens介导的遗传转化研究,揭示影响香榧遗传转化的关键因素,以建立农杆菌介导的香榧幼胚遗传转化体系。【方法】以香榧种子突破种鳞后第8周至第11周的幼胚作为转基因受体材料,比较受体胚龄、农杆菌菌液浓度、侵染时间、抗生素质量浓度对遗传转化效率的影响,并采用潮霉素、绿色荧光蛋白GFP表达及GFP基因聚合酶链式反应对遗传转化香榧幼胚培养物进行阳性筛选。【结果】随着胚龄的增加,香榧幼胚抗逆性增强,污染率降低,成活率提高,第10周和第11周幼胚成活率分别为52.1%和52.3%。农杆菌菌液浓度和侵染时间均对香榧幼胚的污染率、成活率、愈伤组织诱导率以及体胚发生率具有显著影响(P<0.05)。菌液吸光度[D(600)]为0.5时,香榧幼胚愈伤组织诱导率和体胚发生率最高,分别为17.9%和17.3%;侵染时间为10 min时,愈伤组织诱导率和体胚发生率为最高,分别达17.5%和17.1%。羧苄青霉素对于受体材料的农杆菌脱除具有显著影响(P<0.05),当羧苄青霉素质量浓度为300 mg·L^(-1)时,成活率、愈伤组织诱导率和体胚发生率均最高,分别为60.5%、15.8%和17.5%。潮霉素对于香榧幼胚培养物的阳性筛选具有良好的效果,当潮霉素质量浓度为100 mg·L^(-1)时,成活率为30.5%。【结论】不同胚龄香榧幼胚遗传转化效果不同,突破种鳞后第10周香榧幼胚遗传转化效果良好;当农杆菌菌液D(600)为0.5,侵染时间为10 min时,转化效果最佳;300 mg·L^(-1)为羧苄青霉素脱除农杆菌的最佳质量浓度;100 mg·L^(-1)是潮霉素阳性香榧幼胚筛选的最佳质量浓度。图3表5参37。[Objective]The purpose is to explore genetic transformation mediated by Agrobacterium tumefaciens using Torreya grandis‘Merrillii’immature embryos as receptors,so as to reveal key factors affecting its genetic transformation and establish A.tumefaciens mediated transformation system of T.grandis‘Merrillii’embryos.[Method]The immature embryos of T.grandis‘Merrillii’seeds of 8 to 11 weeks after breaking through seed scales were used as transgenic acceptors.The effects of embryo age,A.tumefaciens concentration,infection time and antibiotic concentration on genetic transformation efficiency were compared.Hygromycin,GFP fluorescent expression and GFP gene polymerase chain reaction were used for positive screening and selection of immature embryo culture.[Result]The stress resistance increased,the contamination rate decreased and the survival rate increased with the increase of embryo age.The survival rates of 10-week and 11-week young embryos were 52.1% and 52.3%,respectively.The contamination rate,survival rate,callus induction rate and somatic embryogenesis rate of young embryos were significantly affected by the concentration of A.tumefaciens and infection time(P<0.05).When the liquid optical density[D(600)]was 0.5,the callus induction rate and somatic embryogenesis rate of young embryos were the highest,which were 17.9% and 17.3% respectively.When the infection time was 10 minutes,the callus induction rate and somatic embryogenesis rate were the highest,reaching 17.5% and 17.1%respectively.Carbenicillin had a significant effect on the removal of A.tumefaciens(P<0.05).When the concentration was 300 mg·L^(-1),the survival rate,callus induction rate and somatic embryogenesis rate were the highest,which were 60.5%,15.8% and 17.5% respectively.The concentration of hygromycin in different treatments had a good effect on the positive screening of young embryo culture.The survival rate was 30.5%when the concentration of hygromycin was 100 mg·L^(-1).[Conlusion]Immature embryos at the 10th week after the break

关 键 词：香榧 幼胚 农杆菌介导的遗传转化 羧苄青霉素 潮霉素 

分 类 号：S722.3[农业科学—林木遗传育种]